TM
Pages S1-S148
Gay Crooks, MD Boston, MA, USA Boston, MA, USA
Torrance, CA, USA Boris Fehse, PhD Sjaak Philipsen, PhD
Hamburg, Germany Rotterdam, The Netherlands
Willem Fibbe, MD, PhD Louise Purton, PhD
Camilla Forsberg, PhD
Leiden, The Netherlands Santa Cruz, CA, USA Fitzroy, Victoria, Australia
Jan Jacob Schuringa, PhD
Atsushi Iwama, MD, PhD Kai Fu, MD
Groningen, The Netherlands
Chiba, Japan Omaha, NE, USA
Jianmin Wang, MD
Saghi Ghaffari, MD, PhD Shanghai, China
Toshio Kitamura, MD, PhD New York, NY, USA
Tokyo, Japan Claudia Waskow, PhD
Brian J.P. Huntly, PhD
Jean-Pierre Lévesque, PhD
Cambridge, UK
Dresden, Germany
46th Annual Scientific Meeting of the
Xiaoyan Jiang, MD, PhD
South Brisbane, Australia Vancouver, Canada ISEH – International Society for Experimental Hematology
Anna Rita Migliaccio, PhD Karen Keeshan, PhD
New York, NY, USA Glasgow, Scotland, UK
Bologna, Italy Issay Kitabayashi, PhD
Tokyo, Japan Abstracts
Ellen Rothenberg, PhD Simón Méndez-Ferrer, PhD
Pasadena, CA, USA Cambridge, UK
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ISEH 46th Annual Scientific Meeting/Experimental Hematology 2017; 53 (Suppl 1): S1-S22
TABLE OF CONTENTS
S5 Board of Directors/ISEH Past Presidents Session 10: New Investigators Award Session
S9 Welcome & Opening Remarks Session 14: McCulloch & Till Lecture Award
Friday, 25 August
ABSTRACTS
S9-S13 Session 3: Developmental Hematopoiesis I
S23 Invited Speaker Abstracts
Session 4: HSC Biology I
S41 Short Talk Abstracts
Session 5A: Microenvironment and Signaling
S53 Poster Abstracts
Session 5B: Lineage Differentiation
S137 Author Index
Session 6A: P
luripotent Stem Cells and
Reprogramming
Saturday, 26 August
S3
ISEH 46th Annual Scientific Meeting/Experimental Hematology 2017; 53 (Suppl 1): S1-S22
WELCOME MESSAGE
On behalf of ISEH, the International Society for Experimental Hematology – welcome to the
46th Annual ISEH Scientific Meeting in Frankfurt, Germany. The hallmark of the ISEH Annual
Scientific Meeting is an outstanding schedule of events, blending both educational ses-
sions on basic, translational and clinical hematology as well as unique networking oppor-
tunities for senior scientists and new investigators, and this year’s event is no different. In
2017, the Scientific Program Committee has planned a number of exciting sessions includ-
ing a pre-meeting workshop focused on new investigators, plenary and breakout sessions
featuring the latest science, sponsored technology presentations, and the presentation of
our esteemed Donald Metcalf and McCulloch & Till Awards.
At ISEH’s Annual Scientific Meeting we are showcasing the newest and best research from
around the world, but we always remember the basic biology at the root of our field – it’s all
about the science. Our hope is that this meeting provides you with a multitude of opportuni-
ties to connect with fellow scientists who share your passion, to interact with the brightest
minds in our field, and to explore new product offerings from some of the industry’s leading
vendors.
While in Frankfurt, we hope that you are able to enjoy all that the city and ISEH has to offer.
Some of ISEH’s key initiatives to be on the lookout for this year include:
We sincerely hope that through these initiatives and your enjoyment of 46th Annual ISEH
Scientific Meeting, you will leave with memories of remarkable science, entertaining social
events, and renewed excitement with your ISEH colleagues.
Sincerely,
S4
ISEH 46th Annual Scientific Meeting/Experimental Hematology 2017; 53 (Suppl 1): S1-S22
MEETING COMMITTEES
S6
ISEH 46th Annual Scientific Meeting/Experimental Hematology 2017; 53 (Suppl 1): S1-S22
AWARDS
ISEH DONALD METCALF LECTURE AWARD ISEH MCCULLOCH AND TILL LECTURE AWARD
Irving L. Weissman, MD Trista E. North, PhD
Stanford Institute of Stem Cell Biology and Regenerative Harvard Stem Cell Institute, USA
Medicine, USA Regulation of Vertebrate Hematopoiesis (1034)
Normal and Neoplastic Stem Cell Competition (1001)
S7
ISEH 46th Annual Scientific Meeting/Experimental Hematology 2017; 53 (Suppl 1): S1-S22
S8
ISEH 46th Annual Scientific Meeting/Experimental Hematology 2017; 53 (Suppl 1): S1-S22
*Unless otherwise noted, all functions will take place in the main conference space (Hörsaalzentrum building).
Thursday, 24 August 2017
15:15 – 16:00 Seminar Rooms 1-2 SESSION 1: Donald Metcalf Lecture Award
*Timm Schroeder, ETH Zurich, Switzerland
Irving Weissman, Stanford University, USA (1001)
Normal and neoplastic stem cell competition
16:00 – 16:15 Ground Floor and Coffee Break
First Floor Foyers
Concurrent with Session 5A *Teresa Bowman, Albert Einstein College of Medicine, USA
*Anna Rita Migliaccio, Mount Sinai, USA
Emery Bresnick, University of Wisconsin, USA (1010)
Enhancer mechanisms governing developmental and
regenerative hematopoietic programs
Merav Socolovsky, University of Massachusetts, USA (1011)
Global increase in replication fork speed during
a p57KIP2-regulated erythroid cell fate switch
Helen S. Goodridge, Cedars-Sinai Medical Center, USA (2009)
Independent production of distinct monocyte subsets by
granulocyte-monocyte progenitors (GMPs) and
monocyte-dendritic cell progenitors (MDPs)
Yoon a Kang, Columbia University, USA (2010)
Myeloid-biased multipotent progenitors are secretory cells
that control myelopoiesis
16:15 – 18:00 Seminar Room 1 SESSION 6A: Pluripotent Stem Cells and Reprogramming
Concurrent with Session 6B *Feng Liu, Chinese Academy of Sciences, China
*Isabel Beerman, National Institute on Aging, USA
Ihor Lemischka, Mount Sinai, USA (1012)
Elucidation of the mechanisms of hematopoietic reprogramming
Andrew Elefanty, Murdoch Children’s Research Institute,
Australia (1013)
Modeling human hematopoiesis in pluripotent stem
Andrea Ditadi, San Raffaele - Telethon Institute for Gene
Therapy, Italy (2011)
Dissecting the cellular of Down syndrome TMD and AMKL
Charlotta Böiers, Lund University, Sweden (2012)
A human IPS model implicates embryonic B-myeloid fate restric-
tion as a developmental susceptibility to ETV6-RUNX1
Carlos-Filipe Pereira, Center of Neurosciences and Cell
Biology, Portugal (2013)
Programming antigen-presenting dendritic cells from fibroblasts
Concurrent with Session 6A *Sarah Ellis, Peter Maccallum Cancer Center, Australia
*Tao Cheng, State Key Laboratory of Experimental
Hematology, China
George Vassiliou, Sanger Institute, United Kingdom (1014)
From CRISPR-Cas9 drop-out screens to novel therapeutic
approaches in acute myeloid leukemia
Jan Jacob Schuringa, University of Groningen,
The Netherlands (1015)
Towards identification and targeting of leukemic stem cells and
(epi)genetically distinct subclones using humanized niche xeno-
graft mouse models
Michael Gundry, Baylor College of Medicine, USA (2014)
Nuclear relocalization of mutant NPM1 induces downregulation
of HOX/MEIS1, terminal differentiation, and cell cycle arrest
Luis Carvajal, Albert Einstein College of Medicine, USA (2015)
Dual inhibition of HDMX and HDM2 in acute myeloid leukemia
Pre-meeting Workshop Short Talk Winner II (2016)
8:30 – 10:15 Seminar Room 2 SESSION 7B: Transcriptional and Epigenetic Regulation I
10:35 – 12:05 Seminar Room 2 SESSION 8B: Hematopoiesis During Aging and Stress
BioSpherix
25 Union Street Parish, NY 13131
Phone: +1 315 387-3414
Fax: +1 315 387-3415
www.biospherix.com
BioSpherix will be exhibiting the Xvivo System, world’s first and only cytocentric incubation and processing system opti-
mized for cells. Providing full-time optimization of critical cell process parameters, physiologic simulation and dynamic
control during incubation, it is the only system like it on the market. Enclose your entire process from incubation to
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quality systems for your research or therapy needs!
IBIDI
Am Klopferspitz 19 | 82152 Planegg / Martinsried
Phone: 0800 00 11 11 28
Fax: 0800 00 11 11 29
E-mail: info@ibidi.de
ibidi – cells in focus
ibidi develops, produces, and distributes innovative labware products, instruments, and reagents for live cell analysis
and cell-based assays specifically for high end microscopy. An extensive line of cell-culture biochips—μ-Slides, μ-Dishes,
and μ-Plates—offers solutions for immunofluorescence and basic cell culture, plus complex assays, such as angiogen-
esis, chemotaxis, wound healing, shear stress, and flow. The instrument line includes stage top incubators, a unique
perfusion system that provides continuous flow for the simulation of blood vessels, and a new system for monitoring,
measuring, and controlling the O2 concentration in biological samples. Finally, ibidi’s newest innovation, Fuse-It-mRNA,
allows for the fast and efficient transfer of mRNA into cells, especially primary cells and stem cells.
S19
ISEH 46th Annual Scientific Meeting/Experimental Hematology 2017; 53 (Suppl 1): S1-S22
NanoString Technologies
530 Fairview Avenue N
Seattle, WA
Tel: (888) 358-6266
www.nanostring.com
NanoString® Technologies provides life science tools for translational research and molecular diagnostic products. The
company’s nCounter®Analysis System has been employed in life sciences research since it was first introduced in 2008
and has been cited in more than 1,500 peer-reviewed publications. The nCounter Analysis System offers a cost-effective
way to easily profile the expression of hundreds of genes, proteins, miRNAs, or copy number variations, simultaneously
with high sensitivity and precision, facilitating a wide variety of basic research and translational medicine applications,
including biomarker discovery and validation.
The new PlexSet reagents allow you to run nCounter digital gene expression assays more efficiently and cost-effectively
than ever before for projects ranging from 3 to 24 RNA targets and 96 unique samples on one run, without replicates
and with 15 minutes hands on. PlexSet is compatible with a wide range of sample types including FFPE and cell lysates
that can be processed with a simple lyse-and-go protocol.
PromoCell GmbH
Sickingenstrae 63/65
69126 Heidelberg Germany
Tel: +49 6221 649 34 0
Fax: +49 6221 649 34 40
www.promocell.com
PromoCell is a premier manufacturer of cell culture products. We are known in particular for our broad range of human
primary cells, stem cells and blood cells, optimized cell culture media, and comprehensive line of cell biology research
products.
We believe that our cell culture products are the most suitable ones for biomedical research because they provide
scientists with physiologically accurate models and therefore enable them to obtain better results in their research.
S20
ISEH 46th Annual Scientific Meeting/Experimental Hematology 2017; 53 (Suppl 1): S1-S22
ProteinSimple
3001 Orchard Parkway
San Jose, California, 95134 USA
Toll-Free: (888) 607-9692
Tel: +1 408 510-5500
Fax: +1 408 510-5599
Email: info@proteinsimple.com
ProteinSimple is part of the Protein Platforms division of Bio-Techne. Our goal is simply to help researchers gain a better
understanding of proteins and their role in disease, by making protein analysis simpler, more quantitative and affordable.
We develop and commercialize proprietary systems and consumables for protein analysis that ultimately help reveal
new insights into the true nature of proteins. Our wide-ranging portfolio of tools includes everything from immunoassay
systems that quantify protein expression to systems that probe the structure and purity of protein-based therapeutics.
We have over 200 employees, more than 14,000 systems installed around the world, and our main offices are in San
Jose, California.
S21
ISEH 46th Annual Scientific Meeting/Experimental Hematology 2017; 53 (Suppl 1): S1-S22
SPONSORS
Education Support
BANSS
Stiftung
S22
ISEH 46th Annual Scientific Meeting/Experimental Hematology 2017; 53 (Suppl 1): S23
ABSTRACTS
INVITED SPEAKER
1002 - MOLECULAR AND BIOLOGICAL ANALYSIS OF HUMAN HEMATOPOIETIC STEM CELLS AT SINGLE-CELL
RESOLUTION
Connie Eaves1, D.J.H.F. Knapp2, C.A. Hammond2, A. Hui3, M. van Loenhout1, D. Pellacani2, F. Wang2, P.H. Miller2, A. Lorzadeh3,
N. Aghaeepour2, M. Moksa3, M. Vaninsberghe3, G.M. Rabu2, P.A. Beer2, R.K. Humphries2, S. Bendall4, G.P. Nolan4, C. Hansen3, and M. Hirst3
1
Terry Fox Labratory, Vancouver, Canada; 2BC Cancer Agency, Vancouver, Canada; 3University of British Columbia, Vancouver, Canada; 4Stanford
University, Stanford, United States
Elucidating the properties and processes regulating primitive hematopoietic cell behavior constitutes a clinically important but still elusive goal. A new challenge in addressing
these questions has emerged from experiments in mice that have revealed multiple sources of intrinsically determined heterogeneity in the self renewal potential, lineage compe-
tence and proliferation state of these cells. In addition, it is has been shown that these key properties are not coordinately regulated when the cells are stimulated with different
external cues. These findings have galvanized interest on the power of using index sorting to link functional responses with high-dimensional molecular profiles obtained on single
cells with bioinformatically matched phenotypes. We have now used this approach to analyze the CD34+CD38 CD45RA CD90+CD49f+ subset of normal human cord blood cells
(‘‘CD49f cells’’, 10% pure LT-HSCs) before and after their exposure to different growth factor combinations in vitro. Our initial molecular analyses included measurements of
several signaling intermediates, transcription factors, cell cycle regulators and surface markers measured simultaneously by mass cytometry. The results revealed new features
of intrinsic heterogeneity amongst these very rare human hematopoietic cells and the differential control of their survival, proliferation and regenerative potential. These analyses
also led to the discovery of the identity and hence additional molecular properties of a subset within this CD49f population that exclusively possess durable blood cell output
capacity in serially transplanted immunodeficient mice. These findings set the stage for future examination of how the properties of these critical normal human hematopoietic
cells are affected by development, aging and perturbations that cause disease.
Invited Speaker Abstracts/ Experimental Hematology 53 (2017) S24-S40 S25
1003 - HUMANIZED MICE TO STUDY HUMAN HEMATOPOIETIC STEM CELL FUNCTION IN VIVO
Claudia Waskow
Technical University Dresden, Dresden, Germany
Xenotransplantation models enable the in-depth analysis of human hematopoietic stem cell (HSC) function in vivo.We generated novel mouse models supporting stable HSC
engraftment, which is a prerequisite for the continuous generation of all adult human hematopoietic cell types in mice. By introducing a loss-of-function Kit receptor into
NOD/SCID Il2rg-/- (NSG) mice we generated NOD/SCID Il2rg-/- KitW41/W41 (NSGW41) mice that combine an impaired endogenous HSC compartment with immunodeficiency
that efficiently support stable engraftment of human HSCs in the long-term without the need for any conditioning therapy. As a consequence, multilineage engraftment including
cells of the myeloid and erythroid lineages is highly improved in NSGW41 mice. Mechanistically, endogenous murine HSCs with a defective Kit receptor are largely replaced by
human Kit-proficient donor HSCs. Further, ‘humanization’ results in quantitative and qualitative changes of the mouse bone marrow microenvironment, suggesting that a mutual
cross-talk between human HSCs and the mouse stem cell niche takes place. Finally, using these novel recipients we address effects of manipulations of cell biological functions on
human HSC biology in vivo.
1004 - CELLULAR HIERARCHY AND MOLECULAR MECHANISMS UNDERLYING HAEMATOPOIETIC STEM CELL
DEVELOPMENT
Alexander Medvinsky, Stanislav Rybtsov, Alison McGarvey, Celine Souilhol, and Antoniana Batsivari
University of Edinburgh, Edinburgh, United Kingdom
During embryonic development, adult haematopoietic stem cells (HSCs) emerge preferentially in the ventral domain of the aorta in the aorta–gonad–mesonephros (AGM) region.
Using a combination of ex vivo and in vivo approaches, we found that stage-specific reciprocal dorso–ventral inductive interactions and lateral input from the urogenital ridges are
required to drive HSC development in the aorta. These inductive interactions in the AGM region are mediated by the interplay between spatially polarized signalling pathways,
which are integral parts of the regulatory system involved in the development of HSCs. We also show that at final steps in the AGM region, HSCs begin acquiring properties of
adult bone marrow HSCs as part of their maturation programme.
S26 Invited Speaker Abstracts/ Experimental Hematology 53 (2017) S24-S40
1005 - DECODING HEMATOPOIETIC STEM CELL EMERGENCE IN MOUSE EMBRYOS AT SINGLE-CELL RESOLUTION
Bing Liu
Academy of Military Medical Sciences, Beijing, China (People’s Republic)
Hematopoietic stem cells (HSCs) are generated early from embryonic precursors, such as haemogenic endothelial cells and pre-HSCs located mainly in aorta-gonad-mesonephros
(AGM) region. High level of CD201 expression is employed, together with other surface markers, to capture both CD45- and CD45+ individual pre-HSCs at 30% purity in E11
AGM, as rigorously validated by single-cell-initiated serial transplantation. Intriguingly, single pre-HSCs are capable of generating descendent HSCs with either myeloid-deficient
(g) or lymphomyeloid balanced (b) potential. Moreover, by clonal limiting dilution analysis, we show that the first wave of mature HSCs are composed of mainly myeloid-deficient
(g) HSCs and less the lymphomyeloid balanced (b) subtype, whereas the lymphoid-deficient one (a) is undetectable in 40 long-term repopulated recipients. Therefore, at least two
subtypes of pre-HSCs/HSCs (g and b) are formed during endothelial-to-HSC transition. Subsequently, we use single-cell RNA-Seq technique to analyse endothelial cells, CD45-
and CD45+ pre-HSCs in AGM, and HSCs in fetal liver and adult bone marrow. Pre-HSCs showed unique features in transcriptional machinery, arterial signature, apoptosis, meta-
bolism state, signalling pathway, and transcription factor network. Interestingly, mTOR activation was uncovered indispensable for the emergence of HSCs but not haematopoietic
progenitors. Transcriptome data-based functional analysis revealed remarkable heterogeneity in cell cycle status of pre-HSCs. Finally, core molecular signature of pre-HSCs was
identified. Collectively, our work has successfully constructed the in vivo function-based transcriptome landscape of developing HSCs at single-cell resolution, offering valuable
clues for producing bona fide HSCs in the culture dish.
1008 - REGULATION OF STEM AND PROGENITOR CELL FUNCTION BY THE BONE VASCULATURE
Ralf Adams1,2
1
Max Planck Institute for Molecular Biomedine, Muenster, Germany; 2University of Muenster, Muenster, Germany
In addition to their conventional role as a conduit system for gases, nutrients, waste products or cells, blood vessels in the skeletal control multiple aspects of bone formation and
provide niches for hematopoietic stem cells (HSCs) in bone marrow. Insight into the architecture and function of the vasculature in the skeletal system was previously limited by the
heavily calcified and matrix-rich properties of bone. We have managed to overcome many of these limitations with the help of improved protocols for the processing, immuno-
staining and live imaging of bone (Kusumbe et al. 2015; Bixel et al. 2017). We found that blood vessel growth in bone involves a specialized, tissue-specific form of angiogenesis
that is distinct from other organ systems. Notch signaling promotes endothelial cell proliferation and vascular growth in postnatal long bone, which is the opposite of the well-
established function of Notch and its ligand Dll4 in the endothelium of other organs and tumors. In addition, Notch controls the release of angiocrine signals from the bone endo-
thelium and thereby controls immature perivascular mesenchymal cells and osteoprogenitors. We also discovered that endothelial hypoxia-inducible factor 1a promotes angiogen-
esis in the postnatal skeletal system, leads to the amplification of osteoprogenitor cells and thereby improves bone formation (Kusumbe et al. 2014; Ramasamy et al. 2014; Langen
et al., 2017). Aging is associated with a loss of bone mass and reduced HSC functionality. Our work has uncovered extensive age-related changes in the bone vasculature, which
involve alterations in arteries, blood flow, perivascular osteoprogenitors and other mesenchymal cells as well as in capillary endothelial cell subpopulations. These changes are
associated with a strong reduction in endothelial Notch signaling and, remarkably, endothelial cell-specific genetic strategies leading to a reactivation of the Notch pathway in
aged mice triggered increases in bone formation and HSC niches Kusumbe et al. 2016; Ramasamy et al. 2016). Thus, bone endothelial cells are key regulators of tissue homeostasis
and stem/progenitor cell function in the skeletal system.
S28 Invited Speaker Abstracts/ Experimental Hematology 53 (2017) S24-S40
1011 - GLOBAL INCREASE IN REPLICATION FORK SPEED DURING A P57KIP2-REGULATED ERYTHROID CELL FATE SWITCH
Merav Socolovsky, Yung Hwang, Melinda Futran, and Daniel Hidalgo
University of Massachusetts Medical School, Worcester, United States
Cell cycle regulators are increasingly implicated in cell fate decisions such as the acquisition or loss of pluripotency and self-renewal potential. The cell cycle mechanisms that regulate these
cell fate decisions are largely unknown. Here we studied an S phase- dependent cell fate switch, in which murine early erythroid progenitors transition in vivo from a self-renewal state into a
phase of active erythroid gene transcription and concurrent maturational cell divisions. We found that progenitors are dependent on p57KIP2-mediated slowing of replication forks for self-
renewal, a novel function for cyclin-dependent kinase (CDK) inhibitors. The switch to differentiation entails rapid downregulation of p57KIP2 with a consequent global increase in replication
fork speed and an abruptly shorter S phase. Our work suggests that cell cycles with specialized global DNA replication dynamics are integral to the maintenance of specific cell states and to
cell fate decisions.
1014 - FROM CRISPR-CAS9 DROP-OUT SCREENS TO NOVEL THERAPEUTIC APPROACHES IN ACUTE MYELOID
LEUKAEMIA
George Vassiliou1,2,3
1
Wellcome Trust Sanger Institute, Cambridge, United Kingdom; 2Cambridge Stem Cell Institute, Cambridge, United Kingdom;
3
Cambridge University Hospital NHS Foundation Trust, Cambridge, United Kingdom
Acute myeloid leukaemia (AML) is a devastating disease with a long-term survival of less than 30%. The study of its molecular pathogenesis through rational mechanistic studies
has been at the center of efforts to identify novel therapeutic approaches against the disease, and these efforts have accelerated dramatically in recent years, propelled in part by
advances in cancer genomics. Despite such progress cytarabine, developed more than 50 years ago, represents the last major addition to mainstream anti-AML therapy. An alter-
native and orthogonal approach to the identification of novel therapeutic targets in AML, is through the performance of recessive genetic screens. Such screens, employing RNA
interference (RNAi), led to the identification of BRD4 as a novel therapeutic target in some AML subtypes more than 5 years ago. The recent advent of CRISPR-Cas9 genome
editing technologies and their application to genetic screens has given further impetus to such approaches. We recently described the results of the first systematic genome-wide
CRISPR-Cas9 drop-out screen in AML, which led to the identification of more than 200 potentially druggable genes that are essential to the survival and proliferation of AML
cells. Many of these genes were intuitive and include some, such as MEN1, DOT1L, BCL2, DHODH and PIM1, were already being targeted as therapeutic targets in AML or
related cancers. However, several others represented both novel and unforeseen vulnerabilities. In my talk I will present our studies with two such genes, SRPK1 the gene for
a serine-arginine (SR) protein kinase that phosphorylates SR-rich splicing factors such as SRSF1 and METTL3 a gene that codes for an RNA methyltransferase.
Invited Speaker Abstracts/ Experimental Hematology 53 (2017) S24-S40 S31
1015 - TOWARDS IDENTIFICATION AND TARGETING OF LEUKEMIC STEM CELLS AND (EPI)GENETICALLY DISTINCT
SUBCLONES USING HUMANIZED NICHE XENOGRAFT MOUSE MODELS
Jan Jacob Schuringa
Experimental Hematology, UMCG, Groningen, The Netherlands
Over the past years it has become clear that multiple genetically distinct subclones can co-exist in patients that suffer from acute myeloid leukemia (AML). These subclones often
carry similar founder mutations, but upon leukemic evolution different secondary mutations arise in distinct subclones. Tools to prospectively isolate viable subclones to function-
ally study them are currently lacking. We performed transcriptome and quantitative proteome analysis on large cohorts of primary AML CD34+ samples and healthy CD34+ con-
trols. Integration of these datasets resulted in a list of 60 plasma membrane (PM) proteins that were upregulated in specific subtypes of AML. Many of these were validated in
independent cohorts of patients, and expression of specific markers was correlated to mutation status, disease progression and minimal residual disease. By using infinicyt, expres-
sion of multiple plasma membrane markers was linked to each other and this combinatorial approach was then used to perform principle component analyses in order to determine
which (combination of) PM markers allows to identify distinct subpopulations based on PM expression profiles. While in 30-40% of investigated cases leukemias appear to be
rather monoclonal, we have observed remarkable heterogeneity in clonal distributions in the remaining cases. Prospective sorting and subsequently targeted sequencing of these
populations indeed confirmed the presence of different mutations in these subclones. Transcriptome studies revealed that gene expression profiles were also clearly distinct between
subclones. We have begun to functionally analyse these subclones in vitro and in vivo. First, in order to further improve in vivo xenograft models we have implanted human Mesen-
chymal Stromal Cells (MSCs) with a mixture of matrigel and scaffolds in order to create a human bone marrow-like environment in the mouse. A large cohort of patient samples
successfully engrafted in this model, covering all important genetic and risk subgroups. Furthermore, stem cell self-renewal properties were better maintained as determined by
serial transplantation assays and genome-wide transcriptome studies, and less clonal drift was observed as determined by exome sequencing. The human leukemia xenograft mouse
clinic that we have established will serve as an excellent resource for future studies aimed at exploring novel therapeutic approaches. For instance, we have recently utilized this
model to investigate the role of polycomb group proteins in leukemia, and identified that the non-canonical PRC1.1 complex is critically important for leukemia development. Also,
we have utilized these humanized niche mice to follow the kinetics of prospectively isolated (epi)genetically distinct subclones of individual patients.
1017 - RESOLVING THE FETAL TO ADULT SWITCH IN B LYMPHOPOIESIS AT THE SINGLE CELL LEVEL
Joan Yuan1, Trine Kristiansen1, Stijn Vanhee1, Elin Jaensson-Gyllenb€ack2, Alya Zriwil1, Alexander Doyle1, Ewa Sitnicka Quinn1, Stefan Lang1,
Shamit Soneji1, and David Bryder1
1
Lund University, Lund, Sweden; 2BioInvent, Lund, Sweden
In mice and men, a developmental switch takes place in immune cell output early in life. This is likely designed to accommodate the unique demands of tolerance both in utero and
during neonatal exposure to environmental antigens, while providing protection against common pathogens. In the murine B cell lineage, the output of B-1 cells is predominant in
utero and diminishes within weeks after birth in favor of conventional follicular B-2 cells. B-1 cells constitute a non-redundant aspect of the immune repertoire that controls infec-
tion and inflammation through T-independent secretion of natural antibodies and IL-10. Thus, developmental changes in lymphocyte output contributes to an additional layer of
immunological diversity. However, the basis for this developmental switch remains unclear. It is widely acknowledged that primitive, non self-renewing progenitors that arise prior
to the emergence of definitive hematopoietic stem cells (HSCs) are able give rise to B-1 cells during ontogeny. However, whether definitive HSCs also constitute a source of B-1
cells during later developmental stages has been a subject of debate. To address this, we employed cellular barcoding to track longitudinal changes in HSC behavior at the clonal
level. Our results demonstrate that fetal definitive HSCs give rise to both B-1 and B-2 cells at the clonal level and that initially B-1 potent HSCs become B-2 restricted over time.
Importantly, the loss of B lineage plasticity in adult HSCs was reversible at the clonal level upon ectopic expression of Lin28b; an RNA-binding protein and key regulator of fetal
hematopoiesis. Our findings establish definitive HSCs as a significant source of B-1 cells later during ontogeny and demonstrate that a shift in the molecular program rather than
changes in hematopoietic lineage representation accounts for the postnatal attenuation of B-1 cell output. B-1 cells harbor a self-reactive repertoire enriched for specificities against
common glycolipids that aid in the clearance of foreign microbes as well as self-cellular debris. Since the mature lymphocyte pool is largely shaped by developmental constraints,
we next hypothesized that careful interrogation of the Lin28b induced B-1 cell repertoire may provide key clues to the basis for the developmental switch in B lineage output.
Remarkably, our B cell repertoire profiling results indicate that the threshold for B lineage central tolerance induction is developmentally regulated and more leniently set upon
Lin28b induction, allowing for the maturation of self-reactive B-1 cells. Taken together, our findings peel back the layers of the molecular networks that shape neonatal immunity
and provide fundamental insight into the tailored formation of a diverse yet tolerant immune system.
1021 - GENETIC VARIATION UNDERLIES EPIGENOMIC VARIATION AND THE CONSEQUENCES OF DNMT3A
MUTATION IN HEMATOPOIETIC STEM CELLS
Jennifer Trowbridge1, Rebecca Bell1, Catrina Spruce1, Anna Tyler1, Robyn Ball1, Vivek Philip1, Dan Gatti1, Narayanan Raghupathy1,
Romy Kurasawe2, Kira Young1, Mary Ann Handel1, Michael Stitzel2, Gary Churchill1, Kenneth Paigen1, Petko Petkov1, and Gregory Carter1
1
The Jackson Laboratory, Bar Harbor, United States; 2The Jackson Laboratory for Genomic Medicine, Farmington, United States
Accumulation of somatic mutations in genes including the DNA methyltransferase DNMT3A predisposes many, but not all, individuals to development of clonal hematopoiesis
and acute myeloid leukemia (AML). We hypothesize that individual genetic variation underlies distinct epigenomic patterning, and that this variation alters susceptibility to the
pathogenic consequences of DNMT3A mutations in hematopoietic stem cells (HSCs). Through a collaborative effort to assess epigenome variation in a genetically diverse model
system, we performed an in-depth chromatin analysis of liver cells isolated from 8 classical and wild-derived inbred mouse strains that recapitulate the genetic diversity of the
human population. Comparing histone modification ChIP-seq, RNA-seq, and reduced representation bisulfite sequencing analysis to the sequenced genome of each of these strains
demonstrates that underlying genetic variation leads to alterations in epigenetic patterning and establishment of distinct regulatory regions of the epigenome. To determine whether
distinct epigenomic backgrounds influence response to mutations in DNMT3A, we generated an inducible knock-in mouse model of the most common human AML-associated
DNMT3AR882H allele (mouse Dnmt3aR878H). This mouse was generated on a C57BL/6 background and crossed to the 8 inbred mouse strains described above. Following Mx-Cre
induction of Dnmt3aR878H, we observe variability in HSC expansion and myeloid cell accumulation as a consequence of genetic background. Our work suggests that individual
genetic differences can alter susceptibility to clonal hematopoiesis and AML development following mutation in DNMT3A.
1030 - FIRST-IN-CLASS, ORAL MUTANT IDH2 INHIBITOR REVERSES DIFFERENTIATION BLOCK IN ACUTE MYELOID
LEUKAEMIA TO PRODUCE CLINICALLY MEANINGFUL RESPONSES
Paresh Vyas
University of Oxford
Acute Myeloid Leukaemia (AML) is the most common aggressive human leukaemia. w15% of AML cases have acquired point mutations in the gene Isocitrate Dehydrogenase 2.
Mutant IDH2 (mIDH2) protein has neomorphic gain-of-function enzyme activity resulting in R-2-hydroxyglutarate (2-HG) accumulation. 2-HG competitively inhibits the TET
family of 5-methylcytosine (5mC) hydroxylases and the Jumonji-C domain histone demethylases(. This leads to DNA hypermethylation, increased repressive histone methylation
(6) and a differentiation block. Working with an international collaborative group we have analysed sequential bone marrow and blood samples from patients entering the first
Phase I trial of a first-in-class oral mIDH2 inhibitor AG-221. Single agent AG-221 induces an overall response rate of 40.3% in relapsed/refractory AML. We show the mechanism
of action of the drug in patients is through reversal of differentiation arrest. Mutation analysis shows that primary resistance to AG-221 correlates with higher mutational burden and
gain of function mutations in the RAS pathway. Interestingly, relapse after initial response (secondary resistance) is not due to second site mutations in IDH2 gene or loss of
suppression of 2-HG levels. Single cell analysis of clonal structures shows secondary resistance arises by selection of minor clones that have evolved from parental clones. These
data will hopefully aid rational combination therapy design with AG-221.
Invited Speaker Abstracts/ Experimental Hematology 53 (2017) S24-S40 S39
1032 -
David Scadden
Massachusetts General Hospital
See online program addendum
S40 Invited Speaker Abstracts/ Experimental Hematology 53 (2017) S24-S40
ABSTRACTS
SHORT TALK
PRESENTATIONS
The number of hematopoietic stem cells (HSCs) in humans and the dynamics of how
these contribute to blood over time are critical to our understanding of normal hema-
topoiesis and its subversion in disease. While molecular barcoding or lineage tracing
approaches have been applied to assess the output of single HSCs in animal models,
similar approaches have not been possible in unperturbed human hematopoiesis. To
address this, we investigated hematopoietic dynamics by using somatic mutations as
barcodes. We isolated 200 primary HSCs (lin-CD34+CD38-CD90+CD45RA-) and
progenitor cells (MEPs, CMPs, and GMPs) from a healthy 60 year-old and grew
them until clones were sufficiently large for whole genome sequencing (WGS)
without amplification. We observed 1,000 somatic mutations per clone, half of which
were caused by a previously undescribed mutational process. A phylogeny of the
stem and progenitor cells was reconstructed. We found that bone marrow was highly
polyclonal: 90% of stem and progenitor cells were related to their nearest neighbour
by a common ancestor that occurred in childhood. Furthermore, comparison to
epithelium from the same individual suggested that the most recent common ancestor
of blood occurred early in embryogenesis, perhaps in the fertilised egg. We selected
10,000 of the mutations discovered by WGS for high depth (O10,000X) targeted
sequencing to track clones in fractions of peripheral blood sampled over time. We
found that thousands of HSCs had progeny circulating in the blood at any one
time. This contribution was stable over the 10-month period interrogated at the
time of writing, although additional time-points and fractions of peripheral blood
are currently being investigated. Estimates of the total number of HSCs, their contri-
bution over time and to different lineages of blood will be discussed. Our study re-
veals the phylogenetic relationship between stem cells in the bone marrow and
shows that thousands of stem cells contribute to the blood at any one time in a healthy
human. This has important implications for our understanding of normal hematopoi-
esis, clonal hematopoiesis, and the development of pathological states such as leuke-
mia.
2002 - NEW DELINEATION OF HUMAN CD34+ STEM/ 2003 - MLLT3 SUSTAINS HUMAN HSC SELF-RENEWAL
PROGENITOR CELL HIERARCHICAL ORGANIZATION AND ENGRAFTMENT
Fernando Anjos-Afonso1, Florian Buettner2, and Dominique Bonnet3 Vincenzo Calvanese1, Andrew T. Nguyen2, Timothy Bolan2,
1
ECSCRI, Cardiff, United Kingdom; 2Helmholtz Research Center, Munich, Zoran Galic2, and Hanna Mikkola2
Germany; 3The Francis Crick Institute, London, United Kingdom 1
Department of Molecular, Cell and Developmental Biology, University of California
Los Angeles, Los Angeles, United States; 2UCLA, Los Angeles, United States
Human CD34+ stem/progenitor cell compartment has been recently re-defined and
within the Lin-CD34+CD38-CD45RA- fraction it is possible to separate 4 sub-pop- The use of hematopoietic stem cells (HSC) for treating hematopoietic malignancies
ulations based on CD90 and CD49f expressions (simply refer as CD90+/-CD49f+/-). and genetic disorders is limited by availability of HLA-matched donors. If HSC could
CD90+CD49f+ and CD90-CD49f- cells have been proved to be HSCs and MPPs be expanded in culture or derived from pluripotent stem cells (PSCs), the number of
(multipotent progenitors) respectively. However, it is unclear what is the relationship patients and the spectrum of treatable diseases would greatly increase. To uncover the
between the remaining two sub-populations (CD90+CD49f- and CD90-CD49f+) genetic network that promotes HSC self-renewal, we identified genes whose expres-
with the CD90+CD49f+ HSC and CD90-CD49f- MPP fractions. Using in vivo sion is enriched in self-renewing human hematopoietic stem and progenitor cells
limiting-dilution, secondary and limiting cell dose transplantation assays we ad- (HSPC) in fetal liver (FL), and that are down-regulated in cultured HSPC. Functional
dressed the in vivo relationship of these 4 sub-populations. We uncovered that not testing identified MLLT3, a component of the super elongation complex, as an impor-
only the CD90+CD49f+ but also the CD90-CD49f+ fractions contain long-term tant HSC regulator. shRNA-mediated MLLT3 depletion in human HSPC caused
self-renewing HSCs. More importantly, these two CD49f+ fractions are inter-convert- impaired self-renewal in FL and cord blood HSPC in culture and prevented engraft-
ible and they give rise to CD90+CD49f- cells first then to MPPs and subsequently to ment in immunodeficient NSG mice, implying that MLLT3 expression is essential to
CD45RA+ progenitors in vivo, underlining a sequential hierarchical organization. HSC function. Conversely, MLLT3 overexpression in human HSPC in culture
Further to this, the two CD49f+ fractions have similar engraftment kinetics, lineage enhanced the expansion of immunophenotypic HSPC and their ability to engraft.
outputs, homing capacity, spatial distribution in the mouse bone marrow after trans- This effect was specific to HSPC, as MLLT3 overexpression in committed progeni-
plantation and the capacity to maintain long-term the whole CD34+ compartment. To tors did not confer to them self-renewal properties. Moreover, MLLT3 expression did
further support that CD90-CD49f+ cells are biologically similar to CD90+CD49f+ not alter the ability of expanded HSPC to differentiate into major blood lineages in
HSCs we performed single cell RNA-sequencing (scRNA-Seq) analyses. Using vitro or in engrafted mice. MLLT3 ChIP-seq analysis in human HSPC revealed bind-
gene expression data obtained from total RNA-Seq of the 4 sub-populations and ing to the transcriptional start site of several highly expressed genes, including early
Oral Short Talk Abstracts/ Experimental Hematology 53 (2017) S42-S52 S43
response genes JUNB, FOS and MYC, and known HSC regulators, such as MEIS1, 2005 - HUNDREDS OF EMBRYONIC HEMATOPOIETIC
HOXA9 and BCL11A. MLLT3 overexpression in long-term expanded HSPCs re- PRECURSORS CONTRIBUTE TO LIFE-LONG
vealed that MLLT3 was able to sustain the expression of several genes that are other- HEMATOPOIESIS
wise downregulated in cultured HSPCs. Thus, sustaining MLLT3 expression in
Miguel Ganuza Fernandez, Trent Hall, David Finkelstein,
culture can functionally circumvent HSC ‘‘culture defects’’. This study identifies
MLLT3 as a novel human HSC self-renewal regulator, whose impaired expression
Ashley Chabot, Guolian Kang, and Shannon McKinney-Freeman
St. Jude Children’s Research Hospital, Memphis, United States
in culture contributes to the defective HSC maintenance in vitro. Future studies
will explore if enforced MLLT3 expression using non-integrating delivery methods Current dogma asserts that blood is established by few precursors during mammalian
can help generate HSC for therapeutic use. ontogeny. Here, we revisit this question via a non-invasive approach that employs the
Confetti allele, which contains a cassette in the ROSA26 locus that randomly marks cellular
progeny with GFP, YFP, RFP or CFP when exposed to Cre recombinase. By expanding
limiting dilution replicates of immortalized Confetti+ fibroblasts, we determined that sam-
ple-to-sample variance (SSV) in the distribution of Confetti colors inversely correlates with
the initial number of cells plated. Via this approach, we derived a formula that estimates
‘‘numbers of initiating events’’ using the SSV in Confetti colors that can be applied to any
system or tissue of interest. We validated this formula in vivo via limiting dilution transplan-
tation of Confetti+ bone marrow. The SSV in recipient peripheral blood (PB) Confetti colors
yielded a similar estimate as the frequency of repopulating units calculated via classic
limiting dilution, confirming our formula. Thus, to estimate the numbers of precursors
that establish life-long mammalian hematopoiesis, we analyzed the SSV in the PB Confetti
colors of cohorts of ROSA26(Confetti/+)Flk1(+/Cre) (N57), ROSA26(Confetti/+)VE-cad-
herin(+/Cre) (N512) and ROSA26(Confetti/+) Vav1(+/Cre)(N510) mice. Here, we esti-
mated that 719 mesodermal precursors, 633 endothelial precursors and 545 fetal liver
(FL) hematopoietic precursors contribute to life-long hematopoiesis. These numbers
contrast with previous work, which relied on transplantation and embryo-disruption, that
suggested that very few hematopoietic precursors establish mammalian hematopoiesis.
The similar estimates gleaned with all three mouse models further suggests the existence
of a ‘‘developmental bottleneck’’ after the FL that restricts the numbers of precursors that ul-
timately contribute to life-long hematopoiesis. In sum, we report a novel approach to esti-
mate the complexity of the emerging hematopoietic system without perturbing the
developing embryo. This represents a new platform to identify biologically relevant pro-
cesses that govern the dynamics of different populations during embryonic development.
2004 - SINGLE CELLS UNDERGOING CELL FATE 2006 - MITOCHONDRIAL MORPHOLOGY CONTROLS
CHANGE DURING ENDOTHELIAL-TO-HEMATOPOIETIC HEMATOPOIETIC STEM CELL SELF-RENEWAL AND
CELL TRANSITION SHOW PULSATILE GATA2 CONFERS HSC DIVISIONAL MEMORY
EXPRESSION Marie-Dominique Filippi1, Ashwini Hinge1, Jingyi He1, Jose Javier1,
Elaine Dzierzak1, Christina Eich2, Jochen Arlt1, Chris Vink1, and James Bartram1, Ellen Fjellman1, Hiromi Sesaki2, Lee Grimes1, and
Wiggert van Cappellen2 Nathan Salomonis1
1
University of Edinburgh, Edinburgh, United Kingdom; 2Erasmus MC, Rotterdam, 1
Cincinnati Children’s Hospital Research Foundation, Cincinnati, United States;
2
Netherlands John Hopkins, Baltimore, United States
Underpinning cell behavior during development and differentiation, are fundamental dy- Hematopoietic stem cells (HSC) have extensive regenerative potential but limited self-
namic expression changes that drive cell fate processes away from a pre-existing equilib- renewal ability, since long-term repopulation potential is lost after several divisions and stress
rium. The Gata2 transcription factor is an important regulator of blood cell differentiation hematopoiesis. To understand these mechanisms, we compared the transcriptome of non-
and development, and is pivotal to hematopoietic stem cell generation (HSC). Within a stressed versus stressed HSC [ie before and after transplantation, respectively], using single
short window of developmental time HSCs originate from a natural differentiation of he- cell RNA-sequencing. Interestingly, genes categorized in mitochondrial organization were
mogenic endothelial cells through a process called endothelial to hematopoietic transition markedly downregulated in stressed HSC. Using EGFP-mitochondria reporter mice, which
(EHT). Genetic studies in mice show that Gata2 is required for EHT and HSC generation, labels mitochondria regardless of their membrane potential and activated state, we show that
and that this process is very sensitive to reductions in Gata2 dosage. Surprisingly, nothing HSC had unexpectedly high mitochondria content, (4-fold higher than progenitors). Mito-
is known about the in vivo behavior of Gata2 expression in single cells during develop- chondria were homogeneously organized into a dispersed network. In contrast, mitochon-
ment, and particularly during EHT. We used a recently published Gata2-Venus reporter drial mass was higher in stressed HSC, and mitochondria morphology was abnormal.
system with unperturbed Gata2 expression (Kaimakis et al, Blood, 2016) to follow They were aggregated and less fragmented. Mitochondrial membrane potential was also
Gata2 expression in single cells of the mouse midgestation aorta (the site of EHT and severely reduced in stressed HSC in vivo, indicating that HSC accumulate dysfunctional
HSC generation) by vital confocal time-lapse imaging. EHT subsets that included single mitochondria after replicative stress. During cell division in vitro, mitochondria were un-
hemogenic endothelial cells, bulging cells and intra-aortic hematopoietic cluster cells that equal distributed to daughter cells of single stressed HSC division [one daughter cell had
were imaged over a 10 hour period. Rapid pulsatile Gata2 expression level changes were more aggregated mitochondria], whereas mitochondria were equally partitioned during di-
found in the cells undergoing morphological transition (not cell division), suggesting a vision of non-stressed HSC, suggesting that HSC lose mitochondrial quality control after
highly unstable genetic state during EHT. Moreover, Gata2 pulsatile expression behavior replicative stress. Mitochondrial morphology is maintained through regulated cycles of
was found to be dramatically altered in Gata2 haploinsufficient embryonic aorta cells, fission and fusion, which is essential for maintaining a functional organelle and controls
which undergo fewer endothelial-to-hematopoietic transitions and are known to be mitochondria inheritance during division. We found the activity of the mitochondrial fission
reduced in hematopoietic progenitor and stem cell potential. Our novel finding of the dy- protein Drp1 was reduced in stressed-HSC, perhaps due to impaired mitochondrial-cytoskel-
namic pulsatile expression of Gata2 during the generation of hematopoietic cells in the em- eton association. Genetic loss of Drp1 caused severe mitochondrial aggregation in HSC, and
bryo reinforces the notion that threshold levels of Gata2 influence the transition to impaired HSC self-renewal and repopulation ability in competitive transplant studies.
hematopoietic stem cell fate, and may have implications for Gata2 level-/stability-related Hence, HSC accumulate dysfunctional mitochondria over the course of divisions due to
actions in hematologic dysfunction. lack of mitochondrial fission, which causes gradual loss of self-renewal ability. Mitochon-
drial morphology may confer HSC with divisional memory.
S44 Oral Short Talk Abstracts/ Experimental Hematology 53 (2017) S42-S52
Hematopoiesis is a dynamic process that generates on demand blood cells from hematopoi-
etic stem and progenitor cells (HSPC). The proliferation and differentiation rates of hemato-
poietic stem cells (HSC) and lineage-biased multipotent progenitors (MPP) MPP2, MPP3,
and MPP4 tailor appropriate blood production. However, HSPCs have been largely regarded
as a small reservoir of stem and progenitor cells, and studied as passive receivers of extrinsic
signals, especially cytokines, provided by bone marrow (BM) niche cells. Whether HSPCs
can actively regulate their number and function is currently unexplored. Using a multiplex
platform to screen for cytokine secretion and a single-cell secretion assay, we show that
HSCs and MPPs produce various cytokines upon inflammatory stimulation. However,
each population display a specific secretory pattern and activity, which is highly enhanced
upon stimulation. Among those, myeloid-biased MPP3 have the most robust secretory activ-
ity, correlated with their higher volume of rough endoplasmic reticulum (rER) and up-regu-
lation of unfolded protein response genes. Importantly, we find that conditioned media
containing cytokines secreted by stimulated MPP3 promote the colony forming ability of
HSCs, MPP3, and MPP4, supporting their role in triggering HSPC myeloid differentiation.
Imaging of the BM niche reveals that MPP3 are located nearby HSCs and MPP4, suggesting
that MPP3-secreted cytokines act in a paracrine manner in the native microenvironment. We
also demonstrate that MPP3 are expanded in myeloid disease conditions, and that leukemic
MPP3 have significantly higher rER volume and secretory activity. We identify 11 differen-
tially secreted cytokines, and directly show that conditioned media from leukemic MPP3
promote proliferation of normal HSCs, MPP3, and MPP4, and stimulate myeloid differen-
tiation from HSCs. Our results uncover a novel regulatory function of myeloid-biased MPP3
in blood production, and illuminate new features of the secretory activity of HSPCs during
inflammation and in disease conditions. Together, they indicate that HSPCs directly regulate
their own functionality through paracrine signals in the BM niche.
Oral Short Talk Abstracts/ Experimental Hematology 53 (2017) S42-S52 S45
2012 - A HUMAN IPS MODEL IMPLICATES 2014 - NUCLEAR RELOCALIZATION OF MUTANT NPM1
EMBRYONIC B-MYELOID FATE RESTRICTION AS A INDUCES DOWNREGULATION OF HOX/MEIS1, TERMINAL
DEVELOPMENTAL SUSCEPTIBILITY TO ETV6-RUNX1 DIFFERENTIATION, AND CELL CYCLE ARREST
Charlotta B€oiers1, Simon Richardson2, Alya Zriwil1, Emma Laycock2, Michael Gundry, Lorenzo Brunetti, Hadley Sheppard, Anna Guzman, and
Virginia Turati2, John Brown2, Jason Wray2, Dapeng Wang2, Margaret Goodell
Chela James2, Javier Herrero2, Ewa Sitnicka1, Stefan Karlsson1, Baylor College of Medicine, Houston, United States
Andrew Smith3, Sten Erik Jacobsen4, and Tariq Enver2
1 NPM1 mutated acute myeloid leukemia (AML) is a distinct entity in the WHO classification of
Lund University, Lund, Sweden; 2UCL, Cancer Institute, London, United Kingdom;
3 hematopoietic cancers. It displays a specific phenotype characterized by favorable prognosis
University of Edinburgh, Edinburgh, United Kingdom; 4Karolinska Institute,
and upregulation of HOX cluster genes. NPM1 is a multifunctional nucleolar chaperone.
Stockholm, Sweden
All the mutations in NPM1 described so far in result in cytoplasmic protein localization
Childhood acute lymphoblastic leukemia (cALL) is clinically distinct from that in adults with a (NPM1c) through the acquisition of a nuclear export signal (NES) at the C-terminus. We hy-
higher incidence, better prognosis and distinct mutational spectrum. One hypothesis for this pothesized that NPM1 mutA, the most frequent NPM1 mutation, could be specifically targeted
difference is that cALL arises in transient cells unique to early human development. We using CRISPR to disrupt the C-terminal NES. After transfection of two NPM1 mutated cell
explored this in ETV6-RUNX1 cALL where evidence from twins and neonatal heel prick lines (OCI-AML3 and IMS-M2) with NPM1c sgRNA, we found that the NPM1mutA allele
testing has shown that this mutation arises in utero and is an initiating event. We characterized was edited in 70-90% of cells, while the NPM1wt allele was not targeted. We cloned the edited
B-lymphoid development in first trimester human embryos aiming to identify a transient alleles into a GFP-NPM1 fusion construct, observing nuclear localization in all tested alleles.
compartment vulnerable to the pre-leukemic initiating effect of ETV6-RUNX1, the most com- Nuclear re-localization was followed by significant impairment of cell growth (3.7-4 fold
mon mutation in cALL. The first B cells emerging in human ontogeny uniformly express sur- decrease), colony forming ability (16-20 fold reduction in colonies) and engraftment in xeno-
face IL7 receptor (IL7R) and appear to derive from a CD19 negative IL7R+ progenitor graft models (significant loss of indels at NPM1mutA allele in engrafted cells). The observed
compartment. The IL7R+ progenitor has B and myeloid potential in vitro and single cell immunophenotype and growth defects were preceded by significant downregulation (5 fold) of
qPCR analysis revealed a transition during development from a primarily myeloid to a predom- HOXA genes, HOXB genes and MEIS1. Chromatin profiling revealed that the mechanism of
inantly lymphoid state, transiting through a B-myeloid state that was rare or not present at other downregulation appeared to involve loss of active promoters and enhancers, rather than the
developmental stages investigated. In vitro B cell differentiation of human pluripotent stem deposition of repressive chromatin marks. We reasoned that the dependency of NPM1-mutated
cells (hPSCs) recapitulates these features. Global gene expression analysis further supports cells on the cytoplasmic localization of NPM1c would sensitize them to nuclear export inhib-
that in vitro differentiation of hPSCs closely resembles what is seen during human develop- itors. Indeed, treatment of OCI-AML3 cells with 50nM Selinexor (KPT-330) induced both
ment, thereby providing a tractable and developmentally relevant model in which to study morphologic and immunophenotypic differentiation beginning as early as day 5. Furthermore,
pre-leukemic initiation of cALL. hPSCs were genome engineered to express ETV6- the transcriptional and chromatin changes phenocopied those observed with CRISPR-targeting
RUNX1 from the endogenous ETV6 locus. Differentiation of ETV6-RUNX1+ hPSCs demon- of the mutant allele. By achieving nuclear re-localization of mutant NPM1, we demonstrated
strated impairment of B lineage commitment from the IL7R+ compartment, blocking differ- that cytoplasmic localization of NPM1 is necessary for maintenance of the leukemic pheno-
entiation and producing proB cells that aberrantly co-express B and myeloid genes and that type. Drugs promoting mutant NPM1 nuclear localization are attractive candidates for clinical
retain myeloid potential. We propose that the lineage dynamics of the fetal IL7R+ compart- success in NPM1-mutated AML.
ment are particularly susceptible to dysregulation by ETV6-RUNX1, thereby providing an
explanation for the childhood restriction of cALL.
S46 Oral Short Talk Abstracts/ Experimental Hematology 53 (2017) S42-S52
2015 - DUAL INHIBITION OF HDMX AND HDM2 IN ACUTE 2017 - P57KIP2 EXPANDS AGM HSCS NON-CELL
MYELOID LEUKEMIA AUTONOMOUSLY VIA THE SYMPATHETIC NERVOUS
Luis Carvajal1, Daniela Ben-Neriah1, Adrien Senecal1, Lumie Benard1, SYSTEM
Swathi-Rao Narayanagari1, Charles Kenworhty1, Katrin Ottersbach1, Chrysa Kapeni1, Nicola Wilson2, Berthold Gottgens2,
Victor Thiruthuvanathan1, Vincent Guerlavais2, Allen Annis2, Kristina Kirschner3, and Simon Tomlinson1
1
Boris Bartholdy1, Britta Will1, Jesus Anampa1, Ioannis Mantzaris1, University of Edinburgh, Edinburgh, United Kingdom; 2University of Cambridge,
Manuel Aivado2, Robert Singer1, Robert Coleman1, Amit Verma1, and Cambridge, United Kingdom; 3University of Glasgow, Glasgow, United Kingdom
Ulrich Steidl1 p57Kip2 (Cdkn1c) is a cell cycle inhibitor specifically expressed in quiescent adult hae-
1
Albert Einstein College of Medicine, Bronx, United States; 2Aileron Therapeutics, matopoietic stem cells (HSCs) and crucially required for their self-renewal and mainte-
Boston, United States nance in a cell autonomous manner. In contrast, our group has previously reported that
The tumor suppressor p53 can be inactivated by its interaction with endogenous in- deletion of p57Kip2 during development leads to an expansion of HSCs in the aorta-go-
hibitors HDMX and HDM2. One major limitation of small molecule inhibitors of nads-mesonephros (AGM) region, where HSCs are first generated. We now demonstrate
HDM2 currently undergoing clinical testing is their lack of affinity for HDMX. In that p57Kip2-null embryos have an expanded sympathoadrenal cell compartment, which
this study, we report that HDMX is overexpressed in leukemic stem (Lin- develops around the dorsal aorta at the time of HSC emergence and which we have pre-
CD34+CD38-CD90-) and progenitor (Lin-CD34+CD38+CD123+CD45+) cells in viously shown to promote AGM HSC expansion via the secretion of catecholamines.
AML patients (p ! 0.0001). ALRN-6924 is an alpha helical p53 stapled peptide Indeed, we detected higher expression of the enzyme tyrosine hydroxylase, which catal-
and first-in-class dual HDMX/HDM2 inhibitor which has recently entered clinical yses catecholamine synthesis. In addition, we measured increased levels, specifically in
testing. However, the molecular, cellular, and biochemical effects and mechanisms the AGM, of noradrenaline, which is the only catecholamine present in the AGM at the
of action of ALRN-6924 have not been demonstrated to date. Using an MS2-based time. By employing specific antagonists, we have identified the adrenergic receptor
reporter system combined with live-cell fluorescent microscopy, we found that through which catecholamines promote HSC production in the AGM. Interestingly,
dual HDMX/HDM2 inhibition using ALRN-6924 robustly activated p53-dependent p57Kip2 deletion does not alter HSC numbers in the yolk sac or placenta, demonstrating
transcriptional bursting dynamics at the single cell and single molecule level. that this mechanism is AGM-specific. HSC numbers are also increased in the foetal liver at
ALRN-6924 exhibited strong biochemical and molecular biological on-target activity the initial stages of colonisation; however, since there is no local production of catechol-
in AML cell lines and primary cells in vitro, as well as in a patient who received amines, this expansion is most likely due to increased numbers of AGM HSCs migrating to
ALRN-6924 treatment in the clinic. Dual HDMX/HDM2 inhibition by ALRN- the foetal liver. To gain further insight into the role of the SNS as a signalling centre for
6924 inhibited cellular proliferation by inducing cell cycle arrest and apoptosis in AGM haematopoiesis, we have generated single cell RNA-Seq data which have allowed
cell lines and primary AML patients’ cells, including in leukemic stem cell-enriched us to dissect the heterogeneity of the sympathoadrenal cell compartment, define the differ-
populations. ALRN-6924 disrupted functional clonogenic and serial replating capac- entiation path these cells undergo and identify further soluble factors that are secreted by
ity of AML cells, and showed superiority over HDM2-only inhibition. Most strik- this component of the AGM haematopoietic microenvironment. In summary, our data
ingly, dual HDMX/HDM2 inhibition led to highly significantly improved survival have not only further defined the functional link between the co-developing haemato-
in an AML xenograft model in vivo, with 50% of mice remaining in long-term remis- poietic and SNS, but have also demonstrated that p57Kip2 plays very different roles in em-
sion without signs of disease after 150 days. Our study provides insight into the mo- bryonic vs. adult HSC regulation. This highlights important differences in cell cycle
lecular, cell biological, and in vivo effects of dual HDMX/HDM2 inhibition in AML, control in embryonic and adult HSCs.
and proof-of-concept supporting the continued testing of dual inhibition of HDMX/
HDM2 in AML and other cancers with wild-type p53.
The hematopoietic transcription factor, LIM domain only 2 (LMO2), is essential for the
blood cell formation through activation of the TAL1 transcriptional complex. We iden-
tified a new mechanism of the LMO2 activation. We found that the nicotinamide phos-
phoribosyltransferase (NAMPT) is indispensable for the induction of early stage
hematopoiesis through SIRT2-mediated deacetylation and activation of LMO2.
LMO2 deacetylation enables its interaction with LDB1 and activation of the TAL1 com-
plex leading to early stage hematopoietic differentiation. NAMPT- or SIRT2 inhibition
resulted in the abrogated deacetylation of LMO2, downregulation of the TAL1 complex
target genes expression and markedly diminished hematopoietic differentiation of the
induced pluripotent stem cells (iPSCs) in vitro. Intriguingly, knockdown of NAMPT-
or SIRT2 using specific morpholino dramatically suppressed early hematopoiesis in ze-
brafish embryos in vivo. Moreover, transfection of NAMPT- or SIRT2- deficient zebra-
fish embryos with the deacetylation-mimic mutant of LMO2 completely rescued
defective hematopoiesis, demonstrating the essential role of NAMPT/SIRT2-mediated
LMO2 deacetylation in hematopoietic differentiation in vivo. This new molecular mech-
anism may help explain the pathogenesis of hematopoietic diseases involving the TAL1
complex defects and provide new therapeutic possibilities.
Oral Short Talk Abstracts/ Experimental Hematology 53 (2017) S42-S52 S47
2021 - RECONSTRUCTING BLOOD STEM CELL 2023 - NEOGENIN-1: A NEW RECEPTOR CRITICAL FOR
REGULATORY NETWORK MODELS FROM SINGLE-CELL HEMATOPOIETIC STEM CELL FUNCTION
MOLECULAR PROFILES Gerald de Haan1, Erik Zwart1, Bertien Dethmers-Ausema1,
Fiona Hamey1, Sonia Nestorowa2, Sarah Kinston2, David Kent2, Ronald van Os2, Fiona Hamey3, Berthold G€ottgens3, Leonid Bystrykh1,
Nicola Wilson2, and Berthold G€ottgens2 and Seka Lazare1
1 1
Wellcome Trust - Medical Research Council Cambridge Stem Cell Institute, ERIBA, UMCG, Groningen, The Netherlands; 2MCCA, UMCG, Groningen, The
University of Cambridge, Cambridge, United Kingdom; 2University of Cambridge, Netherlands; 3Cambridge Institute for Medical Research University of Cambridge,
Cambridge, United Kingdom Cambridge, United Kingdom
Adult blood contains a mixture of mature cell types each with specialised functions. Remark- To search for transcripts specifically deregulated during HSC aging we performed low input
ably, the whole blood system can be generated by a single cell, the haematopoietic stem cell RNA-Seq experiments of purified LT-HSCs derived from individual young and aged donors.
(HSC), which is capable of both self-renewal and differentiation into all blood lineages. Dif- We compared our data with 7 previously published datasets. The cell surface receptor Neoge-
ferentiation of HSCs towards alternative lineages must be balanced at the population level nin-1 was the most frequently reported upregulated gene. Downstream signalling pathways
by the fate decisions of individual cells. Transcription factors play a key role in regulating these activated by Neogenin stimulation include Cdc42 and Focal Ahesion Kinase, both implicated
decisions and function within regulatory programs, which can be modelled as transcriptional in HSC quiescence and aging. Remarkably no studies have yet investigated Neogenin in HSCs
regulatory networks. As dysregulation of haematopoietic fate decisions is linked to blood dis- and its role in HSC function has remained unknown. Here we show for the first time that Neo-
orders such as leukaemia, it is vital to understand how these decisions are controlled. Here we genin is critical for murine HSC function. Neogenin-1 expression is enhanced in long-term
present a novel network inference method, which exploits the ability to obtain dynamic infor- HSCs and drastically decreases with diminishing self-renewal capability in short-term
mation from single-cell snapshot expression data based on the expression profiles of 48 genes HSCs and multipotent progenitors. Single cell RNA-Seq data showed Neogenin-1 to be abun-
in 2,167 blood stem and progenitor cells. This allowed us to infer transcriptional regulatory dantly expressed by a subset of the most primitive HSCs. Lentiviral knock-down of Neogenin
network models that recapitulated differentiation of HSCs towards megakaryocyte-erythrocyte in HSCs significantly reduced colony growth in vitro (p! 3.91055E-23). In vivo, HSCs in
progenitors and lymphoid-primed multipotent progenitors. By comparing these two network which Neogenin-1 was lentivirally repressed were unable to contribute to blood production af-
models we identified and subsequently experimentally validated a difference in GATA2 regu- ter transplantation in irradiated recipients. Neogenin-1 is a multifunctional receptor able to
lation of Nfe2 and Cbfa2t3h between these two differentiation trajectories. Our approach cap- regulate cell differentiation, migration and proliferation through multiple ligands including
tures known biology of haematopoiesis, provides new hypotheses about regulation of HSC Repulsive Guidance Molecules, Netrins and Bone Morphogenic Proteins. We show that a sub-
differentiation, and will bewidely applicable to discover regulatory relationships in other differ- set of neogenin ligands are expressed by bone marrow niche cells supporting the relevance for
entiating biological systems. Additionally, we are working to extend our methods to single-cell Neogenin signalling in vivo. We identified multiple genes that were specifically expressed by
RNA-sequencing (scRNA-seq) profiles of blood stem and progenitor cells. scRNA-seq data Neo1hi expressing HSCs, compared to genes expressed by Neo1lo HSCs, including Bmp8a
reveal early progenitor cells from several different lineages as well as quantifying the expres- and Hoxb6. Interestingly, flow cytometry analyses confirmed Neogenin expression on a subset
sion of thousands of genes in individual cells. By linking changes in gene expression with dif- of primitive CD34+CD38- human cord blood HSCs, suggesting that Neogenin signalling may
ferentiation towards specific haematopoietic linages we aim to identify key and novel genes be equally important in human HSCs. Our study reveals the functional relevance of Neo1
involved in regulating the differentiation of HSCs. expression by primitive HSCs and its potential as an HSC marker. Along these lines, ongoing
experiments characterise Neo1hi and Neo1lo HSCs functionally and phenotypically in human
cord blood and mouse bone marrow.
S48 Oral Short Talk Abstracts/ Experimental Hematology 53 (2017) S42-S52
Upon aging, hematopoietic stem cells (HSCs) harbour reduced self-renewal, less efficient bone
marrow (BM)–homing capacity, and show myeloid-lineage skewed differentiation. By cellular
transfer experiments and by analysis of environmental factors, we here tested how extrinsic and
intrinsic factors determine HSC behaviour during aging. We transferred CFSE-labeled young
or aged HSC-containing cell fractions (LKS) into steady-state non-irradiated young or aged
recipients. BM analysis eight weeks later showed that young LKS proliferated faster than
old LKS, independently of their environment. In addition, both young and old LKS were rela-
tively more dormant in an old versus a young environment. To test the HSCs function based on
their divisional histories, we isolated quiescent and various cycling LKS fractions and trans-
planted them into lethally irradiated mice. Quiescent aged HSCs, irrespective of the environ-
ment exposed to, favoured myelopoiesis. In contrast, aged HSCs that were cycling in a young
environment, showed balanced lineage repopulation, similar to young HSCs that were cycling
within a young or an aged environment. Interestingly, serially transplanted and thus experimen-
tally aged HSCs behaved similar as aged HSCs in comparable experimental conditions. To test
extrinsic factors, likely determining biology of ageing HSCs, we performed antibody based
protein arrays and transcriptome analysis with total BM of young and aged animals. Differen-
tial analysis demonstrated that RANTES, MIP-2, IL-1a and IL-1b are upregulated in aged
BM. Further analysis revealed that both, IL-1a and IL-1b drive young HSC towards prolifer-
ation, while this effect is mitigated in aged HSCs. Also, analysis of aged IL1RI KO mice re-
vealed a reduced aging-associated HSCs phenotype. Together, our data demonstrate that
proliferative history imprints a cell-intrinsic dormancy program on HSCs, which is associated
with myeloid-biased differentiation and, at least in natural ageing, with increased IL-1 signal-
ling. Interestingly, this HSC program can in part be ‘‘rejuvenated’’ upon cycling, but not upon
dormancy, in young steady-state environment.
2025 - PRMT5 IS REQUIRED FOR THE MAINTENANCE 2027 - SINGLE UM171 EXPANDED CORD BLOOD
OF GENOMIC INTEGRITY IN HEMATOPOIETIC STEM TRANSPLANT IS FEASIBLE AND SAFE, ACCELERATES
CELLS ENGRAFTMENT, REDUCES HOSPITALIZATION LENGTH
Darren Tan1, Desmond Chin2, Ying Li1, King Pan Ng3, AND MOST IMPORTANTLY IMPROVES HLA MATCHING
Ayako Nakamura-Ishizu1, Henry Yang1, and Toshio Suda1 Sandra Cohen1,2, Jean Roy3, Silvy Lachance3, Anne Marinier4,
1
Cancer Science Institute of Singapore, National University of Singapore, Singapore, Jean Sebastien Delisle3, Denis Claude Roy3, Lambert Busque3,
Singapore; 2Center for Hematology and Regenerative Medicine, Department of Imran Ahmad3, Nadia Bambace3, Lea Bernard3, Thomas Kiss3,
Medicine, Karolinska Institutet, Sweden; 3Cancer and Stem Cell Biology Program, Fannie Larochelle5, Pierre Caudrier6, Severine Landais3,
Duke-NUS Graduate Medical School, Singapore, Singapore
Sebastien Lemieux4, Peter Zandstra7, and Guy Sauvageau4
1
Protein arginine methyltransferase 5 (PRMT5) has been implicated in diverse processes, Department of Hematology, CIUSSS de l’Est de l’Ile de Montreal, Montreal,
including spliceosome assembly. PRMT5 was recently reported to be essential for hemato- Canada; 2University of Montreal, Montreal, Canada; 3CIUSSS de l’Est de l’Ile de
poiesis, and required for splicing in hematopoietic stem/progenitor cells. However, the role of Montreal, Montreal, Canada; 4Institute for Research in Immunology and Cancer,
PRMT5, particularly its importance in splicing regulation, in hematopoietic stem cells Montreal, Canada; 5Center of Excellence in Cellular Therapy, Montreal, Canada;
6
(HSCs) has not been extensively studied. To do so, we employed a loss-of-function approach, ExCellThera, Montreal, Canada; 7University of Toronto, Montreal, Canada
coupled with functional and transcriptome profiling. We report that PRMT5 is essential for
Cord blood (CB) transplants are hampered by low cell dose and high transplant
HSCs. Mx1-Cre Prmt5fl/fl mice (Prmt5D/D) exhibited bone marrow (BM) failure; character-
related mortality (TRM). UM171, a potent agonist of hematopoietic stem cell
ized by rapid reduction in BM cellularity, severe pancytopenia and lethality within 17.5 days.
(HSC) self-renewal could solve this limitation, allowing for CB’s important qualities
Importantly, Prmt5D/D HSCs failed to reconstitute lethally irradiated wild-type recipients.
as lower risk of chronic GVHD and relapse to prevail. Hence, we initiated a clinical
This was associated with transient expansion of the Prmt5D/D HSC compartment, followed
trial to test UM171 expanded CB (eCB). The procedure was designed to be non-labor
by significant loss a week after. Correspondingly, apoptosis was increased among Prmt5D/D
intensive and of short duration for it to be clinically viable. Patients (pts) received a
HSCs, which was associated with p53 activation and aberrant splicing of MDM4; a negative
myeloablative conditioning regimen. On day(D)-7 of transplant, CB was CD34+
regulator of p53. We also obtained similar results in vitro with PRMT5 inhibitor
selected. The CD34- component was cryopreserved and infused at transplant. The
(EPZ015666)-treated EML cell line. We also analyzed the splice variant landscape in
CD34+ component was placed in a closed culture system with UM171 and media
Prmt5D/D HSCs, and identified differential splicing events (DSEs) across several splicing
was injected once a day until D0. This culture system allowed for small culture vol-
classes. Interestingly, DSEs were overrepresented for processes associated with genome
umes, saving cost and labor. Between 9/16-3/17, 7 adult pts received a single eCB.
integrity; e.g. DNA repair. Notably, we show that splicing of key genes involved in base exci-
Median final culture volume was 713mL. Median net CD34 fold expansion was
sion repair, telomere maintenance/repair and Fanconi anemia pathway were dysregulated in
35. Median 1st day of 100 and 500 neutrophils (N) were D+10 and D+18, respec-
Prmt5D/D HSCs. Thus, suggesting Prmt5D/D HSCs are potentially deficient in DNA repair.
tively. As infused (i) CD34 dose is the best predictor of N engraftment with non
In agreement, Prmt5D/D HSCs exhibited elevated gH2A.X levels and DNA strand breaks;
eCB, an algorithm can predict engraftment based on iCD34. We therefore inferred
thus confirming increased DNA damage. Furthermore, Prmt5D/D HSCs also exhibited
that N engraftment improved by a median of 4 days. However, more importantly, pa-
increased replicative stress-associated oxidative DNA lesions. Corroborating these observa-
tients who did not have a N recovery by day 12, had 100 N by day 10 and had clear
tions, Prmt5D/D HSCs were less quiescent and had higher levels of reactive oxygen species.
clinical benefit once 100 N were reached with disappearance of fever and earlier hos-
Thus, loss of PRMT5 renders HSCs vulnerable to DNA damage. Collectively, our study high-
pital discharge. In addition, because cell dose requirements were lower, half received
lights a novel link between PRMT5 and maintenance of genome integrity in HSCs.
a better HLA matched CB. There has been no TRM or relapse. Full donor chimerism
Oral Short Talk Abstracts/ Experimental Hematology 53 (2017) S42-S52 S49
was achieved in all cell subsets. These results favorably compare to other expansion 2029 - CBX7 REGULATES SELF-RENEWAL OF HUMAN
trials which utilize much larger (up to 30L) and longer (up to 21 days) cultures. BENIGN HSC AND INDUCES TERMINAL
Moreover and in contrast to others, we have now initiated a dose reduction study DIFFERENTIATION OF AML CELLS
with smaller, better HLA matched CBs, proven to reduce TRM. Despite very prelim-
Johannes Jung1, Hein Schepers2, Seka Lazare1, Sonja Buisman1,
inary data, a 7 day UM171 single eCB protocol appears feasible and provides clinical
benefit beyond faster engraftment with fewer infectious complications and better
Ellen Weersing1, Bertien Dethmers1, Erik Zwart1, Karin Klauke1,
HLA matching, all the while saving production and hospitalization costs. Raymond Poot3, Leonid Bystrykh1, and Gerald de Haan1
1
European Research Institute for the Biology of Ageing, Groningen, The
Netherlands; 2Experimental Hematology, University Hospital Groningen, Groningen,
The Netherlands; 3Erasmus MC, Rotterdam, The Netherlands
2028 - HEMATOPOIETIC STEM CELLS ARE ACTIVE 2030 - NEOGENIN REGULATES HEMATOPOIETIC STEM
CONTRIBUTORS TO HEMATOPOIESIS IN STEADY STATE CELL QUIESCENCE AND MAINTENANCE
awen1, David Bryder2, Pankaj Mandal3, and Derrick Rossi3
Petter S€ Simom Renders1,2, Pia Sommerkamp1,2, Jasper Panten1,2, Luisa Ladel1,2,
1
Department of Molecular Hematology, Lund University, Malm€o, Sweden; 2Lund Adriana Przybylla1,2, Petra Zeisberger1,2, Daniel Klimmeck1,2,
University, Medical Faculty, Institution for Laboratory Medicine, Division of Katharina Sch€onberger1,2, Nina Cabezas-Wallscheid1,2,3, and
Molecular Hematology, Lund, Sweden; 3Harvard University, Boston Children’s Andreas Trumpp1,2
Hospital, Boston, MA, United States 1
Division of Stem Cells and Cancer, Deutsches Krebsforschungszentrum (DKFZ),
Hematopoietic Stem Cells Are Active Contributors to Hematopoiesis in Steady State Definitive Heidelberg, Germany; 2Heidelberg Institute for Stem Cell Technology and
hematopoietic stem cell (HSC) function has historically been evaluated using transplantation. In Experimental Medicine (HI-STEM gGmbH), Heidelberg, Germany; 3Department of
this setting, self-renewal and potent long-term multilineage contribution can be observed from Cellular and Molecular Immunology, Max Planck Institute of Immunobiology and
even single HSCs. However, recent reports regarding HSC contribution to native hematopoiesis Epigenetics, Freiburg, Germany
have suggested fundamental differences between post-transplantation and steady state hemato- Somatic stem cells harbor the highest self-renewal activity and generate a series of multipotent
poiesis (Sawai et al., 2016; Busch et al., 2015; Sun et al., 2014) and a model with limited HSC progenitors that differentiate into lineage-committed progenitors and subsequently mature
contribution to native hematopoiesis has been proposed. While hematopoietic output has most cells. Recently, we explored the molecular signatures employed by hematopoietic-stem-cells
often been defined as the ability to produce leukocytes, the evaluation of the capacity to generate (HSC) during differentiation by performing quantitative proteome, transcriptome (RNA-seq)
erythroid cells and thrombocytes has often been neglected. Thus, the bulk amount of hemato- and whole genome DNA methylation data analyses on five HSC and multipotent progenitors
poietic cells in need of continuous regeneration has been omitted from such analyses. Here, we populations (MPP1-4) (Cabezas-Wallscheid et al., Cell Stem Cell 2014; Klimmeck et al., Stem
evaluated the kinetics of blood cell generation in adult hematopoiesis using Fgd5-CreERT2 Cell Reports 2014; Lipka et al., Cell Cycle 2014). We identified more than 6,000 expressed
mediated cell tracing (Gazit et al., 2014). Lineage tracing revealed that HSC derived peripheral proteins, 27,000 genes and 15,000 differentially methylated regions. Interestingly, the DCC-
B and T lymphoid cells emerged with very slow kinetics (BOT), while HSC derived granulo- like receptor Neogenin (Neo) was specifically expressed in HSCs. Neogenin can binds
cytes and monocytes emerged with faster kinetics. NK cells were generated from HSCs with different neuronal guidance molecules in a variety of tissues and can function as a BMP co-re-
kinetics more resembling the kinetics for the myeloid lineages. Finally, platelet-tracing revealed ceptor. Based on these observation we hypothesized, that Neo and its ligands may regulate
that this lineage was generated from HSCs not only in a highly robust manner, but also with the HSC maintenance and activation. To investigate its role, we analyzed HSCs isolated from Neo-
fastest kinetics. Detailed kinetic evaluations of defined myeloerythroid progenitors in the bone genin-deficient mice during homeostasis and in reconstitution assays after transplantation.
marrow revealed that megakaryocyte progenitors acquire label earlier than other hematopoietic Interestingly, Neogenin-deficient HSCs gained a competitive repopulation advantage
progenitor subsets. Evaluation of label progression from HSCs to immature Lineage negative, c- compared to control cells in the chimeras. This was associated with reduced stem cell quies-
kit positive and Sca-1 positive (LSK) subfractions revealed that a CD150+CD48+ subset ac- cence. In agreement, RNA-seq analysis of Neogenin-deficient HSCs revealed reduced expres-
quired label faster than other LSK fractions, pointing to a close temporal relationship of sion of dormancy related factors like Egr1, Zfp36 and Tgf-ß signaling. Moreover, Neogenin-
LSKCD150+CD48+ cells and HSCs. The demonstration that HSCs are potent contributors deficient mice presented reduced HSC numbers and potency upon ageing, as well as an
to the megakaryocytic/platelet lineage strongly argues for their physiological importance not increased myeloid differentiation bias. Taken together, our results introduce the Neogenin re-
only upon transplantation, but also in steady state. ceptor as a novel player important for promoting HSC quiescence, while its inhibition increases
reconstitution with exhaustion and loss of HSC self-renewal capacity upon ageing.
S50 Oral Short Talk Abstracts/ Experimental Hematology 53 (2017) S42-S52
2031 - INTERFERON-GAMMA PATHWAY REGULATES 4. RNA sequencing shows that all progenitors have distinct transcriptional sig-
EMERGENCE OF ENGRAFTABLE HEMATOPOIETIC natures with unique expression patterns of transcription factors.
STEM AND PROGENITOR CELLS FROM HUMAN 5. Linking single cell gene expression to FACS indexing we are able to purify exclu-
sively lymphoid and myeloid only progenitors from the current LMP populations.
PLURIPOTENT STEM CELLS
Ryohichi Sugimura1, Clara Soria-Valles1, Deepak Kumar Jha1, In summary, these data define the heterogeneity within existing LMPP, GMP and
Jerry Lee2, and George Daley1 MLP populations and suggest a radically new model of human lympho-myeloid pro-
1
Boston Childrens Hospital, Boston, United States; 2Duke University, Boston, United genitor specification.
States
Derivation of functional human hematopoietic stem cells (HSCs) from autologous hu-
man pluripotent stem cells (PSCs) has been an elusive goal of treatment of hematologic
disorders and malignancy. Building upon recent evidence that HSCs are derived from
definitive hemogenic endothelium (HE), we identified 7TFs (ERG, HOXA5, HOXA9,
HOXA10, LCOR, RUNX1, SPI1) that were sufficient to convert human PSC-derived
HE into engraftable hematopoietic stem and progenitor cells (HSPCs) with long-
term and multilineage capacity (HE-7TF cells, Sugimura, Nature in press). In the cur-
rent study, we attempted to define signaling pathways that further enhance engraftment
of HE-7TF cells. We investigated the source of our long-term engraftable cells by sort-
ing CD34+CD43+CD45+ triple positive cells and CD34+CD43-CD45- single positive
cells from HE-7TF cells. We transplanted these two cell populations into non-irradiated
c-Kit deficient immune-deficient recipients, and monitored engraftment capacity after
8 weeks of transplantation. 2 out of 5 mice transplanted with triple positive cells showed
multi-lineage engraftment, whereas 5 mice transplanted with single positive cells did
not, suggesting that CD34+CD43+CD45+ triple positive cells are the source of long-
term engraftment in our system. We then investigated what signaling pathways regulate
the emergence of CD34+CD43+CD45+ triple positive cells from HE. From high-
throughput screening of 2,000 receptor ligands, cytokines/morphogens, hormone,
and chemical compounds, we observed that interferon-gamma (IFNg) increased
CD34+CD43+CD45+ triple positive cells 6 to 8-fold in 5 independent experiments.
The effect was abrogated by the addition of IFNa. Collectively, these data demonstrate
that the IFNg pathway promotes the emergence of engraftable HSPCs from human
PSC-derived HE.
2032 - SINGLE CELL ASSAYS UNVEIL FUNCTIONAL AND 2033 - CLONAL ANALYSIS OF LINEAGE FATE IN
TRANSCRIPTIONAL HETEROGENEITY OF HUMAN UNPERTURBED HEMATOPOIESIS
HEMOPOIETIC LYMPHO-MYELOID PROGENITORS Alejo Rodriguez-Fraticelli1,2, Samuel Wolock3, Caleb Weinreb3,
Bilyana Stoilova1, Dimitris Karamitros1, Zahra Aboukhalil1, Jialong Sun4, Allon Klein3, and Fernando Camargo4
1
Andreas Reinisch2, Fiona Hamey3, Marina Samitsch1, Lynn Quek1, Stem Cell Program, Boston Children’s Hospital, Boston, United States; 2Harvard
Georg Otto1, Emmanouela Repapi1, Jessica Doondeea1, Stem Cell and Regenerative Biology, Harvard University, Boston, United States;
3
Batchimeg Usukhbayar1, Julien Calvo4, Stephen Taylor1, Harvard Medical School, Boston, United States; 4Boston Children’s Hospital,
Boston, United States
Nicolas Goardon1, Emmanuelle Six5, Francoise Pflumio4,
Catherine Porcher1, Ravindra Majeti2, Berthold Gottgens3, and The classical view of hematopoiesis postulates the existence of branching lineage trees that
Paresh Vyas1 arise from progressively restricted oligopotent progenitor populations. However, these trees
1
MRC Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, have mostly been tested in the contexts of transplantation and in vitro cell culture. Thus, it is
United Kingdom; 2Stanford Institute for Stem Cell Biology and Regenerative unclear whether these branching lineages and such proposed oligopotent progenitors exist
Medicine, Stanford, United States; 3Wellcome Trust-MRC Cambridge Stem Cell under steady state in unperturbed hematopoiesis. Here, we utilize endogenous transposon
Institute, University of Cambridge, Cambridge, United Kingdom; 46UMR967 lineage tracing to study the fate of thousands of progenitors and stem cells over time in order
INSERM/CEA/Universite Paris 7/Universite Paris 11, Paris, France; 57UMR1163, Paris to re-evaluate preexisting hypotheses. Our results describe a novel clonal roadmap where
Descartes–Sorbonne Paris Cite University, Imagine Institute, Paris, France the megakaryocyte replacement is uncoupled to myeloid/erythroid fates. Our data also
demonstrate that blood progenitors are mostly committed to a single lineage, and that
Human lympho-myeloid progenitors (LMPs) downstream of hemopoietic stem cells the truly multilineage contributing cells are restricted to a fraction within the multipotent
remain poorly defined and incompletely purified. Here we purify, functionally (by in progenitor (MPP) compartment. Simultaneous analysis of thousands of stem cell and pro-
vivo transplantation, in vitro single cell, limit dilution and colony assays) and tran- genitor transcriptomes demonstrates that lineage determination starts at the HSC-to-MPP
scriptionally (by population and single cell RNA analysis) characterize 7 human he- transition and identifies a functional hierarchy within these populations that drive hemato-
mopoietic stem and progenitor populations. Focusing on the earliest LMPs, the poiesis. Finally, our results demonstrate that the megakaryocyte lineage branches from
lymphoid-primed multipotent progenitor (LMPP), the granulocyte-macrophage pro- long-term HSCs, which behave physiologically as megakaryocyte progenitors in unper-
genitor (GMP) and the multi-lymphoid progenitor (MLP) we show: turbed conditions. Our data provide evidence for a substantially revised hematopoietic road-
1. The LMPP and MLP are very rare within the mononuclear fraction (1 in 10^4 - map, and highlights unique properties of MPPs and HSCs in situ.
1 in 10^5 cells).
2. In vitro culture of 3806 single cells in 3 different culture conditions and limit dilu-
tion analysis shows that LMPP and GMP are the only progenitors with clonal lym-
pho-myeloid potential (9-15%). The minority of single cells within the populations
are bipotential progenitors but the majority are unipotential (50-80%). The GMP
has mainly myeloid and LMPP and MLP mainly lymphoid potential.
3. When transplanted in vivo using a novel humanized ossicle assay, the LMPP
gives robust myeloid and B cell engraftment, the GMP principally myeloid
engraftment and the MLP mainly B cell output.
Oral Short Talk Abstracts/ Experimental Hematology 53 (2017) S42-S52 S51
2034 - SEVERE CONGENITAL NEUTROPENIA- peripheral blood cells from a JMML patient (Shp2-E78G). Collectively, these data provide ev-
ASSOCIATED MUTATIONS INDUCE ABERRANT STAGE- idence that RUNX1 is an important downstream transcriptional target of activated Shp2 in
SPECIFIC GENETIC PROGRAMS THAT UNDERLIE JMML. RUNX1 is also activated by direct ERK mediated phosphorylation (downstream of
RAS/MEK). We propose that RUNX1 hyperactivation is an important common downstream
BROAD MYELOID DEFECTS
consequence of both activated Shp2 and RAS signaling in JMML, and that RUNX1 inhibition
David E. Muench1, Dietmar J. Kappes2, Nathan Salomonis3, represents a novel therapeutic approach for this disorder.
Kasiani C. Myers4, and H. Leighton Grimes5,6
1
Molecular and Developmental Biology Graduate Program, Cincinnati Children’s
Hospital Medical Center, Cincinnati, United States; 2Fox Chase Cancer Center,
Philadelphia, United States; 3Division of Biomedical Informatics, Cincinnati
Children’s Hospital Medical Center, Cincinnati, United States; 4Division of Bone
Marrow Transplantation and Immune Deficiency, Cincinnati Children’s Hospital
Medical Center, Cincinnati, United States; 5Division of Immunobiology, Cincinnati
Children’s Hospital Medical Center, Cincinnati, United States; 6Division of
Experimental Hematology, Cincinnati Children’s Hospital Medical Center,
Cincinnati, United States
acquisition of copy number alterations affecting the same gene or genes within a pathway, of the KIT-RAS-RAF-MEK-ERK signaling cascade and the downstream cell cycle
thereby resulting in convergent phenotypic evolution; resistance is instead likely driven by a regulator CCND1. Conclusion: As the tumor-suppressive function is independent
pre-existing population of immature cells with a distinct global gene expression signature of patient age or cytogenetics, restoring miR-193b would assure high anti-leukemic
and higher tumor propagating potential. efficacy while preventing the emergence of resistance mechanisms.
ABSTRACTS
POSTER PRESENTATIONS
Poster Presentations
3001 - DECLINED PRESENTATION that of CXCL11 and IL1-b decreased during the progression of leukemia. By
THE NOVEL C/EBPALPHA TARGET GENE EVI2B contrast, the expression of most M2 phenotype-associated genes including ARG1,
REGULATES MYELOID DIFFERENTIATION AND CCL3, CCL17, CD206, IL-10 and TGFb decreased with the infiltration of leukemia
cells whereas MMP9 and VEGFa increased in the late stage of leukemia. Notably,
HEMATOPOIETIC PROGENITOR CELL FUNCTION
high folds increase in iNOS expression, the typical M1 marker, and sustained
Polina Zjablovskaja1, Miroslava Kardosova1, Petr Danek1, decrease in ARG1 expression, the typical M2 marker, were observed. Hence, H-
Pavla Angelisova1, Touati Benoukraf2, Alexander Wurm3, Tomas Kalina4, LAMs showed a more M1-like phenotype. Moreover, H-LAMs expressed a panel
Stephanie Sian2, Martin Balastik5, Ruud Delwel6, Tomas Brdicka1, of cytokines in charge of recruiting inflammatory cells, which contributed to pro-in-
Daniel Tenen7, Gerhard Behre3, Frederic Fiore8, Bernard Malissen9, flammatory microenvironment in liver. These results contributed to a better under-
Vaclav Horejsi1, and Meritxell Alberich-Jorda10 standing of organ-specific macrophage activation and provided a useful reference
1
Institute of Molecular Genetics of the ASCR, Prague, Czech Republic; 2Cancer to the therapy for leukemia.
Science Institute, National University of Singapore, Singapore, Singapore; 3Leipzig
University Hospital, Leipzig, Germany; 4Childhood Leukaemia Investigation Prague,
2nd Faculty of Medicine, Charles University in Prague, University Hospital Motol, Prague,
Czech Republic; 5Institute of Physiology of the ASCR, Prague, Czech Republic;
6
Erasmus University Medical Center, Rotterdam, Netherlands; 7Harvard Stem Cell
Institute, Harvard Medical School, Boston, United States; 8Centre d’Immunophenomique,
Aix Marseille Universite, Inserm, CNRS, Marseille, France; 9Centre d’Immunologie de
Marseille-Luminy, Aix Marseille Universite, Inserm, CNRS, Marseille, France;
10
Institute of Molecular Genetics of the ASCR, Prague, Czech Republic
C/EBPalpha and its target genes are known as important regulators of myeloid differ-
entiation and maintenance of hematopoietic stem cells. Here, we identified a novel C/
EBPalpha target gene, EVI2B, and demonstrated its abundant expression in the ma-
jority of hematopoietic populations, with remarkably high levels in myeloid lineage
cells. We demonstrated that Evi2b depletion alters myeloid lineage development in
vitro, both in murine and human models. Further, we observed that Evi2b-depleted
hematopoietic stem and progenitor cells (HSPC) demonstrate impaired colony form-
ing ability as well as decreased capacity to reconstitute the hematopoietic system of
lethally irradiated mice. In addition, we demonstrated increased apoptosis and
decreased proliferation in progenitor cells with reduced levels of EVI2B, which ex-
plains the affected functionality of these cells. To deepen our understanding of the
mechanistic role of EVI2B in hematopoiesis, we aimed to identify Evi2b interacting
proteins. Using immunoprecipitation followed my mass spectrometry analysis we
identified a list of potential EVI2B binding partners. In within those, CD97, a G pro-
tein-coupled receptor involved in adhesion and migration, was present. The interac-
tion between EVI2B and CD97 was verified by western blot analysis in murine and
human cells. Both EVI2B and CD97 localize in the cellular membrane, and we hy-
pothesize that EVI2B interacts with CD97 and possibly regulates its function. In
addition, alterations in Evi2b-depleted HSPCs might be caused by impaired perfor-
mance of the EVI2B /CD97 complex leading to changes in cell adhesion and/or
migration. Altogether, our work provides an insight into the role of EVI2B in
myeloid differentiation and functionality of hematopoietic progenitors, and suggests
its involvement in cellular adhesion and/or migration.
them IM resistance. To investigate how HNRPDL acts, microarray data comparing 3005 - ANALYSING THE IMPACT OF ERRORS IN SINGLE-
HNRPDL silenced with control K562 cells were generated. The expression of pre- CELL TRACKING EXPERIMENTS
B-cell leukemia homeobox 1 (PBX1) was significantly decreased in HNRPDL Thomas Zerjatke, Ingo Roeder, and Ingmar Glauche
silenced K562 and CML CD34+ cells (n53) compared with their control cells. Acti- TU Dresden, Dresden, Germany
nomycin D treatment impaired the stability of PBX1 upon HNRPDL silencing. The
sequence analysis showed that PBX1 contained a HNRPDL consensus binding motif Time-lapse video microscopy is an increasingly popular method to study the tempo-
(5’- ACUAGC-3’) in its 3’-untranslated region (3’-UTR). A piece of 3’-UTR of ral and spatial behaviour of single cells. It is used for a broad range of applications,
PBX1 covering the consensus motif was sub-cloned in the 3’-end of a luciferase re- e.g. analysing cell migration, proliferation properties, or clonal composition, or re-
porter vector, and it conferred elevated reporter activity in K562 cells than control constructing the complete divisional history of cells that is represented by cellular
vector (6-fold, P ! 0.05, n53). Importantly, PBX1 expression was higher in CML genealogies. A large number of automated methods have been developed for seg-
CD34+ cells (n510) compared with normal control cells (n57) significantly (4- menting and tracking single cells. Although these methods are increasingly sophisti-
fold, P ! 0.05); and PBX1 silencing inhibited the growth of K562 cells, while cated to cope with a broad spectrum of situations they inevitably produce errors in the
PBX1 over-expression rescued the suppressed growth upon HNRPDL silencing. reconstruction of cellular tracks. Using post-processing tools for the manual correc-
Taken together, we have revealed a novel HNRPDL-PBX1 pathway to promote the tion of automatically created tracks can reduce the number of errors. However,
growth of CML cells. ambiguous situations can still occur that lead to different subjective decisions of in-
dividual raters in the assignment of cellular objects. The number of these ambiguous
situations and hence the number of differences in the reconstructed cellular tracks de-
pends on cell type specific properties like migration speed or proliferation rate, as
well as on specific properties of the experimental setting like the spatial and temporal
resolution of the image sequence or the density of seeded cells. To study the inter-
rater variability in single-cell tracking we exemplarily use time-lapse movie experi-
ments of in vitro cultured haematopoietic stem and progenitor cells. We analyse the
impact of the observed variability on the reconstruction of cellular genealogies and
consequently the reliability of statistical measures defined on these data structures.
This analysis is complemented by computer simulations of cell cultures that allow
us to comprehensively mimic a broad range of cell type specific and experimental
properties. Specifically, we aim to quantify maximum error rates that are admissible
to reliably measure particular statistical outcomes. These maximum admissible error
rates can then be accounted for in the design of the experimental set- up and the
choice of the cell tracking procedure.
3004 - INHIBITION OF THE PHOSPHATASE STS1 IN 3006 - TET2 DEFICIENCY IN MESENCHYMAL STEM
ACUTE MYELOID LEUKEMIA CELLS ALTERS THEIR ABILITY TO SUPPORT
Jing Zhang, Olesya Vakhrusheva, and Christian Brandts HEMATOPOIETIC STEM CELL PROLIFERATION AND
Goethe University, Frankfurt am Main, Germany DIFFERENTIATION
The class III receptor tyrosine kinases (RTKs) FLT3 and c-KIT play a central role in
Rong Li1, Zhigang Zhao2, Zizhen Chen1, Wen Xing1, Weiping Yuan1,
normal and malignant hematopoiesis. After binding to their natural ligands, these Fengchun Yang3, Yuan Zhou1, and Mingjiang Xu3
1
RTKs dimerize, become autophosphorylated and activate an intracellular signaling State Key Laboratory of Experimental Hematology, Institute of Hematology and
cascade. Negative RTK regulation by dephosphorylation, ubiquitination and degrada- Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union
tion are equally important to prevent uncontrolled kinase activity and downstream Medical College, Tianjin, China, Tianjin, China (People’s Republic); 2Department of
signaling in normal and malignant hematopoiesis. Recently, we identified STS1/ Hematology and Oncology, Tianjin Medical University Cancer Institute and Hospital,
STS2 as the phosphatases of FLT3 and c-KIT (Zhang J. et al, Stem Cell Reports, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention
2015). Loss of STS1/STS2 causes hyperphosphorylation of FLT3, enhanced AKT and Therapy, Tianjin, China, Tianjin, China (People’s Republic); 3Sylvester
signaling and a strong proliferative advantage of hematopoietic stem and progenitors Comprehensive Cancer Center, Department of Biochemistry and Molecular Biology,
cells in STS1/ STS2 knockout mice. This indicates that STS1 and STS2 serve as University of Miami Miller School of Medicine, Miami, United States
negative regulators for wild-type FLT3 kinase activity and normal hematopoiesis. In- TET2 is a member of TET family which can demethylate 5mC primarily to 5hmC as
ternal tandem duplications of FLT3 (FLT3-ITD) are found in up to 30% of patients well as 5fC and 5caC. TET2 mutations frequently occur in myeloid malignancies.
with acute myeloid leukemia (AML). Here, we investigated the function of STS1/ Although the role of TET2 in HSCs and ESCs are well demonstrated, it remains
STS2 in FLT3-ITD induced malignant transformation. Surprisingly we found that to be explored whether TET2 has important function in stem cells of mesenchymal
loss of STS1 decreased the transforming capacity of the FLT3-ITD mutant, demon- origin. As a vital component of hematopoietic microenvironment, MSCs and their
strated by both reduced cell proliferation and colony forming ability. Importantly, the cellular progenies osteoblasts are essential for HSC maintenance and regulation of
overexpression of STS1 enhanced FLT3-ITD induced cytokine independent cell blood production. To detect the role of TET2 in MSCs, TET2 was knocked down
growth in vitro in a phosphatase-dependent manner. Bone marrow transplantation re- in healthy individual bone marrow derived MSCs (shTET2 MSCs), and empty vector
sults recapitulated the collaboration between FLT3-ITD and STS1/STS2, manifested infected MSCs were used as control. We found that shTET2 MSCs obtained signif-
by the increased engraftment of FLT3-ITD and STS1/STS2 co-expressing cells in icant decrease 5hmC level. Importantly, shTET2 MSCs displayed enhanced self-
vivo. Biological analyses showed prolonged half-life of FLT3-ITD, as well as renewal, proliferation and osteoblast differentiation potential. The hematopoietic sup-
increased downstream signaling in STS1 over-expressing cells. Mechanistically, portive ability of shTET2 MSCs was significantly higher than that of control MSCs.
the E3 ligase activity of CBL was negatively regulated by STS1. In summary, LTC-IC assay revealed that shTET2 MSCs resulted in a skewed differentiation of
STS1/STS2 demonstrated two distinct roles in the context of FLT3wt and FLT3- HSCs towards myeloid cells. Consistently, conditional deletion of Tet2 in MSCs us-
ITD. Our data suggests that the phosphatase inhibition of STS1/STS2 may both ing Prx1–Cre mice demonstrated enhanced proliferation ability, skewed lineage
improve hematopoietic recovery of normal hematopoiesis and decrease the leukemic commitment towards osteoblast and enhanced hematopoietic supportive activity.
potential of FLT3-ITD expressing blasts. Inhibiting STS1 may provide a novel ther- Furthermore, whole genomic 5-hmC profiling and RNA-sequencing analysis showed
apeutic in FLT3-ITD positive AML. that Tet2-/-MSCs exhibited dramatically decreased hydromethylation signatures and
dysregulated gene expression. We confirmed by qPCR 11 dysregulated genes (5 up-
regulated genes Trp63, Fbn2, Sfrp2, Adamts12, Eya1 and 6 down-regulated genes
S56 Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
Hes1, Wnt9a, Syk, Comp, IL-7, Nox4) which could lead to enhanced osteoblast dif- 3008 - TRANSCRIPTIONAL MECHANISMS THAT CONFER
ferentiation potential and 5 down-regulated genes (Ddit4, Pmaip, Ifit3, Clmn and SELF-RENEWING POTENTIAL IN MLL-REARRANGED
Egln) which could contribute to the increased proliferation capacity. Briefly, these re- LEUKEMIA
sults indicate that TET2 loss in MSCs enhances self-renewal, proliferation and oste-
Akihiko Yokoyama1, Hiroshi Okuda1, and Satoshi Takahashi2
oblast differentiation potential, which may in turn alters their ability to support HSC 1
National Cancer Center, Tsuruoka Metabolomics Laboratory, Tsuruoka, Japan;
proliferation and differentiation. These studies provide further insights for the iden- 2
Kyoto University Graduate School of Medicine, Tsuruoka, Japan
tification of additional potential therapeutic targets for patients with TET2 mutated
myeloid malignancies. Cancer stem cells need to self-renew to propagate indefinitely. However, the tran-
scriptional machinery that confers self-renewing potential is poorly understood.
The eleven-nineteen leukemia (ENL) protein family, composed of ENL and AF9,
is a common component of three transcriptional modulators: AF4/ENL/P-TEFb com-
plex (AEP), DOT1L/AF10/ENL complex, and Polycomb repressive complex 1
(PRC1). Each ENL-containing complex associates with chromatin via distinct mech-
anisms, conferring different transcriptional properties including activation, mainte-
nance, and repression. The mixed-lineage leukemia (MLL) gene often fuses with
ENL and AF10 family genes in leukemia. These MLL fusion proteins directly asso-
ciate with some of these ENL-containing complexes to induce leukemia. Therefore, it
is thought that ENL-containing complexes are engaged in transcriptional regulation
to maintain the stemness of cancer stem cell. However, the functional interrelation-
ship among these various ENL-containing complexes in leukemic transformation re-
mains largely elusive. Here, we show that MLL-ENL and MLL-AF10 constitutively
activate transcription by aberrantly inducing both AEP-dependent transcriptional
activation and DOT1L-dependent transcriptional maintenance, mostly in the absence
of PRC1, to confer stemness to leukemia initiating cells. These results reveal a sig-
nificant role of cooperative transcriptional activation mechanism of AEP and DOT1L
in self-renewal and suggest a molecular rationale for the simultaneous inhibition of
the MLL fusion/AF4 complex and DOT1L for better treatment efficacy of MLL-re-
arranged leukemia.
3007 - HSC-INDEPENDENT AND DEPENDENT PATHWAYS 3009 - THE INFLUENCE TO HSC EMERGING BY PTPR
FOR GENERATING PERITONEAL INNATE B-1A CELLS INACTIVATION IN MOUSE EMBRYO
Momoko Yoshimoto1, Michihiro Kobayashi1, and Mervin Yoder2 Tomoko Yamada-Inagawa and Yuko Sekine
1
University of Texas Health Science Center at Houston, Houston, United States; Chiba University, Chiba, Japan
2
Indiana University, Indianapolis, United States
In mouse, adult hematopoietic stem cells (HSCs) emerge in the AGM region at the
Innate immune cells such as tissue resident macrophages have been reported to middle ontogeny. These cells are found in clusters closely associated with the endo-
develop independently from hematopoietic stem cells (HSCs) and persist into post- thelial layer of the aorta. From the complete gene expression profiling of these HSCs
natal life. Similarly, the peritoneal innate B-1a cells have been postulated to be of and their precursors (*1), it was found that receptor protein tyrosine phosphatase
fetal origin because they are not produced from adult bone marrow (BM) HSCs effi- beta/zeta (RPTPb/z) expressed in hemogenic endothelial cells during the endothelial
ciently. Recent reports argue for B-1a cell potential in fetal liver (FL) HSCs by trans- to hematopoietic cell transition (EHT) process. During EHT the shape of endothelial
plantation assay and barcoding system, and these data contradict other papers cell changes from flat to round with migrating toward the lumen of the aorta. The
reporting that B-1a cells emerge in an HSC independent fashion. We have recently EHT process is important for emerging of HSC during ontogeny. RPTPb/z is known
shown that the peritoneal innate B-1 progenitor cells are derived from hemogenic of myelination of neuroaxis, and it is known to be involved in the regulation of self-
endothelial cells at a pre-HSC stage and develop in the fetal liver in the absence renewal and homing of adult HSC and differentiation to blood cells. However, the
of HSCs. However, this evidence supports only the presence of HSC-independent- function of RPTPb/z is unclear in the developmental process of HSC emerging in
B-1 progenitor cells. Therefore, we now ask 1) Are B-1a cells produced from fetal embryo. To elucidate the function of RPTPb/z in the process of EHT, in vitro
liver HSCs and 2) What is the main ontogenic origin of B-1a cells. We transplanted AGM tissue of embryo at E10.5 and E 11 was cultivated with ligand of RPTPb/z
4 populations among FL KSL population based on CD150 and CD48 expression into which disturbs its phosphatase activity. As results in this experiment, the number
NSG neonates and adult BoyJ mice and confirmed the recent report that FL LT-HSCs of each ckit+/CD31+ cells and CD31+ cells were decreased, and CD31-/ckit- cell
do not give rise to B-1a cells efficiently; the CD150-CD48+ population repopulated population was increased. Because RPTPb/z is involved in migration and differenti-
B-1a cells most efficiently. In order to examine if the first HSC in the AGM region ation of cells, it is analysed whether RPTPb/z might transform the morphology of cell
possesses B-1a cell potential, we transplanted 1 or 10 CD45+VEcad+c-kit+EPCR+ for EHT with regulating the expression of identical marker for endothelial cells or
cells into the irradiated adult and neonatal NSG mice, and found that this population HSCs.
contained B-1 cell specific progenitor cells in addition to LT-HSCs that possess both *1 J.Exp.Med 212, 93-106, 2015
B-1a and B-2 lineage potential. Taken together, B-1a cells seem to be derived from
both HSC-independent and dependent pathways, although B-1a cell production from
LT-HSCs is detectable in a very limited developmental time window.
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136 S57
3011 - EFFECTS OF ALLOREACTIVITY ON THE BONE 3013 - UTILIZING ZEBRAFISH FOR NEW INSIGHTS INTO
MARROW MICROENVIRONMENT FOLLOWING THE CELLULAR AND MOLECULAR MECHANISMS OF
ALLOGENEIC HEMATOPOIETIC CELL MICROGLIA ONTOGENY
TRANSPLANTATION Valerie Wittamer1, Giuliano Ferrero1, Eleonore Dupuis1,
Hui Chyn Wong, Stephan Isringhausen, Markus Manz, Christopher Mahony2, Elodie Di Ruggiero1, David Traver3, and
Cesar Nombela-Arrieta, and Antonia M€uller Julien Bertrand2
1
University Hospital Z€
urich, Z€urich, Switzerland Universite Libre de Bruxelles, ULB, Brussels, Belgium; 2University of Geneva,
Geneva, Switzerland; 3University of California, San Diego, La Jolla, United States
Allogeneic hematopoietic cell transplantation (allo-HCT) represents the most powerful
form of cellular therapy for hematological malignancies. Donor T-cell mediated immune Microglia are tissue-resident macrophages in the central nervous system. They represent a
reactions are critical for leukemia cell elimination, but can also target other tissues, causing distinct population of mononuclear phagocytes that serve multiple functions in brain devel-
graft-versus-host-disease. Little attention has been paid to alloreactivity against the bone opment and homeostasis. Previous observations in the human, mouse, avian and zebrafish
marrow (BM) microenvironment and how its structural damage affects hematopoiesis. embryo have demonstrated that the brain parenchyma is seeded by microglial precursors dur-
Here we examined in a MHC-matched, miAg-mismatched mouse model (C3H.SW/B6) ing the early stages of embryonic development. Across vertebrate phyla, myelopoiesis occurs
the effects of lethal radiation conditioning followed by infusion of pure hematopoietic in three distinct waves, beginning with specification of primitive macrophages. Next, oligo-
stem cells (cKIT+Sca1+Lin-) +/- T-cell subsets (total T cells, conventional potent Erythro-Myeloid Progenitors (EMPs) are specified, followed by the generation of mul-
CD4+CD25-, or CD8+) on the BM using FACS and 3D-confocal imaging. Alloreactive tipotent Hematopoietic Stem Cells (HSCs) from aortic hemogenic endothelium. Based on
(but not congenic) T cells severely impaired hematopoiesis and disrupted the non-hemato- fate mapping experiments performed in the mouse model, two putative origins of microglia
poietic compartment: at 2 wks post allo-HCT, B6 mice given pure C3H.SW-HSC showed during vertebrate development have been proposed, namely the primitive macrophages and
prompt recovery of BM cellularity and numbers of endothelial cells, and CXCL12-abun- the EMPs. The relative contribution of each of these populations is however unclear and re-
dant reticular (‘‘CAR’’) cells. Likewise, hematopoietic recovery of myeloid and B-cells mains to be determined. In an effort to provide new insights into microglia ontogeny, we have
was fast. In contrast, recipients of HSC+T cell subsets had significantly lower absolute utilized the unique attributes of the zebrafish to investigate which precursor subsets generate
numbers of stromal subsets and hematopoietic cell populations in the BM (except for brain microglia. Here, we show that microglia arise in two independent waves during embry-
expanded, co-transferred T cells) and their recovery was significantly delayed. 3D- onic development. First, primitive macrophages generate a transient wave of embryonic mi-
confocal imaging confirmed rapid recovery of the extracellular matrix, and sinusoidal croglia. This primitive wave is replaced by definitive microglia that derive from a vascular
vascular structures in the HSC group. In contrast, mice given HSC+T-cell subsets, showed endothelium with hemogenic properties, a concept that is reminiscent of the endothelial
severe disruption of the structural integrity with impaired recovery of the BM microvessels origin of definitive hematopoietic progenitors during embryogenesis (EMPs and HSCs).
at 2 wks post-HCT. Our data shows the impact of alloreactivity on the non-hematopoietic We are currently investigating this hypothesis, using zebrafish hematopoietic mutants. In-
BM compartments in terms of damage and reconstitution of the microenvironment, and sights into the cellular and molecular events that instruct microglia development may ulti-
ultimately hematopoietic recovery. Whether alloreactive T cells directly target these struc- mately contribute to the generation of microglia-like cells in vitro that could potentially
tures or act via an inflammatory milieu needs to be elucidated. serve as a source of replacement microglia for treatment of neurodegenerative diseases.
S58 Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
3015 - DISTURBED IMMATURE HEMATOPOIESIS IN MICE 3017 - THE ACTIN BINDING PROTEIN PLASTIN-3 IS
WITH HEMATOPOIETIC-SPECIFIC WAVE COMPLEX INVOLVED IN THE PATHOGENESIS OF ACUTE MYELOID
DEFICIENCY LEUKEMIA
Eliza Wiercinska1, David de Gorter2, Eva Danner1, Sabine Harenkamp1, Jasmin Wellbrock1, Arne Velthaus1, Kerstin Cornils1, Saskia Gr€ub1,
Anja Schwiebs3, Theresia Stradal4, and Halvard B€onig1 Hauke Stamm1, Daniel Wicklein1, Carsten Bokemeyer1, Michael Heuser2,
1
German Red Cross Blood Donor Service Baden-Wuerttemberg-Hessen, Frankfurt, Sabine Windhorst1, and Walter Fiedler1
Germany; 2Institute of Experimental Musculoskeletal Medicine, University Hospital 1
University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 2Hannover
unster, Germany; 3Institute of General Pharmacology and Toxicology,
M€unster, M€ Medical School, Hannover, Germany
pharmazentrum frankfurt/ZAFES, Clinic of the Goethe University, Frankfurt,
Germany; 4Helmholtz Centre for Infection Research, Department of Cell Biology, Leukemia-initiating cells reside within the bone marrow (BM) in specialized niches where
Braunschweig, Germany they undergo complex interactions with their stromal cells. In order to identify genes being
implicated in the interaction of acute myeloid leukemia (AML) cells and stromal cells, we
WAVE is a heteropentameric complex involved in signal integration between the small performed co-cultures of primary AML cells with endothelial cells and osteoblasts. The
GTPase Rac and downstream ARP2/3-dependent actin polymerization. To study its role in gene expression of co-cultured AML blasts was compared to AML cells grown without stro-
immature hematopoiesis we used a previously described Hem1 knock-out model. Hem1 is mal cells using microarray analysis. Amongst those genes being dysregulated upon co-cul-
strictly hematopoietic specific, but essential for WAVE complex assembly in hematopoietic ture was the actin binding protein plastin-3 (PLS3). Further RT-qPCR analysis revealed an
cells, thus Hem1 knock-out leads to WAVE complex deficiency exclusively in cells of he- endogenous PLS3 expression in about 50% of primary AML samples (n525) but only in 2
matopoietic origin. In Hem1-/- mice, the number of circulating hematopoietic stem and pro- of 12 analyzed AML cell lines. Functional analysis of PLS3 in AML was studied using
genitor cells (HSPC) was strongly elevated, at the same time, HSPC content in the BM of shRNA knockdown and overexpression of PLS3 in Kasumi-1 cells. We revealed that
Hem1-/- mice was reduced by half compared to WT BM, despite strongly induced (presum- PLS3 has an impact on the colony formation capacity of AML cells in vitro as the knock-
ably compensatory) proliferation compared to WT HSPC. Moreover Hem1-/- HSPC failed to down resulted in significantly reduced colony numbers while increased colony growth was
migrate towards the crucial BM niche chemokine CXCL12 in transwell assays, illustrating observed in the Kasumi-1 cells overexpressing PLS3. To investigate the role of PLS3 in vivo,
cell intrinsic defects in HSPC lacking functional WAVE complex. Next we tested the perfor- NSG mice were transplanted with the PLS3 knockdown Kasumi-1 cells. Compared to mice
mance of Hem1-/- HSPC in transplantation assays. Homing, the initial interaction with BM transplanted with Kasumi-1 control cells, the PLS3 knockdown mice survived significantly
environment, was unaffected by Hem1 deficiency. By contrast, engraftment, the ability to longer (median survival time 64 vs. 110 days, respectively). Finally, we investigated
take up normal blood cell production, was strongly diminished in recipients of Hem1-/- whether the expression of PLS3 was associated with AML patients’ outcome using pub-
BM. Neutrophils did not engraft at all, and RBC and platelet engraftment were delayed. lished microarray-based gene expression data (Verhaak et al, Haematologica 2009;94).
Next we tested the competitive repopulation capacity of Hem1-/- vs. WT HSPC. To compen- Clinical data of 290 AML patients were available. Based on the mean gene expression value,
sate for the expected competitive disadvantage of Hem1-/- cells we transplanted an 4:1 excess the patient cohort was divided into high vs low PLS3 expressors. The overall survival was
of Hem1-/- BM cells over WT-GFP+ BM competitor cells; similarly control mice were trans- analyzed in a multivariate Cox proportional hazards model including PLS3 gene expression
planted with 4:1 mix of WT:GFP+ BM. Even under those conditions, Hem1-/- HSPC were and the baseline parameters age, karyotype and FLT3 mutational status. After a stepwise
not able to properly engraft the recipients. Whereas in the control experiments, WT BM out- removal of insignificant terms, the patient’s age and a high PLS3 expression remained as
competed GFP+ BM over time (two months), recipients of Hem1-/- and GFP+ BM were independent prognostic survival markers. In conclusion, our results identify the actin bind-
long-term repopulated solely with GFP+ BM : 2 weeks after transplantation almost all of ing protein PLS3 as potential novel therapeutic target in AML.
B-cells were GFP+. Hem1-/- granulocytes and monocytes were outcompeted in the first 2-
4 weeks after transplantation and Hem1-/- T-cells shortly thereafter. Taken together, Hem1
and WAVE complex are indispensable for proper HSPC function.
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136 S59
3018 - MOLECULAR SIGNALLING FINGERPRINTING OF was significantly reduced, while subpopulations #1, #3 and #4 (including myeloblasts
HUMAN HEMATOPOIETIC STEM CELL FATE and myelocytes) were increased in Rheb1D/D mice. To determine whether Rheb1 af-
Weijia Wang1 and Timm Schroeder2 fects neutrophil maturation intrinsically, we transplanted Rheb1fl/fl or Rheb1D/D
1
Department of Biosystems Science and Engineering, ETH Z€urich, Basel, LKS+ cells into lethally irradiated WT recipients and analyzed the percentage of
Switzerland; 2ETH Z€
urich, Basel, Switzerland donor-derived neutrophils. The donor-derived neutrophils were mostly immature in
mice receiving Rheb1D/D LKS+ cells. These indicate that an intrinsic maturation
Despite decades of research on hematopoietic stem cells (HSCs), little is known about the defect of Rheb1D/D LKS+-derived neutrophils. Since neutrophil maturation defect
molecular mechanisms by which HSCs integrate the signals from their environment and may lead to a reduction in microorganism uptake and bacteria killing, we assessed
determine cell fate (e.g., self-renewal vs. differentiation). It has become clear that the intra- the role of Rheb1 in neutrophil phagocytosis. Neutrophils were isolated and cultured
cellular ‘‘signal processing’’ network is highly interconnected and that the network’s state, with zymosan for live cell imaging. Only approximately 15% of the Rheb1D/D neu-
rather than isolated pathways dictates cellular outcome. On the other hand, HSCs trophils engulfed zymosan, in contrast to 35% of the Rheb1fl/fl neutrophils. The bac-
constantly receive multiple input signals from their environment to maintain hematopoi- terial survival assay showed that Rheb1D/D neutrophils killed less than 10% of the
etic homeostasis. Therefore, systems-level approaches are required to obtain a comprehen- bacteria, while Rheb1fl/fl neutrophils killed more than 60% of the bacteria. In addi-
sive understanding of the molecular control of HSC fate. In addition, the lack of direct tion, Rheb1fl/fl neutrophils released maximal ROS one minute after fMLP stimula-
measurement of both intracellular signalling activity and functional outcome of the tion, while Rheb1D/D neutrophils showed minimal response to the stimulation.
same cell has limited the development of models that can predict cellular responses of a These data collectively indicate that Rheb1 deletion impairs the maturation of neu-
heterogeneous population such as HSCs. To overcome these limitations, interdisciplinary trophils and their phagocytic ability.
methods including live cell imaging, molecular biosensors, microfluidics, and computa-
tional models are used to allow for the direct connection of intracellular signalling dy-
namics with cell fate outputs in human HSCs at the single cell level. Proof-of-principle
experiments have demonstrated the feasibility of introducing translocation-based biosen-
sors into human umbilical cord blood-derived CD34+ cells to enable the continuous and
quantitative measurement of the signalling pathway (e.g., phosphoinositide 3-kinase
(PI3K), extracellular signal-regulated kinase (ERK)) activation in single, live cells. These
single-cell signalling dynamics data, together with data-driven models will be used to
identify key signalling nodes that control the cellular outcomes in human HSCs.
3021 - REGULATION OF HUMAN STEM AND 3023 - IMPAIRMENT OF THE BONE MARROW
PROGENITOR CELLS BY ACUTE MYELOID LEUKAEMIA MICROENVIRONMENT AND HEMATOPOIESIS BY
IN THE HUMAN NICHE WARFARIN
Alexander Waclawiczek, Ashley Hamilton, Kevin Rouault-Pierre, Divij Verma1, Keertik Fulzele2, Rahul Kumar1, Soraya Hoelper3,
Ander Abarrategi, and Dominique Bonnet Paul Ziegler1, Eva Weissenberger1, Sara Chahabi4, Michaela Fontenay4,
The Francis Crick Institute, London, United Kingdom Paola Pajevic2, and Daniela Krause1
1
Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy,
In acute myeloid leukemia (AML) the suppression of normal blood production is a
Frankfurt Am Main, Germany; 2Boston University, Goldman School of Dental Medicine,
major factor contributing to treatment complications and mortality rates. Recently
Boston, United States; 3Institute for Biochemistry II, Goethe University, Frankfurt Am Main,
it was demonstrated that cell cycle inhibition of haematopoietic stem and progenitor
Germany; 4Laboratory of Hematology, H^opital Cochin, Universite Paris Descartes, Paris,
cells (HSPCs) may be the underlying cause of suppression in both AML xenograft
France
and transgenic mouse models. However, the mechanism in human HSPCs is still
poorly understood. Using a fully humanized ex vivo system comprising AML cell The coagulation system is a complex system comprised of various proteases and other factors
lines, umbilical cord blood derived CD34+ HSPCs and bone marrow mesenchymal which result in the formation of a blood clot, and proteases in general are known to regulate
stroma cells (MSCs), we studied the interactions between AML, normal HSPCs mobilization of normal hematopoietic stem cells (HSC) from the bone marrow microenviron-
and their stromal niche. In our ex vivo system, AML but not normal white blood cells ment (BMM). However, whether the proteases of the coagulation system are involved in the
rapidly suppress the proliferation and differentiation of normal HSPCs via cell cycle mobilization or maintenance of hematopoietic stem and progenitor cells (HSPC) is unknown.
inhibition of both (CD34+CD38+) progenitors and (CD34+CD38-CD45RA-CD90+) Hypothesizing that the proteases of the coagulation system play a role in the normal physi-
stem cells. In line with the finding of the in vivo AML models, AML did not induce ology of the BMM and that modulation of coagulation factors may have an effect on HSPC,
apoptosis in HSPCs. Transcriptomic interrogation of a stroma cell line after AML we tested the effects of an anticoagulant warfarin, used in 1% of people worldwide for pro-
exposure identified a hypoxia-response signature and an upregulation of Hypoxia phylactic or therapeutic anticoagulation. Indeed, treatment of wildtype mice with warfarin led
inducible factor -1 alpha (HIF-1a) target genes. Hypoxia-induced stabilization of to a decrease in myeloid cells in peripheral blood, decreased HSC function and mobilization
HIF-1a in MSCs recapitulated AML induced HSPC suppression, which was revers- from the BMM and an almost 8 fold reduction of functional HSC. Intrafemoral co-transplan-
ible by HIF-1a knockdown. Furthermore, we identified Stanniocalcin 1 (STC1), a tation of warfarin-treated, ex-vivo expanded primary murine bone marrow stromal cells with
small secreted glycoprotein, to be upregulated via HIF-1a dependent mechanisms untreated HSPC led to decreased engraftment of HSPC compared to co-transplantation of
in MSCs after AML exposure. Recombinant STC1 alone reduced HSPC proliferation vehicle-treated stroma cells suggesting that warfarin may compromise bone marrow stroma.
and differentiation, using a STC1 neutralizing antibody, we were able to significantly We implicated periostin, a vitamin K dependent factor produced by bone marrow stroma –
reduce the AML-induced cell cycle inhibition of stem cells and increase the number binding to and signaling via integrin ß3 on HSC - as being the cause of impairment of hema-
of progenitors. In summary, we developed a fully humanized ex vivo system to study topoiesis. In addition, warfarin treatment of NOD SCID interleukin-2 receptor gamma
the interaction between AML, normal HSPCs and the stroma niche. We identified knockout (NSG) mice or intrafemoral cotransplantation of human warfarin-treated stroma
HIF-1a stabilization and its downstream target STC1 in MSCs as key contributors cells and untreated human CD34+ HSC led to reduced engraftment of human CD45+ cells.
to the suppression of HSPC differentiation and proliferation, providing a potential In corroboration of our murine data we showed that the total leukocyte and monocyte count,
route to release normal blood production from AML-induced suppression. as well as the percentage of basophils and eosinophils were significantly reduced in 282 pa-
tients receiving the vitamin K antagonist fluindione for anticoagulation. In summary, warfarin
treatment impairs HSC function in humans and mice via modification of the BMM and, spe-
cifically, by the impairment of the periostin/integrin ß3 axis, which we, thereby, implicate also
for the maintenance of normal HSC.
displayed lower densities. Transmigration endothelial assays suggested a correlation 3026 - DECLINED PRESENTATION AND PUBLICATION
of cortactin expression with the capacity of leukemic cells transmigration in response
of CXCL12. Conclusions: Cortactin is over-expressed in ALL and may contribute
the TEM capacity and extramedullary infiltration of CRGs and relapse
3025 - TARGETING HYPOXIA PATHWAY IN A MODEL OF 3027 - MITOCHONDRIA METABOLISM REGULATES THE
ACUTE MYELOID LEUKEMIA ACTIVATION OF HEMATOPOIETIC STEM CELLS
Talia Velasco-Hernandez1, J€org Cammenga2, and David Bryder1 Terumasa Umemoto1, Michihiro Hashimoto1, and Toshio Suda2
1 1
Department of Molecular Hematology, Lund Stem Cell Center, Lund University, International Research Center for Medical Sciences (IRCMS), Kumamoto
Lund, Sweden; 2Department of Hematology, Institute for Clinical and Experimental University, Kumamoto, Japan; 2Cancer Science Institute, National University of
Medicine, Link€
oping University, Link€oping, Sweden Singapore; International Research Center for Medical Sciences (IRCMS), Kumamoto
University, Singapore, Singapore
Acute Myeloid Leukemia (AML) is a hematological disease caused by diverse cytogenetic and
molecular alterations. While new therapeutic approaches are desperately needed, the genetic Hematopoietic stem cells (HSCs) are located in a specialized microenvironment
heterogeneity of AML constitutes a barrier for the development of targeted therapies for this called ‘‘niche’’ within the bone marrow (BM), in which HSC quiescence are mainly
disease. A better understanding of common requirements for all subtypes of AML, for instance maintained through glycolytic metabolism. Nevertheless, HSCs are also activated un-
with regards to tumor environment and metabolism, has the potential to lead to new and more der the stress, such as inflammation or acute blood loss, probably through an increase
broadly applicable therapies. Relapse of leukemia after treatment is most often caused by resis- in cytokines and other humoral factors. Although many report show several mecha-
tance of a subgroup of cells commonly referred to as leukemia initiating cells (LICs). These nisms for regulation of HSC quiescence, the mechanism for HSC activation still re-
cells are thought to harbor this resistance due to specific properties, including their dormant mains unclear. In this study, focusing on the change of energy metabolism, we
and metabolically inactive state, making them less susceptible to genotoxic drugs. The local- elucidate the mechanism for the activation of HSCs from the BM suppression
ization of the LICs to hypoxic niches has been proposed as one of the factors that drive the induced by 5-fluoruracil (5-FU). Since expression patterns of some general HSC
dormancy of the cells. Cellular adaptation to hypoxia is mediated by the transcription factors markers in HSCs are often affected by several stimulations containing 5-FU admin-
HIFs (hypoxia-inducible factors). While the role of HIFs in pathogenesis of solid tumors has istration, we introduced an EPCR phenotyping of HSCs, which is independent of in-
been known for a long time, HIFs have only recently been suggested as key regulators of LICs flammatory stimulations. Using this definition of HSCs, we found that HSCs started
in particular types of leukemia. At present, the topic remains highly controversial, with some to enter cell cycle from 3 to 4 days after 5-FU administration. Importantly, cycling
studies supporting an oncogenic activity of HIFs, while others have pointed to a role for HIFs as HSCs after 5-FU treatment have the BM reconstitution activities, which is equivalent
tumor suppressors. We are currently studying the influence of the hypoxia pathway through the to quiescent HSCs. HSCs showed high membrane potential of mitochondria just
deletion of HIF-1a or its regulators in different aspects of the leukemia initiation and progres- before entering cell cycle, while most of HSCs showed a low activity of mitochondria
sion, including alterations of their metabolism and their susceptibility to chemotherapy. We at steady state. RNA array data also supported that the oxidative metabolism in HSCs
anticipate that a better understanding of the mechanisms that underlie the functions of HIFs enhanced on day 4 after 5-FU. Interestingly, treatment with Dinitrophenol (DNP),
in LICs could identify new therapies aimed at eliminating these resistant cells. known as an uncoupler, led to delayed recovery of BM through impaired HSC expan-
sion after 5-FU, but little affected HSCs at steady state. Moreover, we found that ox-
ygen concentration, cytokines and nucleotides affect the membrane potential of
mitochondria in HSCs after 5-FU administration. Collectively, our results indicate
that the activation of HSCs during the recovery from BM suppression is mediated
by the enhanced membrane potential of mitochondria, which is induced by the
change of surrounding environment. Thus, the change of energy metabolism in
HSCs play a key role in the regulation of cell cycle.
S62 Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
3028 - SMO AND GLI2 ARE KEY REGULATORS 3030 - CYTOKINE-MEDIATED ENDOCYTOSIS IS CRUCIAL
MEDIATING RESISTANCE OF CML STEM/PROGENITOR FOR THE THERAPEUTIC RESISTANCE OF LEUKEMIA-
CELLS TO TYROSINE KINASE INHIBITORS PROPAGATING CELLS
Kelly Turner1,2, Katharina Rothe1, Adrian Woolfson3, and Xiaoyan Jiang1 Cedric Tremblay1, Sung Kai Chiu1, Jesslyn Saw1, Stefan Sonderegger1,
1
British Columbia Cancer Agency, Vancouver, Canada; 2University of British Phillip Robinson2, and David Curtis1
Columbia, Vancouver, Canada; 3Pfizer, Inc., New York, United States 1
Monash University, Melbourne, Australia; 2University of Sydney, Sydney, Australia
Growing evidence suggests that the Hedgehog (HH) pathway plays a key role in the Current therapeutic regimen transiently reduce the tumour burden of patients with T-
survival of primitive chronic myeloid leukemia (CML) cells. However, it is not cell acute lymphoblastic leukemia (T-ALL), but fail to eliminate refractory leukemia-
known whether the expression of key regulators of the pathway in leukemic stem propagating cells (LPCs), which are responsible for relapse. Hence, the eradication of
cells (LSCs) and progenitor cells differ between ABL tyrosine kinase inhibitor LPCs that evade from chemotherapy, will be determinant for achieving long-term
(TKI) imatinib (IM) responders and non-responders. We performed RNA sequencing remission. These resistant LPCs are dependent on the growth factors produced by
analysis of 6 CD34+ chronic phase (CP)-CML samples obtained at diagnosis. 3 pa- the thymic niche to develop and survive following chemotherapy treatment. Using
tients were retrospectively classed as IM responders, and 3 as IM non-responders. We the Lmo2 transgenic mouse model, we have shown previously that these LPCs
compared global gene expression changes between these samples with 3 healthy bone have long-term self-renewal potential and resistance to chemotherapy. We have
marrow (HBM) controls. We identified 27 differentially-expressed (O1.5 fold) HH also shown that growth factors produced by the thymic niche, like IL-7 and Notch1,
pathway genes between CML patients and healthy controls. In particular, Smooth- are crucial for the development and maintenance of these resistant LPCs during the
ened (SMO) and GLI2 were highly upregulated in IM non-responders as compared early stage of the disease. In thymocytes, the activation of these signalling pathways
with responders. We confirmed these results in an additional 18 CP-CML samples is tightly controlled by endocytosis, which is dependent on the GTPase Dynamin 2
and 8 HBM samples using quantitative real-time PCR (p ! 0.05). SMO and GLI2 (DNM2). We found that treatment of Lmo2 transgenic DN3 T-cell progenitors
were most highly expressed in the stem-enriched subpopulation (lin-CD34+38-) as with the specific DNM2 inhibitor Dynole 34-2 blocked IL-7R internalization and
compared with progenitor (lin-CD34+38+) and the more mature (CD34-) subpopula- downstream activation of Stat5, confirming that DNM2 activity is essential for cyto-
tions, especially in IM non-responders compared with IM responders (O50-fold for kine-mediated response in thymocytes. Inhibition of endocytosis with Dynole 34-2
GLI2). We then tested the effects of a highly selective SMO inhibitor PF-04449913 in significantly reduced the frequency and the engraftment of LPCs in transplantation
CD34+ cells from IM responders and IM non-responders. In accordance with the assays. We then treated 2 month-old mice to address the importance of niche-medi-
gene expression results, IM non-responders were more sensitive to SMO inhibition ated signalling in therapeutic resistance. Accordingly, we found that treatment with
as compared with IM responders with regard to viability, induction of apoptosis, Dynole 34-2 sensitized LPCs to the current induction regimen used in the clinic
re-plating potential, and colony-forming ability following long-term (O6-weeks) cul- for T-ALL. Finally, inhibition of DNM2 activity significantly delayed leukemia onset
ture. We also tested a treatment strategy comprising the second generation TKI bo- in mice treated with standard therapeutic agents, suggesting that endocytosis is
sutinib in combination with PF-04449913 in CD34+ IM non-responder cells. This required for the maintenance of treatment-resistant T-ALL. These results provide
was significantly better at reducing colony-forming ability (p ! 0.01) and re-plating the most convincing in vivo evidence that endocytosis is crucial for the maintenance
potential (p ! 0.01) compared with either agent alone. The results suggest that dual and therapeutic resistance of LPCs. Targeting endocytosis may represent an attractive
inhibition of the BCR-ABL and HH pathways may provide a compelling strategy for therapeutic strategy for improving cure rates in refractory leukemias.
targeting drug-insensitive LSCs.
3029 - THE ROLE OF THE H3K4-DEMETHYLASE, KDM5C, 3031 - A STANDARDIZED QUANTIFICATION TOOL FOR
IN MALIGNANT HEMATOPOIESIS BONE MARROW COMPONENTS IN HISTOLOGICAL
Mette Louise Trempenau, Mikkel Schuster, Nicolas Rapin, and Bo Porse SECTIONS
Finsen Laboratory, Rigshospitalet, UCH, Copenhagen N, Denmark Josefine Tratwal1, Chiheb Boussema1, Olivier Burri2, Tereza Koliqi1,
Background: Acute Myeloid Leukemia (AML) is an aggressive blood cancer, char-
Vasco Campos1, Valentina Nardi3, Carmen Barcena4, Rossella Sarro5,
acterized by a rapid accumulation of immature myeloid blasts at the expense of he- Bettina Bisig5, Laurence De Leval5, and Olaia Naveiras1
1
matopoiesis. Unfortunately, only limited improvements in standard treatment and in Laboratory of Regenerative Hematopoiesis, EPFL, Lausanne, Switzerland;
2
overall patient survival have been achieved over the last three to four decades. This Bioimaging and Optics Core Facility, EPFL, Lausanne, Switzerland; 3Massachusetts
emphasizes a need for better understanding of the tumor biology. Epigenetic factors General Hospital, Harvard Medical School, Boston, United States; 4Department of
have been implicated as crucial regulators of tumor biology, which prompted us to Pathology, University Hospital Madrid, Spain; 5University Institute of Pathology,
carry out in vivo shRNA screens to identify novel factors involved in AML progres- CHUV, Lausanne, Switzerland
sion. This led to the identification of Lysine Demethylase 5C (KDM5C) as a tumor The bone marrow (BM) is the seedbed of our blood, existing as hematopoietic or adi-
suppressor in C/EBPa mutated AML. Aim: This project aims to elucidate the role of pocytic marrow heterogeneously distributed with skeletal location, age, and physio-
the epigenetic factor KDM5C in AML. Methods: Using murine AML models and logical condition. Patients who undergo chemotherapy or suffer acute BM failure
flow cytometry assays we investigate the effect of KDM5C in leukemic initiation have a rapid adipocytic conversion of the marrow. After recovery such as upon he-
and progression. To elucidate the mechanisms underlying the effect of KDM5C, matopoietic stem cell transplantation (HSCT) following intensive chemotherapy,
we will identify target genes by a combination of microarray and ChIP-seq and functional hematopoiesis is restored, for which the Gold Standard of quantification
test the effects of these genes on tumor maintenance. Result: Knockdown of is pathologist assessment of BM cellularity in H&E sections. This expertise is not
KDM5C leads to a tumor progressive advantage in C/EBPa mutated AML and pre- accessible to all laboratories, and multi-site standardization of BM cellularity quan-
sents a less differentiated leukemia. Furthermore, microarray analysis showed down- tification is often critical in the clinical and fundamental research setting. In our effort
regulation of AP1 transcription factor genes, known to have a role in myeloid to systematically quantify BM components in histological sections in an unbiased
differentiation. We further show that modulation of c-Jun expression, an AP1 factor, manner, we developed and optimized a semi-automated image analysis tool for Im-
can partially rescue the phenotype of KDM5C knockdown in our AML model. ageJ, MarrowQuant. Area of hematopoietic cells, red blood cells, bone, and adipocyte
Conclusion: We suggest that KDM5C functions as a tumor suppressor in AML in ghosts are identified based on color and texture variations of H&E stains. We find that
part by guiding differentiation through regulation of AP1 gene expression. BM cellularity quantifications correlate directly with scoring by four independent
clinical pathologists from different countries, while quantification of bone and adipo-
cytes compare with microCT volumetric measurements. We have established a
consistent map of cellularity in BM sections of homeostatic C57BL6 mice, and
observe highly predictable hematopoietic-to-adipocytic marrow transitions in the
tibia and caudal tail. With age, BM adiposity increases in the tibia and appears in
the femur. Following HSCT, adipogenesis inversely correlates with kinetics of he-
matopoietic recovery. BM adipocytes reach maximum expansion 17 days post-trans-
plant and hematopoiesis recovers after 25 days, consistent with the exit of severe
neutropenia and recovery of pre-transplant blood counts. MarrowQuant has been
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136 S63
validated on human trephine biopsies and we have observed that it may offer greater growth and NOTCH1 signaling, which might be a mechanism underlying growth
discrimination than pathologist evaluation on extreme cellularities (eg. 0-20% and suppression in CDKN2A-deleted cells. BMI1 inhibitors can be candidates for novel
80-100%), opening avenues for its application in experimental or clinical contexts molecular-targeted therapy.
requiring standardized measurements of BM components.
3032 - BMI1 INHIBITORS SUPPRESS CELL GROWTH AND 3033 - MATRIX METALLOPROTEINASES 2/9 ARE
NOTCH SIGNALLING OF ACUTE LEUKEMIA CELLS NECESSARY FOR INFLAMMATORY-ASSOCIATED
Mika Ohtaka1, Mai Itoh2, and Shuji Tohda2 REGULATION OF HEMATOPOIETIC STEM AND
PROGENITOR CELL PRODUCTION AND FUNCTION
1
Tokyo Medical and Dental University, Dept. of Laboratory Medicine, Tokyo, Japan;
2
Tokyo Medical and Dental University, Tokyo, Japan Lindsay Theodore1, Elliott Hagedorn2, Mauricio Cortes3, Leonard Zon2,
BMI1 is a key component of polycomb repressive complex 1. BMI1 expression is and Trista North3
1
up-regulated in various cancer cells. BMI1 suppresses expression of genes such as Harvard Medical School, Boston, United States; 2Boston Children’s Hospital,
CDKN2A, which encodes p16INK4A/p14ARF, thereby promoting cell prolifera- Harvard Medical School, Boston, United States; 3Beth Israel Deaconess Medical
tion. CDKN2A is often deleted in leukemia cells. To examine whether BMI1 inhib- Center, Harvard Medical School, Boston, United States
itors can be novel molecular-targeted drugs, we examined the effects of BMI1 Hematopoietic stem/progenitor cells (HSPCs) are formed during embryonic development
inhibitors on growth of leukemia cells. Six leukemia cell lines (OCI/AML2, OCI/ from hemogenic endothelium in the ventral wall of the dorsal aorta (VDA), but the physical
AML5, THP-1, and TMD7 derived from AML; Jurkat and KOPT-K1 from T- mechanisms allowing for HSPC migration and maturation are incompletely understood. Ma-
ALL) and normal lymphocytes from healthy volunteers were used. Six cell lines trix metalloproteinases (MMPs) are inflammation-responsive proteins that regulate extracel-
except for TMD7 do not express p16INK4A/p14ARF. Effects of three BMI1 inhib- lular matrix (ECM) remodeling, cell interactions and signaling. Inhibition of vascular-
itors, artemisinin, PTC-209, and PRT4165, on the growth of these cell lines in cul- associated MMP2 function during the window of de novo HSPC production caused accumu-
ture were examined. It is reported that artemisinin and PTC-209 suppress BMI1 lation of fibronectin-rich ECM, retention of runx1/cmyb+ HSPCs in the VDA, and delayed
mRNA expression and that PRT4165 suppresses the function of BMI1 by inhibiting caudal hematopoietic tissue (CHT) colonization. Significantly, this effect was absent in fibro-
the BMI1/RING1A self-ubiquitination. Effects of the inhibitors on protein expres- nectin mutant embryos, indicating that MMP2 activity permits HSPC emergence and migra-
sion was examined by immunoblot analysis. Annexin V apoptosis assay was per- tion through ECM digestion. In contrast, myeloid-associated MMP9 was dispensable for
formed using flow cytometry. Microarray analysis was performed for THP-1 and HSPC budding, being instead required for proper colonization of secondary niches. These
Jurkat cells to screen the changes in comprehensive gene expression by the inhib- migration defects were phenocopied by over-expression and blocked by knockdown of C-
itors. We also performed BMI1 knockdown experiments using small interfering X-C motif chemokine-12 (CXCL12), suggesting that MMP9 controls CHT homeostasis
RNA for Jurkat cells. BMI1 protein was expressed in all cell lines. Artemisinin through chemokine regulation. Both MMP2 and MMP9 are regulated by prostaglandin-
(2 uM) suppressed the growth of Jurkat cells only, whereas PTC-209 (2 uM) and E2 (PGE2) and required downstream of PGE2 for normal HSPC stimulation: co-treatment
PRT4165 (50 uM) suppressed the growth of all cell lines examined. These inhibitors of a stabilized PGE2 derivative (dmPGE2) and an MMP2 inhibitor attenuated PGE2-medi-
induced apoptosis. These inhibitors did not affect the viability of normal lympho- ated enhancement of AGM HSC production, while incubation with dmPGE2 and an MMP9
cytes. Treatment with the inhibitors suppressed the expression of NOTCH1, inhibitor caused enhanced HSPC retention in the CHT at the expense of adult niches. A
HES1, and MYC. Microarray analysis showed that inhibitor treatment suppressed downstream requirement for MMP function was conserved in adults, where chemical inhi-
expression of various genes including NOTCH1, HES1, and MYC. Additionally, bition delayed marrow recovery after sublethal irradiation and blocked the positive effects of
BMI1 knockdown suppressed protein expression of NOTCH1, cleaved NOTCH1, dmPGE2 treatment on repopulation. Intriguingly, a need for MMP2/9 function was recapit-
HES1, and MYC in Jurkat cells. We found that BMI1 inhibitors suppressed cell ulated in the contexts of several inflammatory stimuli, including TNFa, TGFb, and IFNg,
S64 Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
suggestive of an essential role for enzymatic processing in control of developmental HSPC 3035 - P38A PROTECTS HEMATOPOIETIC STEM CELLS
production and migration. Taken together, our findings point to functional intersections be- FROM PHYSIOLOGICAL AND PREMATURE AGING
tween ECM-modifying enzymes, ECM structure and inflammatory cytokines in the onset STRESSES
and maintenance of vertebrate hematopoiesis.
Keiyo Takubo, Yukako Ootomo, and Daiki Karigane
National Center for Global Health and Medicine, Tokyo, Japan
Hematopoietic stem and progenitor cells (HSPCs) maintain the hematopoietic system
for an organism’s entire lifetime under homeostatic or in stress settings. Along with ag-
ing, hematopoietic stem cells (HSCs) show various quantitative and qualitative changes
including accumulation of phenotypic HSCs, transplantation defects, and myeloid skew-
ing. Specific activation of p38MAPK is a candidate pathway that induces HSC aging in
vivo. However, genetic dissection of p38MAPK signaling in HSC aging is still limited.
Recently, we found that conditional deletion of the dominant isozyme of p38MAPK in
hematopoietic cells, p38a, resulted in defective stress response of HSPCs upon acute
stress loading including transplantation. Based on these findings, in this study we
analyzed the hematological effects of p38a loss in physiological and premature aging
models. In aged mice, loss of p38a did not ameliorate the accumulation of phenotypic
HSCs in the bone marrow. In addition, transplantation capacity of aged p38a-deficient
HSCs was defective than aged wild-type HSCs. Also, differentiation capacity of aged
p38a-deficient HSCs was more myeloid-biased than aged wild-type HSCs. Next, we
analyzed the effect of p38a loss in a premature aging model, Ataxia Telangiectasia-
Mutated (Atm) deficient mice. Inducible loss of Atm decreased phenotypic and func-
tional HSCs in vivo. Co-deletion of Atm and p38a induced severe loss of HSCs and
defective transplantation phenotype compared to Atm single deficiency. Neither loss
of Atm nor Atm/p38a changed the apoptosis status and cell cycle quiescence of HSPCs.
Also, generation of reactive oxygen species was comparable among genotypes. These
findings suggest a protective role of p38a on HSC aging.
3034 - TAK1 INHIBITION DISRUPTS A VICIOUS CYCLE 3036 - HIGH EFFICIENCY GENE CORRECTION IN
BETWEEN TUMOR PROGRESSION AND BONE HEMATOPOIETIC CELLS BY DONOR-TEMPLATE-FREE
DESTRUCTION IN MYELOMA CRISPR/CAS9 GENOME EDITING
Masahiro Abe1, Jumpei Teramachi1, Masahiro Hiasa1, Asuka Oda1, Duran S€ur€
un, Nina Kurrle, Hubert Serve, Harald von Melchner, and
Hirofumi Tenshin1, Ryota Amachi1, Takeshi Harada1, Shingen Nakamura1, Frank Schn€utgen
Hirokazu Miki2, Itsuro Endo2, and Toshio Matsumoto1 University Hospital Frankfurt, Frankfurt am Main, Germany
1
Tokushima University, Tokushima, Japan; 2Tokushima University Hospital,
The CRISPR/Cas9 prokaryotic adaptive immune system, and its swift repurposing
Tokushima, Japan
for genome editing enables modification of any prespecified genomic sequence
Multiple myeloma (MM) has a unique propensity to develop and expand almost exclusively with unprecedented accuracy and efficiency, including targeted gene repair. We
in the bone marrow and generates destructive bone disease. MM cells constitutively over- used the CRISPR/Cas9 system for targeted repair of patient-specific point mutations
express Pim-2; cocultures with bone marrow stromal cells (BMSCs) or osteoclasts (OCs) in the Cytochrome b-245 heavy chain gene (CYBB), whose inactivation causes
were found to further upregulate Pim-2 as an anti-apoptotic mediator in MM cells and chronic granulomatous disease (XCGD) - a life-threatening immunodeficiency disor-
induce it also in BMSCs and OCs to progress bone destruction, indicating the critical der characterized by the inability of neutrophils and macrophages to produce micro-
role of Pim-2 in MM growth and bone destruction. We identified TGF-b-activated ki- bicidal reactive oxygen species (ROS). We show that frameshift mutations can be
nase-1 (TAK1) as an upstream mediator responsible for Pim-2 upregulation. In this study, effectively repaired in hematopoietic cells by non-integrating lentiviral vectors car-
we aimed to clarify the role of TAK1 in MM growth and bone destruction and therapeutic rying RNA guided Cas9 endonucleases (RGNs). As about 25% of most inherited
impact of TAK1 inhibition. TAK1 was constitutively overexpressed and phosphorylated in blood disorders are caused by frameshift mutations, our results suggest that up to a
MM cells. The TAK1 inhibitor LLZ1640-2 (LLZ) abolished TNF-a-induced NF-kB, p38 quarter of all patients suffering from monogenic blood disorders could benefit
and ERK activation and IL-6-induced STAT3 activation in MM cells. Importantly, LLZ sup- from gene therapy employing personalized, donor-template free RGNs.
pressed Pim-2 upregulation and induced apoptosis in MM cells even in cocultures with
BMSCs or OCs, while reducing VEGF production and the expression of BCMA and
TACI, receptors for BAFF and APRIL. TAK1 inhibition impaired adhesive interactions be-
tween MM cells and BMSCs along with reducing VCAM-1 and RANKL expression and
IL-6 production by BMSCs, which blunted protective activity of BMSCs for MM cells
against anti-MM agents. The known inhibitors for osteoblastogenesis in MM, including
IL-3, IL-7, TNF-a, TGF-b and activinA, as well as MM cell conditioned media all induced
TAK1 activation and Pim-2 upregulation to suppress their osteoblastogenesis in MC3T3-E1
preosteoblastic cells; however, LLZ abolished the Pim-2 up-regulation and restored their
osteoblastogenesis. Furthermore, although RANKL enhanced osteoclastogenesis along
with activation of the TAK1-Pim-2 signaling, LLZ suppressed the osteoclastogenesis by
RANKL. Finally, treatment with LLZ markedly suppressed MM growth and prevented
bone destruction in mouse MM models. From these results, TAK1 appears to play a pivotal
role in tumor growth and bone destruction in MM. TAK1 inhibition may become a unique
anti-MM therapeutic option with bone anabolic activity.
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136 S65
3037 - PARAOXONASE-2 ALTERS HEMATOPOIETIC STEM 3039 - HIGH PURITY ISOLATION AND IN VIVO KINETICS
CELL DIFFERENTIATION THROUGH REDOX OF A HIERARCHY OF HEMATOPOIETIC PROGENITORS
SIGNALLING MEDIATING RECOVERY FROM IRRADIATION-INDUCED
Lisa Spiecker1, Thomas Kindler2, Sven Horke2, and Ines Witte2 ANEMIA
1
Department of Pharmacology, University Medical Center of the Johannes Sofie Singbrant S€oderberg1,2, Alexander Mattebo3, Tobias Strid4,
Gutenberg, Mainz, Germany; 2University Medical Center of the Johannes Gutenberg, Mikael Sigvardsson4, and Johan Flygare3
Mainz, Germany 1
Lund University, Lund, Sweden; 2Lund Stem Cell Center, Lund, Sweden; 3Division
of Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University,
Intracellular levels of reactive oxygen species (ROS) play important roles in regu-
Lund, Sweden; 4Division of Molecular Hematology, Lund Stem Cell Center, Lund
lating numerous signaling events such as gene expression, cell fate decisions and dif-
University, Lund, Sweden
ferentiation, including hematopoietic stem cell differentiation. The human enzyme
paraoxonase 2 (PON2) is known to have anti-apoptotic and anti-oxidative properties, In the active debate regarding blood development architecture, the progenitors with
among others in leukemia. PON2 locates to the endoplasmic reticulum and mitochon- erythroid and megakaryocytic potential are often neglected since their immunophenotypes
dria where it fulfills vital functions in the control of ROS generation. This is in line are not completely defined, and mature erythrocytes and platelets are difficult to trace in
with the association of PON2 levels with response to front-line therapies in pediatric vivo after transplantation. Precise identification and in vivo validation of progenitor cells
ALL and imatinib resistance in CML patients. These findings and the known impact contributing to steady-state and stress-megakaryo- /erythropoiesis is important in order to
of redox signaling on quiescence, apoptosis, differentiation and self-renewal of HSCs study the regulation of these processes. Here we demonstrate that the combination of sur-
led us to investigate the role of PON2 in the hematopoietic system. First, analysis in face markers CD150, CD9 and Sca1 defines a hierarchy of splenic stress-progenitors dur-
PON2-/- mice showed severe alterations of the HSC compartment, i.e. a significant ing irradiation-induced stress recovery, and provides high purity isolation of the earliest
increase in the LSK fraction as well as at the level of the LT-, ST-HSCs and erythroid progenitor Burst Forming Unit-Erythroid (BFU-Es) both in steady-state bone
MPPs. Further investigations revealed higher percentage of LSK cells in G0 phase marrow and in the spleen during stress-recovery. By transplanting sorted stress-progenitor
of cell cycle and decreased apoptotic rates in old PON2-/- mice. In line with the populations from transgenic mice expressing Kusabira Orange in all cells, including eryth-
anti-oxidative function of the enzyme we observed enhanced superoxide levels in rocytes and platelets, we have for the first time determined their kinetics and full differen-
whole bone marrow as well as enhanced ROS levels in all fractions of the LSK pop- tiation potential in vivo. We demonstrate that splenic CD150+CD9+Sca1- stress-BFU-Es
ulation, which correlates with the expression level of PON2 in WT mice. Transplan- provide a massive but transient erythroid wave, followed by multi-lineage reconstitution
tation of old PON2-/- bone marrow cells (BMCs) resulted in increased numbers of from CD150+CD9+Sca1+ multi-potent stem/progenitor cells. Our findings offer a method
LT-, ST-HSCs and MPPs, that prompted us to more in-depth analysis. Reciprocal for high purity isolation of stress-erythroid progenitors mediating recovery from severe
bone marrow transplantation assays revealed that PON2 functions through intrinsic anemia, with a 100-fold improved enrichment compared to state-of-the-art. This provides
cell signaling rather than the niche. Further, competitive bone marrow transplantation a novel possibility to perform in-depth molecular characterization of these cells, and we
assays exposed significant advantages of PON2-/- BMCs in multi-lineage reconstitu- are currently analyzing genome wide differences in transcriptome and chromatin avail-
tion compared to WT BMCs. To shed some light on, we treated PON2-/- mice with ability between highly pure populations of stress-progenitors and steady-state BFU-Es us-
NAC to uncover whether the observed effects are ROS-dependent. We detected that ing RNA sequencing and ATAC sequencing. The potential to identify new mechanisms
antioxidant-treatment alters the effects caused by increased ROS levels due to PON2 regulating steady-state and stress erythropoiesis is an important step towards understand-
deficiency. Collectively, these studies propose PON2 as crucial redox control enzyme ing and treating anemia.
in HSCs and potential target in anti-leukemia therapies.
3038 - THE ROLE OF THE LNCRNA MEG3 IN THE ADULT 3040 - STUDY OF MYELOFIBROSIS USING JAK2 V617F
HEMATOPOIETIC COMPARTMENT EXPRESSING MACROPHAGES
Pia Sommerkamp1,2, Simon Renders1,2, Luisa Ladel1,2, Anna Silyutina, Irina Gin, Stanislava Prikhodko, Pavel Butylin, and
onberger1,2, Petra Zeisberger1,2, Adriana Przybylla1,2,
Katharina Sch€ Andrey Zaritskey
Nina Cabezas-Wallscheid1,2, and Andreas Trumpp1,2 Almazov Federal North-West Medical Research Centre, Saint-Petersburg, Russia
1
Division of Stem Cells and Cancer, Deutsches Krebsforschungszentrum (DKFZ),
The key role in pathogenesis of Ph-negative chronic myeloproliferative disorders
Heidelberg, Germany; 2Heidelberg Institute for Stem Cell Technology and
(policytemia vera, essential thrombocytemia and primary myelofibrosis) belongs to
Experimental Medicine (HI-STEM gGmbH), Heidelberg, Germany
somatic point mutation JAK2 V617F. CMPD patients with myelofibrosis have the
Hematopoietic stem cells (HSCs) are currently the best characterized adult tissue most unfavorable prognosis. Suggested that the main producers of profibrotic factors
stem cell population in the human and murine system. They represent the centerpiece in bone marrow are clonal megakaryocytes and macrophages. The goal of our study
of hematopoiesis and are located at the top of a tightly regulated hierarchically orga- is estimating expression of profibrotic factors by JAK2 –mutant macrophages cell
nized differentiation cascade. Our lab has recently performed a multi-layer OMICs model in vitro under influence of platelet lysate. THP-1 cell line was modified by len-
analyses on HSCs and progenitor populations to further investigate regulatory pro- tiviral vector. Two cell lines were established: mutant oncogene JAK2 V617F-ex-
cesses (Cabezas-Wallscheid et al., Cell Stem Cell, 2014). This study revealed specific pressing cells (Mut) and wild type (WT) JAK2-overexpressing cells. Cell lines
expression of proteins, splicing variants and long non-coding RNAs (lncRNAs). were differentiated into macrophages by culturing with 80 nM PMA in RPMI supple-
Based on these expression signatures we found that the lncRNA maternally expressed mented with 10% fetal bovine serum (FBS) or 5%, 10% and 15% platelet lysate (PL).
gene 3 (Meg3) is specifically expressed in HSCs. Meg3, which is also known as gene Expression levels of transforming growth factor-beta1 (TGF-beta1), basic fibroblast
trap locus 2 (Gtl2), is encoded within the imprinted Dlk1-Meg3 gene locus. The gene growth factor (bFGF), pentraxin-3, galectin-3, matrix metalloproteinases (MMP) 2,
encodes for a lncRNA that is expressed from the maternal allele. Recent findings 9, 12, 13 and tissue inhibitors of MMP (TIMP) were assessed by specific RT-
show that Meg3 seems to be an important regulator of cellular proliferation and func- qPCR. MMP-2 and -9 activity was analyzed by gelatin zymography. We observed
tions as a tumor suppressor. To address the functional role of Meg3 in adult HSCs, we higher of MMP-2 and MMP-12 expression level in Mut cells compared to normal
used an inducible mouse model to delete Meg3 in adult mice. In contrast to recent cells and WT cells when cultured in presence of 10% HPL. Expression level of
studies where it was shown that Meg3 plays an important functional role in fetal liver MMP-9 also increased in Mut cells in the same culturing condition. But control cells
HSCs, we did not observe impaired hematopoiesis under homeostatic conditions or in showed lower of MMP-9 and MMP-12 expression level when cultured in presence of
primary transplantation settings. Interestingly, we did observe differences in Meg3 different concentration of HPL compared to routine culturing. Gelatin zymography
KO mice under stress conditions, e.g. upon Poly(I:C) injection. This raises the pos- also revealed increased amount of pro-enzymes MMP-2 and its active form in media
sibility that Meg3 might play an important role for HSC activation, an aspect we are conditioned by JAK2 WT-overexpressing and JAK2 V617F-expressing macrophages
currently investigating. compared to control cells. Expression levels of TGF-beta1 and bFGF were signifi-
cantly increased in JAK2 V617F expressing macrophages compared to non-trans-
genic cells only in the presence of 15% PL. From our data we can conclude that
platelet-derived factors could stimulate macrophages to become profibrotic.
S66 Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
3042 - OPTIMAL GENERATION OF DENDRITIC CELLS 3044 - BONE MARROW MICROENVIRONMENT IN ACUTE
FROM APHERESIS SAMPLES OF MULTIPLE MYELOMA MEGAKARYOBLASTIC LEUKEMIA OF CHILDREN WITH
PATIENTS FOR USE IN CANCER IMMUNOTHERAPY DOWN SYNDROME
Prajakta Shinde1, Dr.Sameer Melinkeri2, Vaijayanti Kale1, and Theresa Hack1, Stephanie Sendker2, Nils von Neuhoff1, Dirk Reinhardt1,
Lalita Limaye1 and Mareike Rasche1
1
National Centre for Cell Science, Pune, India; 2Deenanath Mangeshkar Hospital, 1
University Children’s Hospital Essen, Essen, Germany; 2Department of Pediatric
Erandwane, Pune, India Hematology-Oncology, University Children’s Hospital Essen, Essen, Germany
Multiple myeloma (MM) is a malignancy of plasma cells which accounts for 20% of all Children ( ! 4 years) with Down syndrome (DS) are at a markedly increased risk of acute
haematological malignancies. Advances in treatment regimens have increased overall megakaryoblastic leukemia (AMKL) which is linked to transient leukemia (TL) in neo-
survival of MM patients however, they undergo relapses. Cancer immunotherapy using nates. Both diseases are associated with a fibrotic microenvironment (liver and bone
DCs is a safe and feasible way for treatment in MM. However, DCs from MM patients marrow (BM)). We intend to clarify the cell interaction between BM derived human
have some inherent defects. To identify defects in MM-DCs, we generated monocyte- mesenchymal stroma cells (MSC) and leukemic megakaryoblasts. MSCs, derived from
derived DCs from apheresis samples of MM patients and compared them with those BM of healthy donors (n52), DS-, DS-AMKL- (n54) and AMKL- pediatric patients
from healthy donors. The yield of in vitro generated DCs from MM samples was signif- were cocultured with DS-AMKL cell line (CMK) and primary blasts (DS-AMKL and
icantly less than those from healthy donors. Although, parameters like morphology, TL). After indirect and direct coculture for 48 hours at 5%O2 and 21%O2 Colony Forming
phenotype, antigen uptake and allo-T cell stimulatory ability were comparable in Unit (CFU), osteo-, adipo- and chondrogenic differentiation assays, CellTiter-Glo cell
both sets, MM-DCs were compromised in their chemotaxis function. Further, in MM viability assay and microarray analysis of isolated RNA were performed. Colonies of
monocyte-derived DCs:T cell co-cultures the secretion of IFNg in supernatant was CMKs were significantly enhanced after coculture with DS-AMKL and DS derived
significantly lower, as compared to healthy monocyte-derived DCs:T cells co-culture. MSCs compared to those being cocultured with healthy MSCs and CMK control (58.9,
These data suggested that an alternate source is required for DC vaccine preparation. 76.5 vs 30.75, 31; p ! 0.01). MSCs from DS-AMKL, AMKL and DS samples showed
Previously our lab has established a method for large scale generation of homogeneous, markedly increased osteogenic differentiation potential relative to healthy MSCs
mature, and functional DCs from cord blood derived mononuclear cells (MNCs). In the (3.0,2.0,1.5-fold). The effect was abrogated after coculture. Same results after coculture
present study, we examined whether our two-step method is useful in generating clinical with primary blasts confirmed the effect (2.0-fold). Adipogenic differentiation capacity
grade DCs from MNCs of apheresis samples collected from MM patients. These DCs of non-cocultured MSCs was most impressive in healthy MSCs and diminished after
were compared with DCs generated from healthy donors by same method. Our data coculture (0.5-fold). After coculture with DS-AMKL, AMKL, DS and healthy MSCs at
clearly show that, DCs generated from both type of samples were comparable by 5%O2 number of viable CMKs were reduced compared to CMKs, cocultured at 21%O2
morphology and surface marker expression. They had the ability to migrate towards (d15 rel. to d0: 1.22, 1.67, 0.72, 0.119 vs 2.36, 2.36, 1.14, 0.251), non-cocultured
chemokine CCL19 and induced proliferation in T cells. These DCs when pulsed with CMKs revealed inverse effect (0.262 vs 0.112). Gene expression of LIF was enhanced
cell lysate from U266 cell line were able to generate CTLs from na€ıve T cells. CTLs in DS-AMKL MSCs compared to AMKL and healthy MSCs. DS-AMKL and DS
were characterized by high expression of surface activation molecules CD69/CD25 MSCs stimulate the proliferation of cocultured CMKs. Our results of differentiation assays
and increased intracellular levels of granzymes/perforin. CTLs also demonstrated correlate with clinical observations of fibrotic BM in (DS-)AMKL. Coculture assays indi-
high anti-tumor activity as observed by cytotoxicity assay. Taken together, we find cate that cell interaction is bidirectional and further mimics this pattern in healthy MSCs.
that in cancer immunotherapy of MM patients our two-step method of DC generation LIF increases megakaryocyte colony forming capacity, suggesting a decisive role of
may be more appropriate than monocyte-derived DCs MSCs in fibrotic pathogenesis of DS-AMKL.
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136 S67
Erythropoiesis is intimately coupled to cell division, and deletion of the cell cycle Molecular hallmarks of T-ALL are the activation of NOTCH signaling and high ac-
regulator Retinoblastoma protein (pRb) causes anemia in mice. However, at what tivity of the PI3K-AKT-mTOR pathway, constituting potential therapeutic targets.
stage of erythroid development pRb is important, and the precise underlying mech- However, the in vivo performance of mTOR and Notch inhibitors in T-ALL has
anisms, remain elusive. To further elucidate its importance in red blood cell (RBC) yielded mixed results. To investigate the impact of mTOR or Notch inhibition in
development, pRb was conditionally deleted in an erythroid-specific manner (Epor- T-ALL we developed an aggressive murine T-ALL model. In vitro, T-ALL blasts
Cre pRbfl/fl). This resulted in macrocytic anemia, in spite of elevated levels of were highly sensitive to inhibition of AKT, mTOR and Notch signaling. Upon trans-
Epo and accumulation of erythroid progenitors in the bone marrow, a phenotype plantation into secondary recipients treatment with Rapamycin prolonged survival
strongly resembling the refractory anemia seen in Myelodysplastic syndromes (placebo: 27 days, Rapamycin 49 days, p ! 0.001), but eventually all Rapamycin
(MDS). We have now identified a block in terminal erythropoiesis at the orthochro- treated animals succumbed to leukemia despite continuous drug administration.
matic stage, and transcriptional profiling on purified populations immediately around When Rapamycin-resistant blasts were isolated and cultured in petri dishes they
the block revealed that final maturation stages lacking pRb failed to up-regulate large again became susceptible to Rapamycin, demonstrating a context-dependent resis-
clusters of genes critical for mitochondrial function, heme synthesis and iron meta- tance rather than outgrowth of intrinsically resistant clones. We found an upregula-
bolism, including Pgc1b, Alas2, Abcb7, and Tmem14c. In accordance, these cells tion of networks associated with cell-cell interactions in Rapamycin-resistant T-
displayed disturbed mitochondrial membrane potential and heme production. ALL in vivo. Stromal cell support recapitulated the in vivo effect and induced robust
Notably, de-regulated ABCB7 is known to cause ring sideroblastic anemia in resistance to mTOR and Notch-inhibition, which was dependent on direct cell-cell
MDS. And PGC1b, a critical co-activator of mitochondrial biogenesis, is heterozy- interactions. In order to find targets that mediate stroma-induced resistance to Rapa-
gously lost in the most prevalent cytogenetic abnormality of MDS; del5q. Since mycin, we conducted a shRNA screen interrogating more than 150 genes implicated
heme synthesis and iron metabolism are crucial for production of functional RBCs, in organization of cell-cell contacts. This screen showed depletion of shRNAs target-
we asked if enhanced mitochondrial biogenesis could ameliorate the anemia. To ing the IPP-complex upstream of the Rac pathway and the Rac GTPases themselfes.
this end, we transplanted stem/progenitor cells from wildtype and erythroid specific We confirmed our results using independent shRNAs and Clostridium difficile toxin
pRb-deficient mice overexpressing Pgc1b into lethally irradiated recipients, which re- B (TcdBF), that selectively glucosylates and inactivates Rac-GTPases. We show
sulted in a normalization of both RBC counts and red cell size. In conclusion, we show that depletion of Rac2, but not of Rac1, abrogates the stroma-mediated resis-
demonstrate that lack of pRb results in MDS-like anemia with a block in orthochro- tance to mTOR and Notch inhibition in T-ALL without affecting the viability of T-
matic erythroblasts due to failure to up-regulate mitochondrial function and heme ALL cells grown in suspension culture. Altogether, we identify the Rac-GTPases
synthesis. While mitochondrial mutations have been reported in MDS, their relevance as a nexus of stroma-induced drug resistance and show that inhibition of Rac and
for refractory anemia remains elusive. We are currently investigating the role of mito- mTOR is synthetically lethal to T-ALL blasts T-ALL blasts that are in contact
chondrial function in development of human refractory anemia associated with MDS. with stromal cells, paving the way to augment the effectiveness of small molecule
inhibitors in T-ALL.
3049 - PTTG1/SECURIN AS A QUANTITATIVE TRAIT LOCUS ing this, TLR2 surface expression is often (38% of cases) lost upon transformation. Ongoing
CANDIDATE GENE CONTROLLING PROGENITOR experiments are aimed at determining the mechanism by which TLR2 signaling promotes
CELL SURVIVAL AND ORGANISMAL LIFESPAN premalignant cell death.
utz1, Andreas Brown1, Hartmut Geiger2, and
Desiree Sch€
Kalpana Nattamai3
1
Institute of Molecular Medicine, Ulm University, Ulm, Germany; 2EHCB, CCHMC,
Cincinnati, USA, Ulm, Germany; 3EHCB, CCHMC, Cincinnati, USA, Cincinnati,
United States
3050 - TOLL LIKE RECEPTOR 2 (TLR2) SIGNALING 3051 - DECIPHERING THE ONCOGENIC NETWORK OF
PROMOTES APOPTOSIS OF PREMALIGNANT PRC2-LOSS GUIDED LEUKEMOGENESIS
HEMATOPOIETIC STEM AND PROGENITOR CELLS IN A Dorit Schneider1, Adrian Schwarzer2, Sabine Knoess2,
MOUSE MODEL OF MYELODYSPLASTIC SYNDROME Jan-Henning Klusmann2, and Dirk Heckl2
1
Laura Schuettpelz1, Darlene Monlish2, Sima Bhatt2, John Keller2, and Hannover Medical School, Pediatric Hematology and Oncology, Hannover,
Molly Romine2 Germany; 2Hannover Medical School, Hannover, Germany
1
Washington University School of Medicine, Webster Groves, United States;
2 Acute myeloid leukemia is elicited by the sequential acquisition of cooperating mutations
Washington University, St Louis, United States
(mostly three to five) in hematopoietic stem and progenitor cells, which strongly indicates
The myelodysplastic syndromes (MDS) are hematopoietic stem cell (HSC) disorders char- that a single mutation is not sufficient to induce AML. As mutations leading to the loss of
acterized by ineffective hematopoiesis and a high risk of leukemic transformation. The only PRC2 activity - including -7/-7q deletions – frequently occur in human AML, we aimed
curative treatment is stem cell transplantation; thus new therapies are needed. Multiple to decipher the oncogenic network guided by loss of PRC2. Via a subsequent molecular
studies have shown a correlation between MDS and enhanced toll like receptor 2 (TLR2) profiling of these leukemias we sought to unravel novel therapeutic targets in EZH2 loss
signaling, suggesting this might contribute to disease pathogenesis. In fact, an inhibitory AML. To this end, a newly established 96-well based CRISPR-Cas9 in vitro cooperation
TLR2 antibody is currently in clinical trial for use in MDS (OPN-305, Opsona Therapeutics). screening enabled us to efficiently screen for the transforming capacity of different combi-
Despite enthusiasm for TLR2 as a potential drug target, the role of TLR2 in MDS remains nations of sgRNAs in addition to the Ezh2 sgRNA. Four out of six pools (5 genes each), rep-
unclear. Moreover, high TLR2 expression predicts higher, rather than lower, survival rates, resenting 148 possible sgRNA combinations, reproducibly transformed LSK cells with
suggesting TLR2 may actually have a protective role in MDS. Thus, we are exploring how distinct clonal output. The central role of the Ezh2-loss during transformation in this setting
TLR2 signaling regulates cell fate in MDS. To investigate the contribution of TLR2 signaling was highlighted by Ezh2 rescue experiments. The same four pools also induced leukemia in
to MDS pathogenesis, we determined the effects of TLR2 loss in a mouse model of MDS recipient mice after bone marrow transplants. Leukemic phenotypes were confirmed in sec-
(expressing the NUP98-HOXD13 fusion). These ‘‘NHD13’’ mice recapitulate many features ondary transplants. Sequencing for induced mutations in the leukemic cells highlighted loss
of human MDS, and die of leukemia or cytopenias (Lin et al, Blood 2005). Similar to hu- of Runx1, Dnmt3a and Nf1 as potential cooperating events, whereas loss of Cohesin com-
mans, we find increased TLR2 on their HSCs compared to controls. NHD13 mice were plex subunits seemed to be dispensable. To define oncogenic dependencies global gene
crossed to Tlr2-/- mice to generate NHD13+;Tlr2-/-, NHD13+;TLR2+/+, NHD13-;Tlr2-/- expression spectra were analyzed in the in vitro transformed clones revealing the repression
and NHD13-;Tlr2+/+ groups. Interestingly, loss of TLR2 was associated with accelerated of differentiation-associated genes and a distinct expression pattern for each sgRNA combi-
leukemogenesis and worse survival (p50.019). Assessment of the hematopoietic stem nation. However, we could identify overlaps of differentially regulated genes for different
and progenitor cells (HSPCs) of young adult mice revealed that TLR2 deficiency led to sgRNA combinations, which may represent a core Ezh2 signature in AML demonstrating
enhanced cell survival (decreased Annexin V+ HSPCs; p ! .01). Furthermore, the accumu- the existence of oncogenic dependencies and potentially containing novel therapeutic tar-
lated HSPCs in the NHD13;Tlr2-/- mice showed elevated levels of H2AX, suggesting that gets. CRISPR-Cas9 cooperation screenings in vivo and newly established high throughput
TLR2 loss promotes the survival of damaged HSPCs. As apoptosis of HSPCs is character- in vitro screenings proved to have the power to probe oncogenic interaction. Both assays un-
istic of low-risk MDS, these data suggest that TLR2 signaling may be protective against raveled cooperating mutations for the Ezh2-loss and provided valuable cellular resources to
transformation via promotion of apoptosis of damaged premalignant HSCs. Further support- study molecular mechanisms of oncogenic synergies and dependencies.
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136 S69
3052 - ABSENCE OF MCM6 MIMICS HAEMOPOIETIC 3054 - TARGETING ILK IMPAIRS TKI-RESISTANCE OF
AGEING AND HAS IMPLICATIONS FOR LEUKAEMIC QUIESCENT LEUKEMIC STEM CELLS IN VITRO AND IN
TRANSFORMATION VIVO
Evgenia Sarrou1, Brenda Gibson2, and Karen Keeshan1 Katharina Rothe1,2, Artem Babaian1,2, Naoto Nakamichi1,2, Min Chen1,
1
University of Glasgow, Glasgow, United Kingdom; 2Royal Hospital for Sick Donna Forrest3, Shoukat Dedhar4, Connie Eaves1, and Xiaoyan Jiang1,2,5
1
Children, Glasgow, United Kingdom TFL, BC Cancer Agency, Vancouver, BC, Canada; 2Department of Medical
Genetics, UBC, Vancouver, BC, Canada; 3Leukemia/BMT program of BC, BC
Maintenance of genomic stability is critical for tumour suppression. Deregulation of the DNA
Cancer Agency, Vancouver, BC, Canada; 4Department of Biochemistry and
replication machinery leads to accumulation of replication stress and increased mutational
Molecular Biology, UBC; Genetics Unit, Integrative Oncology, BC Cancer Agency,
events often leading to oncogenesis. Replication stress occurs during normal cell ageing;
Vancouver, BC, Canada; 5Department of Medicine, UBC, Vancouver, BC, Canada
aged cells acquire more background mutations and show inefficient DNA damage repair
response (DDRR). Here, we aimed to investigatewhether background mutations affect haemo- Quiescent leukemic stem cells (LSCs) in chronic myeloid leukemia (CML) patients
poietic stem and progenitor cell (HSPC) functionality and leukaemic transformation. We respond poorly to tyrosine kinase inhibitors (TKIs) and are thought to be the major cause
generated an ageing-like haemopoietic model using CRISPR lentiviral vectors to knockout of disease recurrence. In this study, we sought to pinpoint mechanisms of LSC interac-
(KO) Mcm6, a key factor of the DNA replication machinery whose expression decreases tions with the bone marrow (BM) niche that could potentially be targeted to enhance their
with age. Murine HSPCs were transduced with CRISPR empty vector (EV) or carrying guide sensitivity to TKIs. Transcriptome analyses of CD34+ CML patient cells (n533), lin-
RNAs (gRNAs) targeting Mcm6. HSPC functionality was assessed in colony forming cell as- CD34+CD38- stem cell-enriched CML subpopulations (n58) and quiescent LSCs
says. KO cells formed fewer, but morphologically bigger and more compact colonies indi- treated with the frontline drug Dasatinib (DA), revealed in comparison to controls a sig-
cating a defect in self-renewal/stemness. Flow cytometry analysis revealed that KO cells nificant increase in the expression of Integrin-linked kinase (ILK), PINCH and Parvin (all
expressed significantly lower levels of ter119 and c-Kit markers and higher levels of CD71 sug- p ! 0.01), a core focal adhesion complex implicated in mediating interactions and cross-
gesting a block in erythropoiesis in vitro. To elucidate Mcm6 KO effects in vivo, transduced talk with surrounding niche elements in the BM. To investigate whether targeting ILK
HSPCs were injected into lethally irradiated mice. Mice that received KO cells (KO chimeras) affects survival responses of primitive CML cells from TKI-nonresponders, we per-
had lower levels of CD19 B cell marker and higher CD11b and Gr-1 myeloid markers in the formed genetic and pharmacological inhibition of ILK. Stable ILK suppression resulted
peripheral blood. Bone marrow cells isolated from the KO chimeric mice accumulated repli- in decreased cell viability (30-80%) and proliferation (2-12-fold), and a significant reduc-
cation stress evident by an increase in gH2AX, and showed poor DDRR supported by the lower tion in clonal short- and long-term colony assays in the presence of stromal fibroblasts
expression of key DDR genes. Finally, using EV and KO transduced 32D cells, we assessed (80-90%) compared to control-transduced cells. Moreover, long-term engraftment of
leukaemic transformation following MLL-AF9 (MA9) oncogene expression. 32D-KO- ILK knockdown (KD) CML cells was strongly impaired for up to 25 weeks in trans-
MA9+ cells exhibited a proliferative advantage and expressed lower levels of the c-kit progen- planted NRG and NRG-3GS mice (w10-fold, p ! 0.05) and was not detectable in sec-
itor marker compared to controls indicating that DNA replication stress as a result of Mcm6 ondary recipient mice (up to 30 weeks post-transplantation) in contrast to recipients of
deficiency changes the leukaemic phenotype. Collectively, our data indicates that replication control-transduced cells. Western blotting showed that ILK KD also decreased PINCH
stress alters both haemopoietic differentiation and leukaemic transformation, reminiscent of and Parvin protein expression. Pharmacological inhibition of ILK led to a significant
HSPC aged cells and the increased incidence of AML in the aged. reduction in quiescent LSCs and synergistic effects in combination with DA in both in
vitro and in vivo assays. RNA sequencing of quiescent vs. dividing patient cells (300
cells/sample) suggests that novel signalling hubs are affected in response to ILK inhibitor
vs. DA treatment. Together, our findings indicate that ILK plays a critical role in regu-
lating therapeutic responses of CML stem cells and their survival in the BM niche.
progenitor cells (HSPCs) strictly depended on the presence of Gaq, and the ablation 3057 - MECHANISM OF LONG-TERM ROBUST HUMAN
of Gaq/11 in transplanted Flt3-ITD-transduced HSPCs from conditional Gaq/11 HSC ENGRAFTMENT NSGW41 MICE
knock-out mice delayed leukemic burden. These results place Gaq as an important Susann Rahmig, Nicole Mende, and Claudia Waskow
target for antileukemic strategies to overcome receptor tyrosine kinase resistance Regeneration in Hematopoiesis, Institute for Immunology, TU Dresden, Dresden,
mechanisms. Germany
Xenotransplantation models enable the in-depth analysis of human hematopoietic stem cell
(HSC) function in vivo.However, human HSC engraftment and self-renewal are limited in xen-
otransplantation settings restricting the study of mechanisms regulating human HSC function.
By introducing a loss-of-function KITreceptor into NOD/SCID Il2rg-/- (NSG) micewe created
a novel mouse strain, NOD/SCID Il2rg-/- KitW41/W41 (NSGW41), which combines an impaired
endogenous HSC compartment with immunodeficiency. This mouse strain efficiently supports
stable long-term engraftment of human HSCs without the need for conditioning and shows
highly improved multilineage engraftment, including the myeloid and megakaryocyte-
erythroid lineages. Thus, we postulate that stable human HSC engraftment is a major prereq-
uisite for the continuous differentiation of all hematopoietic lineages. To investigate whether
self-renewal und functionality of engrafted human HSCs is conserved in NSGW41 mice we
transplanted titrated numbers of human HSCs in primary and secondary recipients and could
show enhanced pick-up, maintenance and expansion of human HSCs in vivo compared to con-
ventional KIT-proficient NSG mice. We hypothesized that endogenous HSCs with a defective
KIT receptor have a competitive disadvantage compared to human KIT-proficient HSCs and
thus allow for their stable engraftment. In fact, in NSGW41 mice the endogenous HSC pool is
significantly decreased after engraftment of human HSPCs, supporting our hypothesis that
endogenous HSCs are actively replaced from their niche. Analysis of the murine bone marrow
niche revealed a significant expansion of mesenchymal stromal cells (MSCs) after transplan-
tation of human cells. The increase in MSCs strictly correlated with the number of engrafted
human HSCs, suggesting that human donor HSCs modulate their niche cells. The transcrip-
tional profile of murine MSCs is also strongly altered after humanization, favoring the expres-
sion of genes involved in blood formation and differentiation. We suggest that MSCs provide a
niche for human HSCs in mice and that human HSCs actively modify the murine bone marrow
niche after xenotransplantation.
KLH and KLH arms, respectively (p50.42). Immune response analysis revealed a 3060 - ERNAS: A POTENTIAL PROXY FOR ENHANCER
greater change in immune response genes in the Id-KLH arm after transplant, and a sig- STATE DURING BLOOD DIFFERENTIATION AT THE
nificant decrease in mRNA expression of immune inhibitory genes by NanoString SINGLE-CELL LEVEL
nCounter in the Id-KLH group who had achieved near complete remission or better.
Blanca Pijuan-Sala, Fernando Calero-Nieto, Nicola Wilson, and
Sixteen (100%) and 19 (95%) patients went on to receive maintenance therapy in the
Id-KLH and KLH arms, respectively (p50.42). After a median follow up of 27.4 months,
Berthold G€ottgens
University of Cambridge, Cambridge, United Kingdom
2-year PFS was 64% and 84% in Id-KLH and KLH arms, respectively (p50.10).
Conclusion: Id-KLH vaccine and vaccine-primed costimulated T cells can be safely Blood differentiation is a dynamic process governed by a series of regulatory events
administered in the setting of auto-HCT. Gene expression profile showed a correlation leading to continuous changes in gene expression. It is widely accepted that this process
between the activity of immune response genes and the depth of clinical response. is mainly orchestrated by enhancers, inter- or intra-genic DNA regions where transcrip-
tion factors bind to promote the activation of gene expression. Recently, it has been
shown that enhancers can also function as transcriptional units, which produce short
and unstable transcripts, called enhancer RNAs or eRNAs. While their functionality is
still controversial, some studies have shown that enhancer expression correlates with
enhancer activity as well as with its target gene expression. These findings open exciting
avenues in the field of gene regulation: can we use eRNAs as a surrogate for enhancer
state? If so, could we capture cells before they start expressing key blood-related genes
and study the differentiation pathway in more detail? Unfortunately, all the aforemen-
tioned results have been inferred from bulk experiments, which average gene expression
profiles from multiple cells, thus obscuring the heterogeneity that can exist within cell
populations. Therefore, a single-cell approach to assess the feasibility of eRNAs as pre-
dictors of enhancer state at the single-cell level is needed. The goal of this project is to
assess the enhancer RNA expression for a few blood-related genes, such as Tal1, Gata1
and Gata2, and to evaluate how well eRNAs correlate with gene expression and whether
they may be used as a predictor to infer novel progenitor cell states. Preliminary results
suggest that eRNA expression can be captured at the single-cell level with single-cell
RNA-seq and that, due to their low abundance, increasing the sequencing depth could
potentially boost their detection. With my poster presentation, I will provide an update
on the questions highlighted above, with a particular focus on potential insights into
eRNA biology that may be obtained through the use of single-cell techniques.
3059 - HLX REGULATES HEMATOPOIESIS BY 3061 - ALTERED HSC FATE UNDERLIES ABERRANT
MODULATING CELL METABOLISM BLOOD PHENOTYPES IN RHEUMATOID ARTHRITIS
Indre Piragyte1, Thomas Clapes1, Ramon Klein Geltink1, Na Yin1, Eric Pietras, Giovanny Hernandez, Susan Kuldanek, Rabe Jennifer,
Aikaterini Polyzou1, Javier Langa Oliva2, Cora Beckmann3, Erika Pearce1, Gregory Kirkpatrick, Leila Noetzli, Sumitra Acharya, Biniam Adane,
Angelika Rambold1, Freidrich Kapp3, Marina Mione4, Alexander Polyzos5, Craig Jordan, Jorge DiPaola, Michael Holers, and Nirmal Banda
and Eirini Trompouki1 University of Colorado Anschutz Medical Campus, Aurora, United States
1
MPI of Immunobiology and Epigenetics, Freiburg, Germany; 2University of Bern,
Rheumatoid arthritis (RA) is a debilitating chronic autoimmune disorder character-
Bern, Switzerland; 3University of Freiburg, Freiburg, Germany; 4University of
ized by joint inflammation and progressive degradation, particularly in the hands
Trento, Trento, Italy; 5Biomedical Research Foundation Academy of Athens, Athens,
and feet. Notably, RA is also characterized by a systemic inflammatory phenotype
Greece
that includes increased production of pro-inflammatory cytokines such as interleukin
Upregulation of transcription factor HLX (H2.0-like Homeobox) is frequently observed in pa- (IL)-1 and TNF, and blood system dysfunction typified by chronic anemia of disease,
tients with AML. Since developmental pathways are often reactivated in cancer, we asked impaired lymphopoiesis and production of tissue-damaging myeloid cells. However,
whether hlx1 plays a role during hematopoietic development in zebrafish. Endothelial cell the direct impact and mechanism of RA-driven inflammation on hematopoietic stem
(EC) specific overexpression of human HLX led to aberrant production of hematopoietic cell fate and function has not been well characterized. In the present study, we used a
stem and progenitor cells (HSPCs) and increased number of immature myeloid cells at 48 mouse collagen-induced arthritis (CIA) model of human RA, which faithfully reca-
hpf mimicking a preleukemic phenotype. On the other hand, hlx1 morphants had diminished pitulates many features of human RA. Strikingly, hematopoietic output was pro-
numbers of HSPCs, a defect that could be rescued by overexpressing human HLX from either foundly altered in CIA mice, with concurrent expansion of myeloid cells in the
an EC or HSPCs specific promoter. Edu-labelling of EC, the precursors of HSPCs, from over- bone marrow at the expense of lymphoid and erythroid lineages. Consistent with
expression or morphant fish showed hyper-or-hypo-proliferation of these cells respectively. these observations, we observed a significant decrease in the number of erythroid
RNA-seq analysis of ECs revealed that overexpression of HLX led to downregulation of mito- and lymphoid progenitors (CLP) in the bone marrow and thymus. On the other
chondrial electron chain enzymes (e.g., NDUFS4 and SDHD) and upregulation of the perox- hand, we observed a significant and specific increase in the number of myeloid-
isome proliferator activated receptor family members (PPARs) together with enzymes biased MPP3. Notably, these changes occur in concert with a significant remodeling
important for fatty acid oxidation (FAO). Downregulation of hlx1 caused upregulation of of the bone marrow stroma, resulting in reduced numbers of endothelial, mesen-
the mitochondrial chain enzymes and downregulation of PPARs. These results suggested chymal and osteoblast-lineage cells in the bone. Consistent with these observations,
that HLX regulates the proliferative potential of hematopoietic cells by fine tuning mitochon- molecular analyses of HSCs from CIA mice indicates the activation of key myeloid
drial potential and FAO. Indeed PPAR agonists could rescue HSPC generation in zebrafish lineage determinants that may drive myeloid overproduction. Current investigations
morphants, while PPAR antagonists reversed the differentiation block of myeloid cells in ze- are now addressing the mechanism of myeloid expansion, the contribution of key
brafish. To test whether role of HLX is conserved, we used human progenitor CD34+ cells. In pro-inflammatory cytokines to this phenotype, and the impact of aberrant autoim-
accordance with our results in zebrafish, overexpression of HLX led to increased number of mune processes in initiating inflammatory hematopoiesis prior to the onset of clini-
CFU-C, decreased oxidative phosphorylation capacity and mitochondrial potential, whereas cally apparent arthritis. Altogether, our findings demonstrate a profound
downregulation had the opposite effect. ChIP-seq experiments revealed that HLX binds to hematopoietic remodeling in the context of RA that likely fuels the disease process,
mitochondrial genes (e.g., NDUFS4 and OPA1), and also genes involved in lipid metabolism and identifies potential mechanisms by which current and future therapeutic ap-
(e.g., PPARD, and CPT2). In summary we establish HLX as a direct regulator of the prolifer- proaches can modulate blood production to prevent/delay disease or improve patient
ative potential of HSPCs by modulating their metabolic status. outcomes.
S72 Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
3063 - EMBRYO DERIVED MACROPHAGES REGULATE 3065 - HIERARCHY OF SCA-1POSITIVE AND SCA-
DENDRITIC CELL POOL SIZE IN THE ADULT SPLEEN 1NEGATIVE HEMATOPOIETIC STEM AND PROGENITOR
Gulce Percin, Jiri Eitler, and Claudia Waskow CELLS AND THEIR CONTRIBUTION TO BLOOD CELL
Dresden, Germany PRODUCTION
Petr Paral, Katerina Faltusova, Martin Molik, Nicol Renesova, Ludek Sefc,
Dendritic cells (DCs) are potent antigen presenting cells that depend on Flk2-medi-
ated signals for their differentiation in vivo. However, in Flk2-deficient mice a small and Emanuel Necas
but functionally active fraction of DCs remains. We show here that the combined Institute of Pathological Physiology, 1st Faculty of Medicine, Charles University,
deficiency for Flk2 and Csf1r, another member of the same family of receptor tyro- Prague, Czech Republic
sine kinases, results in natural DC null mice. In contrast to Flk2, Csf1r-mediated sig- The hematopoiesis steadily generates a large number of blood cells by intensive cell
nals affect DC differentiation by a cell extrinsic, non-hematopoietic mechanism that proliferation. A hierarchy of hematopoietic stem and progenitor cells (HSPCs) has
does not require Csf1r signaling within the entire adult mouse. During embryonic been well characterized in mouse. The aim of our study was to find out to which
development Csf1r activation is crucial for the generation of macrophages, which extent these distinct populations contributed to blood cell production. We estimated
emerge from erythro-myeloid progenitors in the yolk sac before the presence of the production rates of various types of HSPCs according to the percentage of S
definitive hematopoietic stem cells (dHSCs). Tissue-resident red pulp macrophages phase cells, their number in bone marrow, and the S-phase duration. Flow cytometry
(RPMp) in the spleen are such embryonic macrophages, and we show here that was used for immunophenotyping of HSPCs according to Lin markers, c-Kit, Sca-1,
Csf1r signaling is required for their differentiation. In the absence of Flk2-mediated CD48, CD150, CD34, CD16/32, CD71, and IL7R expression. The dual BrdU/EdU
signals, loss of Csf1r expression results in complete ablation of spleen DCs in vivo labelling technique was applied to determine the S-phase duration and the percentage
due to the lack of embryonic macrophages. RPMps have been implicated in the main- of DNA-synthesizing (S-phase) HSPCs. The HSPC apoptotic rate was detected by
tenance of tissue hemostasis, removal of pathogens and clearance of cellular debris. Ghost DyeÔ Red 780 staining. The S-phase fraction significantly differed in various
We assign here a novel important role to RPMps in the maintenance of tissue homeo- types of HSPCs. The values ranged from z 1 % to z 90 % of cells in S-phase, and
stasis by regulating the pool size of dHSC-derived myeloid cells in the spleen. were a characterizing feature of particular types of HSPCs. The CD71 (transferrin re-
ceptor) expression level closely correlated with the proliferation rate in all types of
HSPCs. The highest S-phase fraction was found in the Lin c Kit+Sca-1- progenitors
committed to the megakaryocyte erythroid development, followed by the progenitors
committed to the granulocyte macrophage development. The HSPCs lacking the Sca-
1 antigen generated approximately 22 times more cells than the Sca 1 expressing
ones.
Research was supported by the Grant Agency of the Czech Republic (GACR 17-
01897S).
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136 S73
Objectives: The caudal-type homeobox (CDX) gene family regulates embryonic hemato- Primary effusion lymphoma (PEL) is an incurable hematological malignancy and novel bio-
poiesis via downstream HOX genes and interactions with the WNT signaling pathway. logical-based treatments are urgently necessitated in the clinical settings. Shikonin (SHK), a
CDX2 expression is not detected in healthy bone marrow (BM) cells but present in natural naphthoquinone, has been reported to trigger cell death and overcome drug resistance
O80% of human acute myeloid (AML) and lymphoid leukemia (ALL). Ectopic activation in anti-tumour therapy. In the present study, we investigated the effectiveness and molecular
in murine BM cells induces myeloid leukemia. Here, we explore the functional role and mo- mechanism of SHK in treatment with PEL both in vitro and in animal model. Treatment of
lecular targets of CDX2 in human leukemia. Methods: CDX2 expression was genetically PEL cells with SHK resulted in profound induction of apoptosis in a dose dependent manner
modulated (via lentiviral or siRNA treatment) in healthy human cord blood-derived CD34+ accompanied by rapid generation of reactive oxygen species (ROS), striking activation of c-
cells (for CDX2 overexpression) and in human AML cell lines (SKM1, EOL-1) and primary Jun-N-terminal kinase (JNK) and p38, marked decrease the mitochondrial membrane poten-
patient-derived cells (both for CDX2 knockdown). CDX2 modified and control cells were tial (Djm), activation of caspase-9, -8 and -3. Scavenging of ROS in the presence of N-ace-
subjected to growth, colony forming (CFU), cell cycle, flow cytometry, adhesion and tylcysteine (NAC) almost blocked the JNK activation, caspase-3 cleavage, the loss of Djm as
qRT-PCR assays and analyzed in vivo upon xenotransplantation in NOD/SCID/IL2Rgnull well as the induction of apoptosis in the SHK treated PEL cells. SP600125, a specific inhib-
(NSG) mice for bone marrow (BM) homing and leukemogenesis. Results: CDX2 knock- itor of JNK rescued the cells from the apoptosis of SHK, indicated that JNK pathway plays
down in AML cells strongly reduced clonogenicity while leaving proliferation, apoptosis main role for SHK dependent apoptosis. In a PEL xenograft Nude-Rag2/Jak3 double defi-
and cell cycle unaltered. Importantly, CDX2 knockdown profoundly suppressed in vivo cient mice model, SHK treatment reduced the amount of ascites without showing significant
leukemogenic properties. Gene set enrichment analyses (GSEA) of microarray data systemic toxicity. These findings demonstrated that SHK would be a potential anti-tumor
collected on CDX2 overexpressing versus control leukemic cells revealed cell adhesion agent for the therapeutic treatment of PEL.
genes as one of the pathways most prominently regulated by CDX2. Furthermore in vitro
adhesion assays of human AML cell lines or respectively primary patient-derived AML cells
on murine MS5 stromal cells revealed CDX2 to positively regulate leukemic cell adhesion to
stroma. These data suggest that CDX2 expression might promote leukemogenicity by
enhancing AML cell adherence to protective BM niches. Consistently, high CDX2 expres-
sion levels associated with enhanced homing of AML cells to the BM in in vivo assays.
Conclusion: Our data suggest that CDX2 plays important roles in human AML cells dur-
ing in vivo leukemogenesis by inducing clonogenicity and BM niche adherence.
3067 - DECLINED PRESENTATION 3069 - A NOVEL STRATEGY FOR ABL TYROSINE KINASE
THE STANDARD TREATMENT OF ACUTE INHIBITOR RESISTANT PH-CHROMOSOME POSITIVE
LYMPHOBLASTIC LEUKEMIA (ALL) IS LEUKEMIA CELLS
BASED ON POLYCHEMOTHERAPY Seiichi Okabe, Tetsuzo Tauchi, Yuko Tanaka, and Kazuma Ohyashiki
€
Erbey Ziya Ozdemir 1
, Sarah Ebinger2, Christoph Ziegenhain3, Tokyo Medical University, Tokyo, Japan
Wolfgang Enard3, and Irmela Jeremias2 Background: Although ABL tyrosine kinase inhibitors (TKIs) such as imatinib have demon-
1
Department of Gene Vectors, Helmholtz Zentrum M€unchen, Munich, Germany;
2 strated the potency against Philadelphia chromosome (Ph)-positive leukemia patients, resis-
Department of Gene Vectors, Helmholtz Zentrum M€unchen, Germany, Munich, Germany;
3 tance to ABL TKI can develop in chronic myeloid leukemia (CML) patients. Aims: We
Department Biology II, Ludwig-Maximilians-Universit€at M€unchen, Munich, Germany
hypothesized that ABL TKI resistance may often happen due to additional somatic mutations
The standard treatment of acute lymphoblastic leukemia (ALL) is based on polychemotherapy. in the oncogene. Methods: We established several TKI-resistant in vitro cell line models. We
Often high tumor burdens can be reduced and patients reach remission. However, potentially also investigated model to evaluate the next-generation sequencing (NGS) panel. Results: We
remaining leukemic cells after therapy, e.g., in the disease stage of minimal residual disease established ABL TKI resistant cell lines (K562 imatinib-R, K562 nilotinib-R, K562 dasatinib-
(MRD), indicate a high risk for relapse resulting in a bad prognosis. Novel treatment strategies R, K562 ponatinib-R, Ba/F3 T315I and Ba/F3 ponatinib-R) in this study. BCR-ABL expression
are urgently needed and have to target the clinically challenging MRD cells. However, so far no levels were not increased. We could not detect the BCR-ABL point mutation. However, the
preclinical model was available to develop novel approaches for targeting infinitesimal exon 4 deletion in the BCR-ABL gene was found in K562 ponatinib-R cells. In contrast, com-
amounts of leukemic cells surviving chemotherapy. In this study we established two distinct pound mutations in BCR-ABL were found in Ba/F3 ponatinib-R cells. We next evaluated the
patient-derived xenograft (PDX) mouse models for the identification and analysis of minor NGS panel (GeneRead DNAseq Targeted Panels V2) to investigate the mutation. We found that
ALL populations associated with challenging features of dormancy and chemotherapy resis- several somatic mutations in TET2, FLT3, RB1, TP53, SETBP1, ASXL1, and BCORL1 in
tance. Genetic engineering of the PDX cells facilitated the detection and isolation of these cells parental K562 cells. We also found that additional somatic mutations in K562 imatinib-R
from the murine bone marrow by expression of reporter genes and a luciferase. In the first PDX (IDH1 and KRAS), K562 dasatinib-R (IDH1) and K562 ponatinib-R (SF3A1). We could
model, a long-term dormant subpopulation of ALL cells growing in mice was identified by not detect additional mutation in K562 nilotinib-R cells. Combined treatment of ABLTKI resis-
CFSE stainings. In the second PDX model, a therapy-resistant ALL population was generated tant K562 with imatinib or dasatinib and MEK inhibitor or IDH1 inhibitor caused more cyto-
by application of a combination chemotherapy over several weeks in order to reduce high leu- toxicity. Because aberrant activation of PI3K signaling pathway and deregulation of HDAC
kemia burden to MRD levels of less than 0.1% of PDX cells in the murine bone marrow. Func- activity may be a cause of malignant disease in humans, we examined the PI3K and HDAC
tional studies showed high plasticity and similar features between the both critical ALL inhibitor in ABL TKI resistant cells. The inhibitor of class I PI3K as well as class I and II
populations in the two PDX models. The identified dormant ALL cells were chemotherapy HDAC enzymes, CUDC-907 exhibits cell growth inhibition. Summary: Our study indicated
resistant in vivo and the generated chemotherapy resistant ALL cells displayed a dormant that leukemia cells have acquired resistance through somatic mutation or exon 4 deletion in the
phenotype. Furthermore, no of the both critical ALL populations were enriched for stemness BCR-ABL gene, suggested that individual based investigations may be important to evaluate
and chemotherapy resistance compared to the untreated dividing controls. Gene expression the ABL TKI resistance. We also provide the promising clinical relevance as a candidate
profiles (GEP), obtained by bulk and single cell RNA-seq, revealed high similarities between drug for treatment of ABL TKI resistant leukemia patients.
dormant and chemotherapy resistant PDX populations. The clinical relevance of these cells
was shown by high correlation of their GEP with primary GEP, obtained from patients in
MRD. This two established PDX models provide an unique platform to develop novel treat-
ment strategies against MRD cells.
S74 Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
3074 - CELL PROPERTIES OF SIDE POPULATION CELLS 3076 - THERAPEUTIC TARGETING OF A9B1 AND A4B1
DERIVED FROM XENOPUS LAEVIS HEMATOPOIETIC INTEGRINS FOR CHEMOSENSITIZATION OF DRUG
ORGANS RESISTANT ACUTE LEUKAEMIA
Ikki Nomura, Yuta Tanizaki, and Tkashi Kato Susie Nilsson1,2, Benjamin Cao3, Wing Yan Leung3, Brenda Williams3,
Department of Biology, School of Education, Waseda University, Tokyo, Shinjuku, Jess Hatwell-Humble3, Melanie Domingues3, Luke Jones4, Linda Bendall5,
Japan and Richard Lock4
1
CSIRO Manufacturing, Melbourne, Australia; 2ARMI, Monash University,
To understand the hematopoiesis conserved among the vertebrates properly, we have
Melbourne, Australia; 3CSIRO, Melbourne, Australia; 4Children’s Cancer Institute,
used amphibian Xenopus laevis as a model animal to elucidate common hematopoietic
Sydney, Australia; 5The University of Sydney, Sydney, Australia
system. We previous reported Xenopus maintained hematopoiesis predominantly in the
liver. Thrombocytes also produced in the spleen; however, a method for isolating he- Hematopoietic stem cells (HSC) reside in the bone marrow (BM) niche, which is comprised of
matopoietic stem cells (HSCs) from these hematopoietic organs in Xenopus has not multiple cell types that regulate stem cell quiescence, proliferation and differentiation. Several
been established due to the unavailability of adequate cell surface markers and anti- interactions have been identified as important HSC regulators, including CXCR4/SDF-1 and
bodies to them. Here we established a procedure for the isolation of side population the integrins a9b1 and a4b1, which interact with thrombin cleaved osteopontin (tcOPN). Like-
(SP) cells from hematopoietic organs, including the liver and the spleen in Xenopus. wise, leukemic cells also express CXCR4 and a4b1, which allow similar interactions with the
The conditions were; 7.5 mg/mL Hoechst33342 (Ho) corresponding to the report BM microenvironment and have previously been shown to mediate leukemic cell quiescence
isolating SP cells of tetraploid zebrafish, 500 mM verapamil (ABC transporter Bcrp1/ and drug resistance. However, while the role of integrin a9b1 in HSC regulation is well docu-
ABCG2 inhibitor) at pH 7.9 and 26 C, optimized with the SP cells collection efficiency mented, the role of a9b1 in leukemia regulation and drug resistance is not known. Phenotypic
and the proportion of dead cells. Each of the temperature and the pH affection to Ho screening of patient B-cell precursor ALL (BCP-ALL) BM demonstrated that although all
staining was higher than the concentration of the Ho. The morphology of SP cells samples expressed CXCR4 and integrin a4b1, only 40% co-expressed a9b1. Notably, co-
resembled typical mammalian HSCs distinguished by high nuclear/cytoplasm ratio. expression of a9b1 was correlated with poor prognosis and was also associated with chemo-
The SP cells, which comprised 0.07560.02% of the liver cells obtained by perfusion resistance in vitro. Using the small molecule integrin antagonist ‘‘BOP’’, which potently in-
with collagenase, expressed the counterpart genes of mammalian undifferentiated hibits both a4b1 and a9b1, we show that ALL can be effectively sensitized to
markers significantly, including pou5f3.1, pou5f3.2, gata2, and sox2, but gata1, cd41, chemotherapy in vitro. In addition, synergistic chemosensitization was observed when BOP
and mpo did not. Furthermore, the SP cells partially overlapped with a subpopulation was used in combination with the CXCR4 antagonist AMD3100. Furthermore, using a pa-
of Mpl positive hematopoietic stem and progenitor cells (HSPCs), we previously re- tient-derived xenograft model, we demonstrate BOP can effectively bind to minimal residual
ported in ISEH 2014, in Xenopus. These results suggest that the Mpl expression in disease in BM during remission and rapidly dislodges these cells from their protective micro-
HSPCs is conserved in the course of evolution among vertebrates. The SP cells in environment in vivo. In addition, our preliminary results show that co-administration of BOP
the HSPCs of the liver were only 0.006%. The rate of content is almost comparable with AMD3100 reduces ALL burden when used in conjunction with a standard chemotherapy
to murine HSCs; however, an unidentified Mpl negative HSPCs subpopulation may regime consisting of vincristine/dexamethasone/L-asparaginase. Together, our results validate
exist in the SP cells. In conjunction with proteomics analysis, we discussed comparative integrin a9b1 as a drug target in ALL and demonstrates that concomitant inhibition of a4b1/
cellular characteristics of the primitive cells in the liver and the spleen. a9b1/CXCR4 using BOP plus AMD3100 or other CXCR4 antagonists may be an effective
strategy for chemosensitizing drug resistant ALL.
3075 - CHROMATIN REMODELING FACTOR BRM 3077 - LOSS OF THE SELECTIVE AUTOPHAGY
MAINTAINS HSC VIA EPIGENETIC REGULATION RECEPTOR P62 DELAYS LEUKEMIA DEVELOPMENT
Eriko Nitta1, Masayuki Yamashita2, Motohiko Oshima1, Atsushi Iwama1, AND IMPAIRS MITOPHAGY
and Toshio Suda2 Vu Thu Nguyen1, Shabnam Shaid1, and Christian Brandts2
1 1
Department of Cellular and Molecular Medicine, Chiba University Graduate school Department of Medicine, Hematology/Oncology, Translational Oncology, Goethe
of Medicine, Chiba, Japan; 2Department of Cell Differentiation, the Sakaguchi University, Frankfurt, Germany, Frankfurt am Main, Germany; 2Department of
Laboratory of Developmental Biology, Keio University School of Medicine, Tokyo, Medicine, Hematology/Oncology, Translational Oncology, University Cancer Center
Japan (UCT) Frankfurt, Goethe University, Frankfurt, Frankfurt am Main, Germany
Recent observations shed light on epigenetic regulation systems as the great contributors to The lysosomal degradation pathway of autophagy has a tumor suppressor function during ma-
the decision of hematopoietic stem cell (HSC) fates; self-renew or differentiation to respec- lignant transformation and a survival mechanism in established tumors. Recently, autophagy
tive lineages. HSC soundness is fine-tuned, but its dysregulation leads to the development of has been shown to be fundamental for maintaining hematopoietic stem cell (HSC) integrity and
hematological diseases, including bone marrow failure and hematological malignancies. preventing malignant transformation. Besides the bulk degradation process, autophagy also
Among many players in the epigenetic regulation, the SWI/SNFATP-dependent chromatin serves as an intracellular quality control by selective removal of protein aggregates or damaged
remodeling factor BRG1 has recently been demonstrated to be essential for leukemic stem organelles. This process is referred to as selective autophagy, is mediated by substrate ubiqui-
cell (LSC) maintenance. Whereas BRG1 is implicated in the pathogenesis of Fanconi ane- tination and requires autophagy receptors such as p62 that specifically bridge the ubiquitinated
mia and bone marrow failure, BRM, the homologue of BRG1, is expressed more specif- cargos with the inner membrane of autophagosome. Here, we investigate the function of p62 in
ically in HSCs compared with BRG1 and appeared to be involved in physiological the development of acute myeloid leukemia (AML) and demonstrate that knock-down of p62
regulation of HSCs. We have elucidated an essential role of BRM in the maintenance of impaired expansion and colony-forming ability of the oncogene-transformed cells in vitro.
HSCs using genetically modified BRM-null mice. BRM-null HSCs showed profoundly Consistently, transplantation of p62-/- HSCs expressing the leukemia oncogene MN1 signifi-
impaired reconstitution capacity in competitive BMT assays and exhibited more activate cantly reduced latency in mice compared to WT HSCs. To understand the underlying mech-
cell cycling than wild type HSCs after bone marrow transplantation, suggesting that anism of p62-dependent malignant transformation, we performed a SILAC interactome
BRM plays a role in reversion of cycling HSCs into a quiescent state. Latest studies have analysis and identified a strong interaction of p62 with mitochondrial proteins in MN1-driven
revealed that SWI/SNF chromatin remodeling complex has a critical role in both develop- leukemia cells. Therefore, we analyzed the numbers and function of mitochondria in p62-/-
ment and disease by opposing Polycomb group (PcG) proteins by rapid ATP-dependent cells and found a significant accumulation of mitochondria with increased mitochondrial su-
eviction, leading to the formation of accessible chromatin. PcG proteins maintain the peroxide anions as well as defective basal and maximal mitochondrial respiration. Moreover,
self-renewal capacity of HSCs by keeping differentiation programs poised for activation we observed a significantly delayed removal of defective mitochondria by autophagy (mitoph-
in HSCs by repressing a cohort of hematopoietic developmental regulator genes via bivalent agy) caused by loss of p62. Rescue experiments with deletion mutants of p62 indicate that the
chromatin domains. These aspects prompt us to support the hypothesis that BRM maintain autophagy-dependent functions of p62 are relevant for malignant transformation. Our results
self-renewal capacity of HSCs by controlling the transcriptional regulation of stem cell fac- demonstrate a prominent role of p62 as a selective autophagy receptor in leukemia develop-
tors or differential genes by PcG proteins. Now we are identifying the epigenetic status on ment and open new therapeutic opportunities to target selective autophagy in the treatment
target genes of BRM by executing ChIP- and ATAC-sequences and will demonstrate the of AML.
molecular mechanisms underlying HSC regulation by BRM.
S76 Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
3078 - CHRONIC EXPOSURE TO HIGH ERYTHROPOIETIN 3080 - GFI1B REGULATES THE LEVEL OF
LEVELS DIFFERENTIALLY MODULATES THE WNT/B-CATENIN SIGNALING IN HEMATOPOIETIC STEM
HEMATOPOIETIC STEM CELL COMPARTMENT IN MICE CELLS AND MEGAKARYOCYTES
Marta Murray1, Rashim Singh2, Tatyana Grinenko2, and Ben Wielockx2 Tarik Moroy1,2 and Peiman Shooshtarizadeh3
1
TU Dresden, Faculty of Medicine - IKL, Dresden, Germany; 2Institute for Clinical 1
Institut de recherches cliniques de Montreal, Montreal, Canada; 2Quebec, Montreal,
Chemistry and Laboratory Medicine, TU Dresden, Dresden, Germany Canada; 3IRCM, Montreal, Canada
The hematopoietic stem cell (HSC) compartment consists of a small but heteroge- Gfi1b is a DNA binding transcriptional repressor highly expressed in hematopoietic
neous pool of cells, which is able to replenish all blood cells. Although it is generally stem cells (HSCs) and in megakaryocytes (MKs). Gfi1b deficiency leads to expansion
accepted that the hematopoietic system is assembled in a hierarchical organization of both cell types and abrogates the ability of MKs to respond to integrin substrates
under steady-state conditions, evidence for distinct differentiation pathways as a with spreading and movement. Here we show that Gfi1b is present in complexes with
response to stress are emerging. However, it remains unclear how the different mem- b-catenin and the b-catenin co-factors Pontin52, CHD8, TLE3 and CtBP1 and is
bers of the stem cell and progenitor compartment behave under sustained chronic required for the activation of b-catenin/TCF dependent transcription. The activation
stress. Here, using adult transgenic mice over-expressing erythropoietin (Epo), we of TCF-dependent transcription by Gfi1b is increased after treatment with Wnt3a and
reveal for the first time a differential response of the HSCs and their closely related requires the interaction between Gfi1b and lysine demethylase 1 (LSD1). This sug-
multipotent progenitors (MPPs). HSCs exhibit a vastly committed erythroid progen- gests that a tripartite b-catenin/Gfi1b/LSD1 complex exists that regulates expression
itor profile that is linked to enhanced cell division and metabolic activity. In contrast, of Wnt/b-catenin target genes. Consistently, expression of many canonical Wnt/b-
MPPs display opposing cell signatures (erythroid and myeloid) and an accumulation catenin target genes is decreased in Gfi1b-deficient cells and many of these target
of uncommitted cells. Together, our results identify HSCs as the master regulator of genes are co-occupied by Gfi1b, b-catenin and LSD1 at promoter sites. When
chronic stress erythropoiesis, bypassing the hierarchical differentiation-detour Gfi1b deficient cells were treated with Wnt3a, their normal cellularity was restored
and the Gfi1b-deficient MKs regained their ability to spread on integrin substrates.
This suggests that Gfi1b controls both the cellularity and functional integrity of
HSCs and MKs by regulating Wnt/b-catenin target gene expression.
3079 - DISSECTING THE ORIGIN OF DENDRITIC CELL 3081 - SCA-1 EXPRESSION LEVEL IDENTIFIES
AND MACROPHAGE SUBSETS IN HUMAN QUIESCENT HEMATOPOIETIC STEM AND PROGENITOR
HEMATOPOIESIS CELLS
Florian Murke1, Andre G€orgens2, Peter Horn1, and Bernd Giebel1 Mina Morcos1, Kristina Schoedel1, Anja Hoppe1, Rayk Behrendt1,
1
University Hospital Essen, Institute for Transfusion Medicine, Essen, Germany; Onur Basak2, Hans Clevers2, Axel Roers1, and Alexander Gerbaulet1
2 1
Karolinska Institutet, Department of Laboratory Medicine, Stockholm, Sweden Institute for Immunology, Faculty of Medicine, TU Dresden, Dresden, Germany;
2
Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and
According to recent findings multipotent progenitors (MPPs) do not create common lympho-
University Medical Center Utrecht, Utrecht, Netherlands
cyte (CLP) and common myeloid progenitors (CMP) as suggested by the classical model of
hematopoiesis. Instead, they create lymphoid-primed multipotent (LMPP) and erythro- The hematopoietic system is a highly regenerative system responsible for the contin-
myeloid progenitors (EMP). Thus, subsets of myeloid cells derive from both branches. uous production of all different lineages of mature blood cells. This continuous blood
We previously showed that neutrophils are derivatives of LMPPs and eosinophils and baso- supply is known to be driven by the proliferation and differentiation of the hemato-
phils of EMPs. Monocytes/Macrophages arise from progenitors of both branches. Without poietic stem cells (HSCs) residing at the top of the hierarchy of the hematopoietic
dissecting their concrete origin, dendritic cells (DCs) have been classically discriminated system, which then give rise to multipotent progenitor populations, lineage
into lymphoid [plasmacytoid DC (pDC)] and myeloid DCs [monocyte-related DC committed progenitors and finally terminally differentiated blood cells. Early he-
(MoDC), myeloid DC1 (mDC1), myeloid DC2 (mDC2)]. Now, the novel hematopoietic matopoietic stem and progenitor cells (HSPCs) express the surface molecule Stem
lineage relationships raise questions on the origin and complexity of the myeloid compart- cell antigen-1 (Sca-1/LY6A), a glycosyl phosphatidylinositol-anchored cell surface
ment. Consequently, we aim to unravel the exact origin the different DC subsets and to char- protein, and constitute the Lineage-Sca-1+Kit+ (LSK) population of the murine
acterize LMPP- and EMP-derived macrophage subsets. We have adopted an in vitro bone marrow. Analysis of Histone-2B red fluorescent fusion protein (H2B-RFP)
differentiation assay allowing the generation of all current DC subsets. By including retention revealed a positive correlation between Sca-1 expression and quiescence
HLA-DR and CD11c in our analysis, we were able to increase the phenotypic resolution of HSCs as well as hematopoietic progenitor cells. This finding was further
of in vitro generated DC subsets. For now, we identify pDCs as HLA-DRdimCD14- confirmed by analyzing Ki67-RFP cell cycle reporter mice, in which actively cycling
CD1c-CD303+CD123+, mDC1s as HLA-DRdim/+CD14-CD1c+CD11c+CD141dim, HSPCs displayed lower levels of Sca-1 expression while their quiescent counterparts
and mDC2s as HLA-DRdim/+CD14-CD1c+CD11clowCD141+CLEC9a+ cells. As showed higher Sca-1 expression. Purification and transplantation of HSPCs according
pDCs and mDC2 were generated in low quantities only, we have started to compare the to their Sca-1 expression level revealed higher repopulation activity of various Sca-
DC formation on different stromal cells. In parallel, we modulate the cytokine conditions 1hi LSK subpopulations. Although the function of Sca-1 is poorly understood, it is
to increase the generation of lymphoid-myeloid cell fates. In a first set of experiments, higher known to be strongly up-regulated by type I interferon (IFN). We show however
pDC and mDC2 yields were obtained. The current condition supported MPPs to create all that the link between high Sca-1 expression and quiescence as well as repopulation
DC subtypes as well as macrophages, neutrophils, NK cells and B cells. In the future, we activity among HSPCs is type I interferon independent. Our finding that quiescent
will further optimize this DC assay for single cell analysis and characterize the DC potential subpopulations of HSPCs are identified by differential Sca-1 expression easily allows
of distinct lympho-myeloid and erythro-myeloid progenitors. Furthermore, we plan to for refined purification and analysis strategies. We are currently investigating the
compare macrophages obtained from different progenitors at the molecular and functional lineal relationship and differentiation pattern of LSK subpopulation with diverse
level. Sca-1 expression by lineage tracing experiments.
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136 S77
Haematopoietic stem cells (HSCs) maintain the vertebrate blood system throughout Adult acute myeloid leukemia (AML) is a rapidly progressing blood cancer, with low sur-
life. They arise during embryogenesis from the haemogenic endothelium (HE), vival rates. This is due to inexplicable intrinsic resistance mechanisms that act in the rare and
located in the floor of the dorsal aorta. Our understanding of HE specification remains highly regenerative Leukemia Stem Cells (LSCs) which persist in the course of chemo-
incomplete, but regulation of Gata2 is crucial for its programming. Here we investi- therapy and initiate leukemia relapse. A lack of tools to directly isolate viable human
gate whether gata2a is required for HSC emergence in zebrafish. We found that the LSCs precludes their comprehensive study and efficient targeting. To that end, we devel-
endothelial-specific intronic gata2a enhancer (i4 enhancer) is conserved throughout oped a lentiviral reporter system that pinpoints LSCs by exploiting a stem-like genetic pro-
vertebrates and established a transgenic line driven by this enhancer to monitor the gram driven by the combinatorial binding of the heptad transcription factors (SCL/TAL1,
activity of gata2a in vivo. Homozygous deletion of the i4 enhancer (gata2aDi4/Di4) LMO2, LYL1, ERG, FLI1, RUNX1, GATA2) aberrantly activated in a wide-range of leu-
led to endothelial-specific loss of gata2a expression, including the HE. This corre- kemias. To generate a reporter, we cloned the binding sites of the heptad from the endoge-
lated with a decrease in gata2b and runx1 in the HE of gata2aDi4/Di4 mutants, sug- nous ERG+85 stem cell enhancer upstream of the Blue Fluorescent Protein (BFP). Using a
gesting that Gata2a is an upstream regulator of the gata2b/runx1 axis that broad panel of human leukemia cell lines we revealed highly heterogeneous reporter activ-
programmes the HE to become haematopoietic. Thus, endothelial Gata2a activity ity. Remarkably, high reporter activity cells (BFPhigh) demonstrated increased clonogenic-
is required to programme the HE and generate HSCs. ity and resistance to chemotherapy and irradiation relative to their BFPlow counterparts.
Moreover, BFPhigh cells’ growth was selectively arrested by the retinoic acid treatment,
reinforcing the distinct transcriptional status of these cells. Importantly, we revealed
BFPhigh cells as a minor population in primary AML samples. Currently, we use transcrip-
tomics, proteomics and chemogenomics approaches to systematically characterize regula-
tors that drive a stem-like program in BFPhigh leukemic cells. We hope that the utilization
of this novel and versatile research tool will identify crucial determinants of leukemia resis-
tance that might be used as prognostic markers, but most importantly, will provide the foun-
dation for their targeting and thus also their cure.
cKITY900, which serves as docking site for the adaptors CrkL/CrkII that associate the were able to confirm that the MDS clonal population had engrafted in our mice. Next,
receptor with c-Cbl to undergo ubiquitination, incorporation into lysosomes and degra- we developed an in vitro 2D co-culture system as an alternative tool to in vivo. Using
dation. 145KDa-cKIT degradation activates de-novo synthesis of 120KDa-cKIT which our in vitro system, we were been able to co-culture CD34+ cells from MDS patient
by N-linked glycosylation mature into 145KDa-cKIT to be exposed to the cell-mem- (n59) bone marrow, on auto- and/or allogenic MSCs, over 4 weeks with a fold expansion
brane. In spite of the overwhelming evidence demonstrating the importance of SCF/ of up to 600 times. It is important to note that these expanded cells conserved their MDS
cKIT interaction in erythropoiesis, cKIT signaling in human erythroid cells is still clonal architecture. Using SNP karyotyping (Affymetrix) on pre and post cultured cells for
poorly known. In cultures containing SCF, progenitor cells from polycythemia vera 3 patients, we were able to show that the MDS clones were stable during the co-culture and
(PV) generate similar numbers of erythroid cells (Erys) as those from adult (AB) and did not induce any additional chromosomal abnormalities therefore maintaining their
cord (CB) blood by day 10. However, by day 13 PV cells generate significantly genomic integrity. Thus, our ex vivo culture system, which lasts for only 4 weeks and re-
more Erys than AB and CB. SCF concentration/proliferation curves indicate that differ- quires low number of human CD34+ cells, provides a robust preclinical assessment model
ences in proliferation are associated with greater response at lower concentration of to test therapeutic effects of different drugs and other approaches on the MDS clonality
Erys from PV (and CB) than those from AB, suggesting that in PV the JAK2V617F prior to treatment of MDS patients as well as providing a model to better dissect the
mutation increases Ery response to SCF. To test this hypothesis, we exploited the power cross-talk between MSCs and the malignant clones.
of phosphoproteomic analyses determining the phosphoproteomic landscaping of day-
10 Erys from PV, AB and CB by Reverse Phase Protein Array (RPPA) using as target
O160 signaling events (see http://capmm.gmu.edu/data). Overall, 40 proteins were sta-
tistically different between PV and AB and 30 proteins were statistically different be-
tween CB and AB. Pathway analyses of significant hits identified that PV and CB
Erys differ from the AB ones in the activation states of 1-2 proteins involved in stemness
and cell cycle control inferring that there is no major change in their cycling or differ-
entiation state. By contrast, the 3 populations showed numerous differences in cKIT
signaling. PV Ery differed from AB cells by expressing greater levels of cKITY719
and cKIT703, which were reduced to barely detectable levels by the pan-JAK inhibitor
Ruxolitinib, and of elements of PI3K (eNOS/NosIII, PDK1 and PKCd) and MAPK
(pMARCKS, MSK1, AMPKa1 and b1 and p38 MAPK) signaling downstream, respec-
tively, to cKITY719 and cKIT703. PV Ery expressed also greater levels of JAK2Y1007/
1008 and of its downstream target STAT3Y705. CB Ery showed lower levels of cKIT,
cKITY703, cKITY719 and CD63, a member of tetraspanin superfamily that binds
cKITY719 switching its intracellular fate from recycling to lysosome degradation,
greater phosphorylation of proteins of MAPK (pMARCKS, MSK1, PTEN and Src)
and PI3K (PKCd, mTOR, p70S6K and panPKC/bII) signaling than AB Erys. These re-
sults were stoichiometrically validated by WB and indicate that PV Erys undergo
greater cKIT activation in response to SCF than AB Erys. This hypothesis was tested
by RPPA analyses (and stoichiometric validated by WB) of Erys from PV, AB and
CB growth factor deprived (GFD) and then stimulated with SCF for 15’ and 2h.
GFD altered the activation state of 25 proteins (22 de-activated and 3 activated) in
PV, of 12 proteins (10 de-activated and 2 activated) in AB and 8 proteins (4 de-activated
and 4 activated) in CB. SCF altered the activation state of 36 proteins in PV (18 acti-
vated and 18 de-activated), 23 proteins in CB (all activations) and 6 proteins in AB (all
activations). In PV and CB Ery, GFD decreased cKITY719 and cKITY703 and the acti-
vation state of their downstream targets JAK2Y1007/1008, MAPKs and mTOR while
SCF increased the stoichiometric levels of cKITY719 and cKITY703 and the activation
of mTOR. SCF also increased cKITY703 and cKITY719 but did not activate mTOR in
Ery from AB. Screening of 97 inhibitors against targets analysed by RPPA approved for
clinical use by FDA revealed that growth of PV Ery was more sensitive than that of AB
both to JAK and cKIT inhibitors. These results provide the first phosphoproteomic land-
scaping of cKIT signalling in erythroid cells and indicate that SCF activates a prolonged
non-canonical cKIT signaling (involving activation of PLCg1 and suppression of DEP-
TOR) in PV but, respectively, transient and prolonged canonical cKIT signaling in AB
and CB and confirm cKIT as a potential therapeutic target for PV.
Myelodysplastic syndromes (MDS) represent a heterogeneous group of clonal hematopoi- The identification of novel therapies for acute myeloid leukemia (AML) requires robust
etic stem cell disorders with diverse phenotypes, characterized by ineffective hematopoi- experimental systems where the effect of specific perturbations can be rapidly and reliably
esis and bone marrow morphological dysplasia. Here, we have tested the bone marrow measured. Here, we report the development of an approach where we could perturb, in vivo
cells from 38 MDS patients covering all risk groups into two immuno-deficient mouse and in parallel, 20,611 genes using CRISPR technology and measure the relevance of each
models (NSG and NSG-SGM3). Injection of mononuclear cells (MNCs) or enriched gene on leukemic growth. Two murine AML lines with high leukemic stem cell (LSC) con-
CD34+ cells into the NSG and/or NSG-SGM3 mice yielded engraftment of hCD45+ cells tent were generated by retrovirally transducing, with the MLL/AF9 or HoxA9/Meis1 onco-
ranging from 0.01% to 15%. We then co-injected mesenchymal stromal cells (MSCs) genes, myeloid progenitors isolated from Rosa26-Cas9/GFP knock-in mice. The two lines
along with MNCs or CD34+ cells but we did not observe improved level of engraftment were expanded ex vivo and transduced with two sgRNA libraries containing 130,209 se-
in either models. Interestingly, the level of engraftment was patient specific with no cor- quences in total. Following transduction, the cells were transplanted in cohorts of murine re-
relation to any specific MDS risk group. Furthermore using high-depth sequencing, we cipients or grown in vitro. Following development of AML in the recipients, genomic DNA
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136 S79
was harvested from the cells and the sgRNA proviral region was deep-sequenced to assess 3089 - A SOMATIC MUTATION IN ERYTHRO-MYELOID
changes in sgRNA abundance between leukemic samples, pre-transplant populations and the PROGENITORS CAUSES NEURODEGENERATIVE
plasmid library. By pooling the sgRNA sequencing counts of the resultant AML from mul- DISEASE
tiple recipients, we could overcome issues related to stochastic engraftment in individual re-
Elvira Mass1, Christian Jacome-Galarza1, Thomas Blank2, Tomi Lazarov1,
cipients. By comparing enrichment or depletion of targeting sgRNAs against the background
of non-targeting controls in the library, we could identify genes that have a statistically sig-
Alessandro Pastore1, Marius Schwabenland2, Benjamin Durham1,
nificant effect on AML growth. We then validated the effect of the top 1034 genes on Marco Prinz3, Omar Abdel-Wahab1, and Frederic Geissmann1
1
leukemic cells and normal hematopoietic progenitors isolated from Cas9 knock-in mice us- MSKCC, New York, United States; 2University of Freiburg, Freiburg, Germany;
3
ing a second library. Our screen allowed us to identify genes specifically depleted in the in University of Freiburg, Freiburg, United States
vivo arm of the screen: top candidates include genes related to cell adhesion, immune The pathophysiology of neurodegenerative diseases is poorly understood, and therapeutic op-
response or oxidative phosphorylation. We are currently validating lead candidates specific tions are few. Neurodegenerative diseases are hallmarked by progressive neuronal dysfunction
to AML growth in vivo using genetic and pharmacological tools. and loss, associated with chronic glial and immune activation. Microglia are the myeloid glial
cells of the central nervous system, important for synaptic plasticity and clearance of cellular
debris, and their activation is viewed in general as a secondary process in neurodegeneration.
Late-onset neurodegenerative disease is frequently observed in patients with histiocytoses,
which are clonal myeloid diseases associated with somatic mutations in the RAS/MEK/
ERK pathway such as BRAFV600E. This suggests a possible role of somatic mutations in
myeloid cells in neurodegeneration, yet expression of BRAFV600E in the hematopoietic
stem cell (HSC) lineage does not cause neurodegenerative disease. Recent work indicates
that microglia belong to a lineage of adult tissue-resident myeloid cells that develop during
organogenesis from yolk sac erythro-myeloid progenitors (EMP) distinct from HSC. We
thus hypothesized that somatic BRAFV600E mutations in this resident macrophage lineage
may cause neurodegeneration. We found that while BRAFV600E expression in the HSC line-
age causes leukemic and tumoral diseases, its expression in the EMP lineage instead results in a
severe late-onset neurodegenerative disorder. Neurobehavioral signs, neuronal death, astroglio-
sis, immune activation, and amyloid precursor protein deposition were driven by ERK-acti-
vated microglia clones and were preventable by RAF inhibition. These results identify the
yolk sac precursors of tissue-resident macrophages as a potential cell-of-origin for histiocyto-
ses, and demonstrate that a somatic mutation in the EMP lineage can drive late-onset neuro-
degeneration. Moreover, these data identify activation of the MAP kinase pathway in
microglia as a cause of neurodegenerative disease, and provide opportunities for therapeutic
intervention aimed at preventing neuronal death in neurodegenerative diseases.
3088 - A NOVEL TUBB1 MUTATION IDENTIFIED IN 3090 - THE LEUKEMIC ONCOGENE AML1-ETO BLOCKS
FAMILIAL PLATELET DISORDER LEADS TO DIFFERENTIATION BY ALTERING THE
IMPAIRED PROPLATELET FORMATION AND TRANSCRIPTOME AND EPIGENOME DURING HUMAN
GENOME INSTABILITY IPSC-BASED HEMATOPOIETIC DEVELOPMENT
Takayoshi Matsumura, Ayako Ishizu, Motomi Osato, and Toshio Suda Joost Martens, Esther Tijchon, Amit Mandoli, Jos Smits,
Cancer Science Institute of Singapore, Singapore, Singapore Francesco Ferrari, and Falco Wijnen
Radboud University Nijmegen, Nijmegen, The Netherlands
Familial platelet disorder (FPD) is a genetically heterogeneous disease entity characterized
by thrombocytopenia of varying severity, and some subsets of FPD show a higher risk of he- Acute myeloid leukemia (AML) is characterized by a block in myeloid differentia-
matological malignancy. While there are some well-characterized entities including MYH9- tion, increased self-renewal and decreased apoptosis. These effects are dependent
related disease, Bernard-Soulier syndrome and FPD with predisposition to acute myeloid on transforming oncogenes such as the AML1-ETO fusion protein which is a result
leukemia due to germline RUNX1 mutations, the genetic cause of many cases remains un- of the t(8;21) translocation and initiates the most common form of human AML. Here
known. We studied a family with FPD and propensity to myeloid malignancy. Whole-exome we study the differentiation of human iPS cells expressing an inducible AML1-ETO
sequencing of genomic DNA from 3 patients and 1 healthy sibling among this family iden- gene into myeloid cells as a model. This AML1-ETO-iPSC model allows to investi-
tified a novel TUBB1 mutation. TUBB1 codes for beta1-tubulin, a major component of mi- gate leukemic transformation by the expression of a single oncogene as well as in-
crotubules abundant especially in megakaryocytes. Crystal structure analysis using duction of oncogenic hits at specific stages during development, before and after
previously published data showed this mutation disrupted a normal conformation of a devel- the formation of hematopoietic cells. We found that this differentiation system faith-
opmentally conserved alpha-helix of this protein. Immunocytochemistry using a human fully recapitulates various stages of blood cell development and produces myeloid
acute megakaryoblastic leukemia cell line CMK115 and mouse fetal liver-derived megakar- precursor cells as well as mature monocytes/macrophages and granulocytes.
yocytes revealed mutant beta1-tubulin did not participate in proper assembly of microtu- AML1-ETO expression inhibited granulocytic differentiation, while effects on mono-
bules, and mutant beta1-tubulin-induced mouse megakaryocytes have impaired activity of cyte/macrophage differentiation were limited. At the transcriptome level AML1-ETO
proplatelet formation, which may suggest the cause of thrombocytopenia in this family. interfered with both activating and repressive activities during normal myeloid differ-
Next, to understand how microtubule dysfunction causes genetic instability leading to ma- entiation with most target genes repressed. These repressed genes displayed reduced
lignant transformation, CMK115 cells were infected with lentivirus expressing shRNA for histone acetylation levels at associated promoter and enhancer regions and were pri-
TUBB1. TUBB1 suppression led to slower cell growth and increased apoptosis. DNA dam- marily linked to granulocytic differentiation. The response of the transcriptional
age repair after radiation or etoposide, as evaluated with a DNA double strand break marker network to AML1-ETO expression was developmental stage specific, with an early
gamma-H2AX, was severely impaired by TUBB1 RNAi. We found p21 induction after oncogenic hit resulting in reduced expression of granulocyte differentiation driving
DNA damage is inhibited in TUBB1-deficient cells. Consistent with this, inhibitors of micro- genes. Together, these results showed that our AML1-ETO-iPSC model mimics
tubule dynamics, vincristine and paclitaxel, are reported to suppress p21 induction by inhib- oncogenic effects that also occur in vivo, and that observed effects can be attributed
iting p53 nuclear translocation after DNA damage. Thus we propose a novel mechanism that to AML1-ETO expression and not to other oncogenic events. Moreover, AML1-ETO
TUBB1 suppression leads to decreased p21 induction after DNA damage, and this may expression alters the transcriptional output within target cells thereby altering their
explain abnormal DNA damage response and subsequent genome instability in these cells. developmental potential.
These studies will lead us to a better understanding of how microtubule dysfunction can lead
to accumulation of DNA damage, genetic instability, and finally to leukemogenesis.
S80 Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
3091 - THE IMPORTANCE OF BEING A MACROPHAGE 3093 - MIR-128A, MIR-130B AND MIR-155 ARE
DURING HEMATOPOIETIC STEM CELL GENERATION CO-DRIVERS OF T(4;11) MLL-AF4 ACUTE
Samanta Mariani, Zhuan Li, Chris Vink, Siobhan Rice, and LYMPHOBLASTIC LEUKAEMIA
Elaine Dzierzak Camille Malouf1, Franziska Sahm2, Chrysa Kapeni3, and
The University of Edinburgh, Edinburgh, United Kingdom Katrin Ottersbach1
1
University of Edinburgh, Edinburgh, United Kingdom; 2Technische Universit€at
The first hematopoietic stem cells (HSCs) arise in the mouse aorta-gonad-mesonephros
Berlin, Berlin, Germany; 3University of Cambridge, Cambridge, United Kingdom
(AGM) at E10.5 but little is known about the microenvironment supporting their generation.
Interestingly, macrophages (Mo) are present in the embryo before the production of HSCs, T(4;11) MLL-AF4 pro-B acute lymphoblastic leukaemia (ALL) is an aggressive hae-
leading to the hypothesis that yolk sac-derived cells could be involved in the HSC niche. Recent matological malignancy initiated in utero. This disease lacks cooperating mutations,
studies in zebrafish revealed that Mo have a role in hematopoiesis by affecting the migration of but displays a deregulated gene expression profile. We focused our attention on micro-
hematopoietic progenitors from the aorta to the caudal hematopoietic tissue. To investigate RNAs and performed expression profiling of t(4;11)+ paediatric leukaemia patients. We
whether this is conserved in mice, we used the MacGreen mouse model that expresses found an upregulation of miR-128a, miR-130b and miR-155 in patients. To assess their
EGFP under the control of the Csf1r promoter. Flow cytometry confirmed that all EGFP+ cells role in leukemogenesis, we modulated their activity in our Mll-AF4+ pre-leukaemia
in the embryo (E8-11) are phenotypic Mo (CD45+CD11b+F4/80+MerTK+CD16/32+Ly6C- mouse model (Barrett & Malouf et al., Cell Reports 2016) and assessed how this
Ly6G-). Time-lapse imaging on AGM sections showed that embryonic Mo are highly motile affected clonogenic, proliferation and differentiation potentials through colony-form-
cells that undergo mitosis, intravasate into the aorta and interact with CD31+cKit+ cells. Mo ing assays and transplantation in mice. In E14 foetal liver, Mll-AF4+ LSK show a higher
numbers in the AGM increase over time, peaking at E10-10.5 just before the production of the engraftment and self-renewal potential compared to Mll-AF4- LSK, but also a strong B
first HSCs. We tested whether chemokine receptors/ligands may affect Mo recruitment to the lymphoid bias in the Flt3+ fraction (LMPP), suggesting it is the stage when disease ini-
AGM by flow cytometry. AGM Mo were positive for CCR2, CCR5, CXCR4 and CX3CR1 tiates. E14 foetal liver LSK upregulate miR-155 upon Mll-AF4 expression, but not
expression. qrtPCR on sorted AGM endothelial cells, showed CX3CL1 and CXCL12 expres- miR-128a or miR-130b. When miR-155 activity is inhibited, the B lymphoid potential
sion, suggesting that these chemokines can mediate Mo recruitment to the aorta. CX3CR1 defi- decreases and cells acquire a more mature phenotype (IgM+). There is also an increase
cient embryos showed a decreased number of Mo in the AGM confirming the involvement of in myeloid clonogenic potential. When miR-128a or miR-130b are activated in Mll-
the CX3CL1/CX3CR1 axis in Mo migration to the AGM. Moreover, AGM explant experi- AF4+ LSK, the B lymphoid clonogenic potential and proliferation increase. The
ments revealed a 50% decrease in the number of CFU-GEMMs upon treatment with clodro- engraftment of Mll-AF4+ LSK in mice is unaffected by the modulation of miR-128a,
nate liposomes that ablate 80% of AGM Mo. CSF-1RCre:RosaDTA embryos, in which more miR-130b or miR-155 activity, and the lineage output recapitulates the colony-forming
than 95% of Mo are depleted, showed the absence of CFU-GEMMs. Two populations of AGM assay results. Therefore, all three microRNAs participate in the B lymphoid bias and
Mo differentially expressing CD206 were found. CD206+ Mo expressed higher levels of met- differentiation arrest observed in patients. In SEM cells (Mll-AF4+ leukaemia cell),
alloproteinases and inflammatory cytokines. Together, these results support a role for Mo in the inhibition of miR-128a and miR-130b also leads to a decrease in cell proliferation
regulating hematopoiesis in the mouse embryo and future experiments will focus on the mech- and a higher cell death, respectively. We identified three co-drivers of t(4;11) MLL-AF4
anisms by which the interacting cells affect the establishment of definitive hematopoiesis. pro-B ALL development that modulate the B lymphoid clonogenic potential, differen-
tiation and survival/proliferation in early (miR-155) and advanced (miR-128a and miR-
130b) stages of disease. This study is a step forward in understanding the development
of t(4;11) MLL-AF4 pro-B ALL.
Teratomas generated from both normal and CML derived hiPSCs were able to pro- 3096 - MBD3/NURD-MEDIATED CHROMATIN
duce granulopoietic, erythroid and mixed colonies in standard GF-supplemented REMODELLING CONTROLS LYMPHOID CELL FATE
methylcellulose cultures providing evidence of functionally competent human he- AND INHIBITS TUMORIGENESIS BY OPPOSING
matopoietic progenitors. These experiments lay the foundation for a more detailed
TRANSCRIPTIONAL PIONEERING DURING B CELL
investigation of the conditions required for the generation of normal and diseased
HSCs and their properties.
PROGRAMMING
Stephen Loughran1, Federico Comoglio1, Alice Giustacchini2,
Youssef Errami1, Eleanor Earp1, Fiona Hamey1, Berthold Gottgens1,
Sten Eirik Jacobsen3, Adam Mead2, Brian Hendrich1, and Anthony Green1
1
University of Cambridge, Cambridge, United Kingdom; 2University of Oxford,
Oxford, United Kingdom; 3Karolinska Institutet, Stockholm, Sweden
3095 - DECELLULARIZED BONE MARROW REVEALS A 3097 - ASYMMETRIC CELL DIVISION OF HEMATOPOIETIC
NOVEL NICHE PROTEIN THAT MODULATE ADHESION STEM CELLS: ASYMMETRIC INHERITANCE OF CELL
Troy Lund1, Ashley Kramer2, Mandy Taisto1, Amanda Blake1, and FATE DETERMINANTS AND ITS ROLE IN CELL FATE
Michael Lehrke3 DECISIONS
1
University of Minnesota, Minneapolis, United States; 2Washington University in St. Dirk Loeffler1 and Timm Schroeder2
Louis, St. Louis, United States; 3Mayo School of Medicine, Rochester, United States 1
Cell Systems Dynamics, Department of Biosystems Science and Engineering
(D-BSSE), ETH Zurich, Basel, Switzerland; 2ETH Z€urich, Basel, Switzerland
The hematopoietic marrow microenvironment is composed of multiple cell types embedded in
an extracellular matrix (ECM). Decellurization of murine long bones was used to isolate ECM. Hematopoietic stem cells (HSCs) maintain their numbers while also differentiating to replenish
The proteome signature was determined using mass spectrometry which identified over 240 the entire blood system lifelong. Asymmetric cell division is often discussed as the underlying
proteins including Dermatopontin (DPT), a small non-collagenous ECM protein. To under- mechanism balancing HSC self-renewal and differentiation. In this model, future daughter cell
stand DPT’s role in the niche, we generated constitutional DPT-/- mice that were viable and fates are prospectively determined by a mechanism linked to mitosis, e.g. the unequal inheri-
had no peripheral lympho-hematopoietic abnormalities. Marrow composition of wild-type tance of cell fate determinants in the two daughters. This leads to the differentiation of one
and DPT-/- mice was equivalent in terms of cellularity, CFU-C, LSK (Lineage-, SCA-1+, daughter while the other retains its stem cell properties. However, this mechanism could never
KIT+) and LSK-SLAM (LSK, CD48-, CD150+) frequencies. Interesting, when DPT-/- been shown directly. It is therefore also possible that HSCs do not divide asymmetrically, and
mice were used as recipients in adoptive transfer experiments, they displayed a 51% increase commitment to differentiation is determined by mechanisms not related to division. The asym-
in donor cell homing 20 hours following lethal irradiation (n 5 9 mice/group, p ! 0.05); this metric inheritance of cell fate determinants hasbeen reported during invertebrate and vertebrate
translated to greater long term engraftment after primary and secondary transplants (sublethally progenitor divisions. However, the low frequency of HSCs and the technical challenges asso-
irradiated, DPT-/- recipients engrafted at 60% vs 40% in wild-type (n 5 12, p ! 0.01). ciated with reliable continuous quantitative analysis of suspension cells have hampered the
Conversely, pre-treating mice with recombinant DPT showed a 90% reduction (n 5 12, p study of events during HSC mitosis. Few studies therefore provide evidence for asymmetric
5 0.02) and a 40% reduction (n 5 6, p ! 0.05) in whole marrow and LSK cells that migrated inheritance of proteins during hematopoietic stem and progenitor cell (HSPC) division, and
to the marrow 20 hours post-transplant, respectively. This reduction in homing translated to de- none was able to demonstrate that future daughter cell fates correlate with the asymmetric in-
creases in short- and long-term multilineage engraftment. Furthermore, we also determined heritance of cell fate determinants. Using novel continuous and quantitative live cell imaging,
DPT can bind endothelial cells and DPT-/- mice displayed increased endothelial permeability. we now identified molecules asymmetrically inherited to HSC daughters. We demonstrate that
These data suggest that DPT plays a novel role in vascular integrity and cell adherence, but is their asymmetric inheritance predicts future daughter cell behavior. We therefore provide for
not required for steady state hematopoiesis invivo. As well, there are likely overlapping cellular the first time direct evidence that HSPCs utilize asymmetric cell division and that HSPC
adhesion mechanisms that can compensate to maintain the hematopoietic niche in the absence daughter cells fate can actually be determined prospectively by asymmetric molecule inheri-
of DPT. tance during mitosis.
S82 Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
3098 - SMAD4 HAS A CRITICAL ROLE IN 3100 - CONVERSION OF ADULT ENDOTHELIUM INTO
ERYTHROPOIESIS AND HSC FORMATION DURING IMMUNE-COMPETENT HAEMATOPOIETIC STEM CELLS
EMBRYOGENESIS Raphael Lis1, Charles Karrasch1, Micheal Poulos1, Balvir Kunar1,
Carlos Lizama, Kelly Cautivo, and Ann Zovein David Redmond1, Jose Gabriel Barcia Duran1, Chaitanya Badwe1,
University of California, San Francisco, San Francisco, CA, United States Micheal Ginsberg2, William Schachterle1, Jenny Xiang1,
Hematopoietic stem cells (HSC) are generated during embryogenesis from specialized
Arash Rafii Tabrizi3, Koji Shido1, Zev Rosenwaks1, Olivier Elemento1,
vascular beds termed hemogenic endothelium (HE), which includes the aortic endothelium Nancy Speck4, Jason Butler1, Joseph Scandura1, and Shahin Rafii1
1
of the aortic-gonado-mesonephros (AGM) region. In the aorta, pre-HSCs are organized in Weill Cornell Medicine, New York, United States; 2Angiocrine Bioscience, San
clusters attached to the vascular endothelium. The TGF signaling pathway has a pivotal Diego, United States; 3Weill Cornell Medicine-Qatar, Doha, Qatar; 4University of
role in both arterial specification and HSC formation. Previous endothelial SMAD4 loss- Pennsylvania, Philadelphia, United States
of-function studies in mice using a Tie2cre have demonstrated a critical role of this protein Developmental pathways choreographing the ephemeral transition of endothelial
in the endothelial to hematopoietic transition (EHT), with notable phenotypes of increased cells (ECs) into haematopoietic stem cells (HSCs) remain undefined. Here, we devel-
cluster formation and embryonic lethality at E10.5. In order to bypass the early lethality seen oped a tractable approach for converting adult murine ECs to HSCs (rEC-HSCs) by
in other studies, we used an inducible arterial Cre to delete SMAD4 to evaluate the contri- transiently expressing transcription factors FosB, Gfi1, Runx1, and Spi1 (FGRS) and
bution of the TGF signaling pathway to later EHT events, as well as HSC migration, and angiocrine stimulation by vascular-niche. Induction phase (day 0-8) of conversion is
colonization of adult hematopoietic organs. We noted embryonic lethality between E13.5- initiated by expressing FGRS in mature ECs resulting in endogenous Runx1 expres-
E17.5 in animals with SMAD4 loss of function in arterial endothelial cells. The mutants ex- sion. During specification phase (day 8-20), endogenous Runx1+ FGRS-transduced
hibited decreased HSC numbers, enlarged fetal livers, erythropoiesis defects, and the ECs commit to a haematopoietic fate and no longer require FGRS expression. The
inability to engraft adult recipients. In addition, we evaluated the deletion of SMAD4 using vascular-niche drives robust self-renewal and expansion of rEC-HSCs (day 21-28).
NG2cre which is expressed within the sub-aortic mesenchyme during EHT. The mutant an- rEC-HSCs have a transcriptome and long-term self-renewal capacity similar to adult
imals did not exhibit any hematopoietic abnormalities and survived until adult life. Our data HSCs, are competent for clonal engraftment and multi-lineage reconstituting poten-
suggest that the TGF signaling pathway in arterial endothelial cells is critical for definitive tial, including antigen-dependent adaptive immune function. Inhibition of TGF-b and
hematopoiesis; however other non-endothelial sources of TGF do not contribute to the endo- Cxcr7 or activation of Bmp and Cxcr4 signaling enhanced rEC-HSCs generation.
thelial to hematopoietic transition or definitive hematopoiesis. Conversion of ECs into autologous authentic HSCs promises treatment of haemato-
logical disorders.
3099 - RE-EXPRESSION OF KLF4 IMPAIRS GROWTH OF 3101 - RAS-PATHWAY MUTATION PATTERNS DEFINE
PATIENT-DERIVED ACUTE LYMPHOMA LEUKEMIA EPIGENETIC SUBCLASSES IN JUVENILE
CELLS IN VIVO AND SENSITIZES TOWARDS MYELOMONOCYTIC LEUKEMIA
CHEMOTHERAPY Daniel Lipka1, Tania Witte2, Reka Toth2, Jing Yang2, Manuel Wiesenfarth2,
Wen-Hsin Liu1,2 and Irmela Jeremias1 Peter N€ollke3, Alexandra Fischer4, David Brocks2, Zuguang Gu2,
1
unchen, Munich, Germany; 2Bayern, Munich, Germany
Helmholtz Zentrum M€ Jeongbin Park2, Brigitte Strahm3, Marcin Wlodarski3, Ayami Yoshimi3,
Rainer Claus5, Michael L€ubbert5, Hauke Busch2, Melanie B€orries2,
Kr€uppel-like factor 4 (KLF4) is a zinc-finger transcription factor which acts as an
oncogene or tumor suppressor in a tissue-dependent manner. Acute lymphoblastic Albert Catala6, Barbara De Moerloose7, Michael Dworzak8,
leukemia (ALL) is associated with chemoresistance causing relapse with poor Henrik Hasle9, Franco Locatelli10, Riccardo Masetti11, Owen Smith12,
outcome. However, downregulation of KLF4 in ALL has been reported and we found Markus Schmugge13, Jan Stary14, Marek Ussowicz15,
the most prominently reduction in minimal residual disease by gene expression Marry van den Heuvel-Eibrink16, Yassen Assenov2, Matthias Schlesner2,
profiling of primary ALL cells (Cancer Cell 2016). Here, we aimed at deciphering Charlotte Niemeyer3, Christian Flotho3, and Christoph Plass2
1
the role of decreased KFL4 for growth behavior and chemosensitivity in patient- German Cancer Research Center (DKFZ), Heidelberg, Germany; 2DKFZ,
derived xenograft (PDX) ALL cells in vivo. We transplanted primary ALL patients’ Heidelberg, Germany; 3Department of Pediatrics and Adolescent Medicine,
sample into immune-compromised mice to generate PDX cells. Lentiviral transduc- University of Freiburg Medical Center, Freiburg, Germany; 4Department of
tion allowed genetic engineering to re-introduce wildtype (wt) KLF4 and a zinc- Pediatrics and Adolescent Medicine, Heidelberg, Germany; 5Division of
finger DNA binding domain truncated form of KLF4 (mut). As wtKLF4 might impair Hematology, Oncology and Stem Cell Transplantation, University of Freiburg
ALL growth and despite major technical challenges, we established a tetracycline Medical Center, Freiburg, Germany; 6Department of Hematology and Oncology,
inducible expression system in PDX ALL cells. Coupling the transgenes to a fluoro- Hospital Sant Joan de Deu, Barcelona, Spain; 7Department of Pediatric Hematology-
chromic marker gene and optimizing concentrations of Doxycycline (DOX) in vivo, Oncology and Stem Cell Transplantation, Ghent University Hospital, Ghent,
we were able to re-express KLF4 at physiological levels in an on and off manner. Belgium; 8St. Anna Children’s Hospital and Children’s Cancer Research Institute,
Upon re-expression of wt, but not mutKLF4, spontaneous proliferation of PDX Medical University of Vienna, Vienna, Austria; 9Department of Pediatrics, Aarhus
ALL cells was significantly diminished in two different ALL patient samples in University Hospital Skejby, Aarhus, Denmark; 10Department of Pediatric Hematology and
vivo. Re-expression of KLF4 caused cell cycle arrest and induced apoptosis signaling Oncology, Bambino Gesu Children’s Hospital, Rome, Italy; 11Department of Pediatric
including Caspase-3 and PARP cleavage. To investigate the effect of re-expressed Oncology and Hematology, University of Bologna, Bologna, Italy; 12Paediatric Oncology
KLF4 on chemosensitivity, mice were treated both with Dox and additionally with and Haematology, Our Lady’s Children’s Hospital Crumlin, Dublin, Ireland; 13Department
the conventional chemotherapeutic drug Vincristine (VCR). KLF4-expressing cells of Hematology and Oncology, University Children’s Hospital, Zurich, Switzerland;
are eliminated by VCR with much higher efficiency than control cells suggesting 14
Department of Pediatric Hematology and Oncology, Charles University and University
that chemosensitivity for VCR was significantly increased upon re-expressing Hospital Motol, Prague, Czech Republic; 15Department of Pediatric Hematology,
KLF4. Taken together, our data suggest that re-expression of KLF4 to physiological Oncology, and BMT, Wrozlaw Medical University, Wrozlaw, Poland; 16Princess Maxima
levels impairs tumor growth in ALL and sensitizes cells towards treatment. We Center for Pediatric Oncology/Hematology, University of Utrecht, Utrecht, Netherlands
conclude that drugs increasing KLF4 levels such as APTO-253 (Aptose Biosciences)
should be developed towards clinical use for the benefit of patients with ALL, espe- Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative dis-
cially at minimal residual disease. order of early childhood. Hematopoietic stem cell transplantation is the only curative
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136 S83
treatment option, but still, 5-year event-free survival reaches only about 50%. Hyper- 3103 - MITOCHONDRIA ARE IMPLICATED IN THE
active RAS signaling caused by genetic alterations in CBL, KRAS, NF1, NRAS or REGULATION OF TERMINAL ERYTHROPOIESIS
PTPN11 is the main driving event in JMML. So far, there is no clear understanding Raymond Liang1 and Saghi Ghaffari2
of how RAS pathway mutations relate to the heterogeneous disease biology and var- 1
Icahn School of Medicine at Mount Sinai, Astoria, United States; 2Icahn School of
iable clinical outcome observed. We hypothesized that DNA methylation profiling, Medicine at Mount Sinai, New York, United States
alone or in combination with genetic alterations, might provide a molecular basis
for disease classification. DNA methylome analysis of 167 JMML samples (plus Erythroblast enucleation, the process of nucleus removal, is a critical step for red blood cell
exome sequencing and expression profiling in 50 and 15 cases, respectively) identi- (RBC) formation and function. Our data suggests a novel mechanism required for enucleation,
fied three subgroups with low, intermediate and high methylation levels (LM, IM and which involves mitochondrial activity. Outside of their role in generating heme, mitochondrial
HM). The HM group was enriched for patients with somatic PTPN11 mutations and function during other erythroid specific processes is unknown. We observed that prior to murine
includes poor outcome cases while the LM group was enriched for somatic NRAS & erythroblast enucleation, mitochondria migrate distal to the extruding nucleus. Immunofluores-
CBL mutations as well as for Noonan-patients and represents the good prognosis cence images of FACS sorted erythroblasts at distinct stages of maturation show increasing
group. The IM group was enriched for cases with monosomy 7 and somatic KRAS mitochondrial polarization with maturation. This observation was further confirmed by Image-
mutations. Differentially methylated sites were enriched for regions decorated with Stream analysis that combines microfluidics with imaging to allow high-throughput imaging of
H3K27me3 or PRC2 components and for genes associated with oncogenic RAS- single cells. With ImageStream we could distinguish cells in the process of enucleating depen-
signaling. Integrative analysis revealed frequent co-occurrence of $2 mutations acti- dent on the degree of nucleus extrusion. We found a significant positive correlation between
vating the RAS-RAF-MEK-ERK pathway in the HM & IM groups. This finding was erythroblast enucleation and mitochondrial polarization and predicted that mitochondria func-
paralleled by a significant up-regulation of DNMT1 & DNMT3B suggesting aberrant tion might relate to the energetic demands of the enucleation process. To test this, we tracked
activation of the DNA methylation machinery in this context. Together, we identified total mitochondria abundance and mitochondrial membrane potential (MMP), a measure of
three JMML subgroups characterized by distinct clinical and biological features. We mitochondrial activity, using fluorescent probes. Although mitochondrial abundance and
further provide evidence for a molecular mechanism by which additional genetic MMP decrease as erythroblasts mature, a transient increase in MMP is associated with enucle-
events, presumably further activating the RAS-RAF-MEK-ERK pathway, mediate ating cells. We also sorted primary late stage erythroblasts and allowed them to mature towards
DNA hypermethylation via up-regulation of DNMTs in aggressive JMML cases. enucleated RBCs in the presence of inhibitors of the electron transport chain (ETC), which lead
to decreased ATP production. Treatment with these drugs led to a significant decrease in eryth-
roblast enucleation rates compared to DMSO control treated erythroblasts. The treatment was
specific to enucleation and not other erythroblast maturation processes. Notably, this effect was
not due to increased cell death and was reversible through a wash step. Overall our data support a
novel role for mitochondria in promoting the last stages of erythroblast enucleation, potentially
through ATP generation and/or other co-factors produced through the ETC.
3102 - GATA-3 SUPPRESSES IRF8 TO PROMOTE HUMAN 3104 - A ROLE FOR MICROGLIA IN HEMATOPOIETIC
T-CELL LINEAGE COMMITMENT STEM/PROGENITOR CELL EMERGENCE IN THE
Kai Ling Liang, Inge Van de Walle, Laurentijn Tilleman, EMBRYONIC BRAIN
Filip Van Nieuwerburgh, and Tom Taghon Zhuan Li1, Samanta Mariani2, Chris Vink2, and Elaine Dzierzak2
1
Ghent University, Ghent, Belgium QMRI, Edinburgh, United Kingdom; 2The University of Edinburgh, Edinburgh,
United Kingdom
The thymus is an organ where T-cell development takes place. However, human
thymic progenitors have the capacity to differentiate into cells of other haemato- The mouse embryonic head is one of the sites for haematopoietic stem and progenitor cell
poietic lineages. This multi-lineage differentiation potential is lost progressively as (HSPC) development. Transplantation of subdissected head region cells has shown HSC local-
thymic progenitors undergo sequential T-cell lineage specification and commitment. ization in the hindbrain and branchial arches (HBA). Recent studies reveal a dynamic interac-
The former is dependent on Notch1 signalling and results in loss of myeloid and B- tion of primitive macrophages with aortic HSPCs in zebrafish embryos. It is becoming clear by
cell potential. At the commitment stage, T-cell specific transcription factors, rather fate mapping that under physiological conditions, microglia cells (macrophages in the head,
than Notch signalling, are essential to suppress residual non-T potential. Recently, MV) are derived from the yolk sac. However, it is unknown what role(s) MV play in
we reported that GATA-3 is a key driver in human T-lineage commitment by repres- HSPC emergence in the head. In our study using MacGreen (Csf1r-GFP) embryos, we found
sing natural killer (NK) cell fate through down-modulation of Notch signalling. Now, in the HBA (E9.5-11) that more than 95% of MV (CD45+F4/80+CD11b+Gr1-) are GFP+.
transcriptomic analyses of GATA-3 transduced human haematopoietic progenitors al- Flow cytometry showed that most MV in the E10 HBA express CX3CR1 and CXCR4.
lowed us to identify IRF8 as a novel target gene. In comparison to the hypomorphic qRTPCR analysis showed that E10 HBA endothelial cells (CD31+CD45-CD41-, EC) express
KRR-mutant of GATA-3, enforced expression of GATA-3 wild type significantly CX3CL1, which encodes the ligand of CX3CR1. In CX3CR1-/- embryos, the frequency of
downregulated endogenous IRF8 RNA and protein expression. The effect was MV in the HBA is reduced. Interestingly, at E10 the percentage of haematopoietic progenitors
reversed in a GATA-3 knockdown setting. IRF8 is a member of interferon regulatory (cKit+CD41lo) is decreased in the CX3CR1 deficient HBA, but it is not decreased in the E11
factor family that plays an important role in human haematopoiesis. In human, IRF8 HBA. To test if MV play a role in endothelial cell-to-haematopoietic cell-transition (EHT),
is highly expressed in all haematopoietic lineages except T-cells. Dysregulation of HBA EC were cultured with/without microglia in an OP9-DL1 coculture system. The presence
IRF8 has been implicated in human leukaemia and mutations in IRF8 are clinically of MV boosted the HPC output from EC more than 2-fold compared to the control group (EC
associated with human dendritic cell (DC) and NK cell immunodeficiency. As such, only). qRTPCR experiments showed MV (MacGreen+) express significantly higher levels of
we hypothesize that negative regulation of IRF8 by GATA-3 is important to promote pro-inflammatory factor genes (IL-1b, IL-1c, TNFb) compared to MacGreen- cells. Interest-
human T-lineage commitment. Using multi-coloured flow cytometry, we showed that ingly, EC expressed higher levels of receptor genes (IL1R1, TNFbR1) than CD41+/CD45+ or
IRF8 expression is restricted in ex vivo uncommitted (CD34+CD1a-) human thymic CD31-CD41-CD45- cells, suggesting that MV are involved in hematopoietic cell emergence
progenitors. Developmental trajectory of human thymic progenitors, defined by early in the HBA through the IL-1 and/or TNFa pathway. To examine if MV function in vivo, MV
T-cell markers, revealed dynamic and sequential expressions of IRF8 and GATA-3. were specifically ablated in Csf1r-Cre;RosaDTA mice. In the HBA (E9.5-11), all the MV were
This enabled us to identify, within the uncommitted pool of thymic progenitors, depleted. Methylcellulose culture data showed the numbers of hematopoietic progenitors were
new subpopulations that reflect successive extinction of DC and NK lineage choices. reduced significantly after MV ablation in the HBA. In summary, these data suggest that mi-
Using in vitro co-culture assay, we recapitulated this previously uncharacterized tran- croglia cells play important roles in hematopoietic cell emergence in HBA.
sitional cell populations during human T-lineage commitment.
S84 Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
3105 - DIFFERENT ABERRANT EXPRESSION PATTERN OF the expressing levels of PD-1 and Foxp3 genes in the gd T cells sorting by MACS
IMMUNE CHECKPOINT RECEPTOR IN PATIENTS WITH technique from peripheral blood of AML patients and HI. Clinical relevance of gd
T-NHL AND NK/T-CL T cells and their subsets in AML patients were also analyzed. A decreasing trend
of gd T cells expression was found in AML patients compared with HI controls.
Ziwei Liao1, Xuewen Lv2, Sichu Liu3, Zifan He4, Lijian Yang4,
Expression levels of PD-1high gd T cells、Foxp3+ gd T cells, and PD1high
Shaohua Chen4, Liang Wang5, Wenyu Li3, and Yangqiu Li4 Foxp3+ gd T cells in AML patients were significant higher than that in HI. The
1
Medical College of Jinan University, Guangzhou, Guangzhou, China (People’s
expression proportions of PD-1high gd T cells and Foxp3+ gd T cells had positive
Republic); 2Key Laboratory for Regenerative Medicine of Ministry of Education, Institute
correlation to PD1high Foxp3+ gd T cells. Expression levels of PD-1 gene in gd
of Hematology, Guangzhou, China (People’s Republic); 3Guangdong General Hospital &
T cells had positive correlation to the Foxp3 gene in AML patients. Moreover, the
Guangdong Academy of Medical Sciences, Guangzhou, China (People’s Republic); 4Key
curative effects analysis of AML patients indicated high expression proportions of
Laboratory for Regenerative Medicine of Ministry of Education, Institute of Hematology,
Foxp3+ gd T cells and PD-1high Foxp3+ gd T cells were correlated with poor prog-
Jinan University, Guangzhou, China (People’s Republic); 5Department of Oncology, First
nosis. In conclusion, a novel regulatory cell subsets of PD1high Foxp3+ gd T cells
Affiliated Hospital, Jinan University, Guangzhou, China (People’s Republic)
were found in de novo AML patients. High expression proportion of PD1high
T-cell immunodeficiency plays a crucial role in the development and progression of tumor. Im- Foxp3+ gd T cells might influence the outcome of AML disease.
mune checkpoint (co-inhibitory receptors) and TCR signal pathway regulate T-cell function in
opposite way. Our previous study showed the decreasing trend in T cell activation related TCR
signal pathway expression in peripheral blood mononuclear cells (PBMCs) from T-cell non-
Hodgkin lymphomas (T-NHL) and NK/T-cell lymphoma (NK/T-CL). However, how immu-
nosuppression status regulated by the co-inhibitory receptors, like programmed death 1 (PD-1),
cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), B/T lymphocyte attenuator (BTLA)
expanded to Lymphocyte-activation gene 3 (LAG-3), T cell immunoglobulin-3 (TIM-3), T cell
immunoglobulin and ITIM domain (TIGIT) remains unclear. In this study, we characterized
the expression pattern of all these immunosuppressive receptors in PBMCs form T-NHL
and NK/T-CL patient by real-time qPCR. Significantly higher PD-1 expression level was de-
tected in both T-NHL (n514) and NK/T-CL (n58) when compare to HI (healthy individuals,
n522,), while the expression level of its ligand PD-L1 had no statistic difference in both groups
in comparison to HI group. The expression of BTLA, LAG-3, TIM-3, ICOS and TIGIT signif-
icantly increased in T-NHL group, but not in NK/T-CL group. While higher expression of
CTLA-4 was found only in NK/T-CL group. When disease stage was considered in, BTLA
is only up-regulating in advanced stage (stage III and IV), most patients with high ICOS expres-
sion level are in stage I and II. Moreover, the positive correlation between the expression level
of LAG-3, TIM3 and TIGIT gene in HI group was impacted when it comes to either T-NHL or
NK/T-CL patients. In conclusions, aberrant expression pattern of T-lymphocyte inhibitory re-
ceptors is a common characteristic in patients with T- NHL or NK/T-CL, which is one of rea-
sons of immunosuppression, moreover, the different upregulating of such inhibitory receptors
between T-NHL and NK/T-CL might be as immune biomarkers for evaluation the immuno-
suppression status and help to establish the precision target of immunotherapy.
3106 - EXPRESSION OF NOVEL REGULATORY CELL 3107 - HIGHER TIM-3 EXPRESSION CONCURRENT
SUBSETS OF PD-1HIGH FOXP3+ gd T CELLS IN DE NOVO WITH PD-1 IN EXHAUSTED CD4+ AND CD8+T CELLS
AML IN PATIENTS WITH ACUTE MYELOID LEUKEMIA
Zhenyi Jin1, Dan Qiu1, Qiang Luo1, Jie Chen2, Shaohua Chen1, Bo Li3, Jiaxiong Tan1, Shaohua Chen2, Danlin Yao2, Yikai Zhang2, Lijian Yang2,
Xiuli Wu4, and Yangqiu Li5 Jing Lai3, Kaikai Huang3, Jie Chen3, Zhi Yu3, Jun Zhong3, Yuhong Lu3,
1
Institute of Hematology, Jinan University, Guangzhou, China (People’s Republic); and Yangqiu Li2
2 1
Department of Hematology, First Affiliated Hospital, Jinan University, Guangzhou, Medical college of Jinan University, Guangzhou, Guang Zhou, China (People’s
China (People’s Republic); 3Key Laboratory for Regenerative Medicine of Ministry Republic); 2Jinan University, Guangzhou, China (People’s Republic); 3First
of Education, Institute of Hematology, Jinan University, Guangzhou, China (People’s Affiliated Hospital of Jinan University, Guangzhou, China (People’s Republic)
Republic); 4Institute of Hematology, Key Laboratory for Regenerative Medicine of
Ministry of Education, Jinan University, Guangzhou, China (People’s Republic); T cell dysfunctionality is a characteristic of patients with leukemia and plays an
5
Institute of Hematology, Department of Hematology, First Affiliated Hospital, Key important role in leukemia progression. Increasing data showed immune checkpoint
Laboratory for Regenerative Medicine of Ministry of Education, Jinan University, which mediate suppression and exhaustion of T cells play the crucial role in tumor
Guangzhou, China (People’s Republic) immunosuppression. Upregulating Tim-3 and PD-1 could be identified on exhausted
T cell in mice with acute myeloid leukemia (AML). Our previous study also showed
Acute myeloid leukemia (AML) is one of adult leukemia with overall survival rates co-expression of PD-1 and CD244,CD57-exhausted T cells in patients with AML,
of 40% at 5 years. gd T cells, representing 1-10% of T cells in human peripheral and found a particular influence on CD8+T cells.In this study,we further investigated
blood, can recognize specific antigen without MHC-restriction and play a critical the characteristics of the distribution of TIM,and the co-expression with PD-1,
role in anti-leukemia response. But gd T cells have different functional subsets CD244, and CD57 on CD3+, CD4+ and CD8+ T cells from patients with de novo
like regulatory cell subsets. Relevant regulatory mechanism is still unknown. Pro- AML and AML in complete remission (CR). Surface expression of PD-1, TIM-3
grammed death-1 (PD-1), one of T cell inhibitory receptors may mediate T cell sup- and CD244 and CD57 on CD3+, CD4+ and CD8+ T cells from peripheral blood sam-
pression and dysfunction in leukemia. It reported that PD-1 pathway might relate to ples from 18 newly diagnosed and 10 cases with AML-CR was analyzed by flow cy-
general regulatory T cells which express transcription factor Foxp3. So we supposed tometry. 16 healthy individuals served as control. Significantly higher percentages of
that PD-1 pathway was involved in regulation of regulatory Foxp3+ gd T cells. In this TIM-3+CD3+, TIM-3+CD4+T cells were found in AML group compared with
study,we first investigated the expression of regulatory cell subsets of PD-1high healthy controls. Moreover, higher numbers of TIM-3+CD244+,TIM-
Foxp3+ gd T cells in patients with de novo AML. The flow cytometry was used to 3+CD57+CD4+T cells were identified in AML group, increased levels of TIM-
detect the expression levels of gd T cells and their subsets in 14 de novo AML pa- 3+CD8+, TIM-3+CD244+, TIM-3+CD57+CD8+T cells were also found in AML
tients and 15 healthy individuals (HI). Real-time quantitative PCR was used to detect group, however, the difference was not statistical significant. Interesting, when we
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136 S85
analyzed the coexpression of TIM-3 and PD-1 on CD4+ and CD8+ T cells, signifi- TCR repertoire, we identified TRBV11-2/TRBD1/TRBJ1-1 T cell clone special for
cant higher levels of TIM-3+PD-1+CD4+ and TIM-3+PD-1+CD8+T cells were iden- WT1 peptide induction which was similar to to the T cell clone for CML associated
tified either in de novo AML or AML-CR groups. Among the AML patients, one antigen in CML patients, this kind of WT1 induced T cell clone may be considered
AML-M2 case with poor outcome, who had bladder tumor and underwent partial cys- for constitution of donor derived T cell immune therapy for CML patients. And the
tectomy and chemotherapy in 7 years ago, showed highest TIM-3 expression in global expression profile of TCR repertoire between different T cell immune statuses
almost T cell subsets. In conclusion, we characterized for the first time major T could be characterized by high throughput TCR sequencing.
cell defects, including co-expression of TIM-3, PD-1 and CD244,CD57-exhausted
T cells in patients with de novo AML and found that TIM-3 may particular influence
on CD4+T cells, while immunesupression with TIM-3 and PD-1 coexpression may
involve on both CD4+ and CD8+ T cells, suggesting a poor anti-leukemia immune
response in these patients.
3108 - IDENTIFICATION OF TRBV11-2/TRBD1/TRBJ1-1 T 3109 - LOWER TSCM AND TCM WITH HIGHER TEM
CELL CLONE SPECIAL FOR WT1 IN DONOR T CELLS AND TEF CELLS IN PATIENTS WITH ACUTE MYELOID
BY HIGH THROUGHPUT T-CELL RECEPTOR BETA- LEUKEMIA
CHAIN SEQUENCING Danlin Yao1, Ling Xu2, Jiaxiong Tan2, Yikai Zhang2, Jing Lai2,
Zhang Yikai1, Xu Ling1, Yao Danlin1, Lu shuai2, Chen Shaohua1, Yuhong Lu2, Lijian Yang2, Xianfeng Zha2, Shaohua Chen3, and Yangqiu Li2
1
Yang Lijian1, Wu Xiuli1, Zha Xianfeng3, and Li Yangqiu4 Institute of Hematology, Jinan University, Guangzhou, China; Department of
1 Hematology, First Affiliated Hospital, Jinan University, Guangzhou, China;
Institute of Hematology, School of Medicine, Jinan University, Guangzhou, China
(People’s Republic); 2Key Laboratory for Regenerative Medicine of Ministry of Department of Clinical Laboratory, First Affiliated Hospital, Jinan University, G,
Education, Jinan University, Guangzhou, China (People’s Republic); 3First Affiliated Guangzhou, China (People’s Republic); 2Institute of Hematology, Jinan University,
Hospital of Jinan University, Guangzhou, China (People’s Republic); 4Key Guangzhou, China; Department of Hematology, First Affiliated Hospital, Jinan
Laboratory for Regenerative Medicine of Ministry of Education, Institute of University, Guangzhou, China; Department of clinical laboratory, First Affiliated
Hematology, Jinan University, Guangzhou, China (People’s Republic) Hospital, Jinan University, Guangzhou, China; Key Laboratory for Regenerative
Medicine of Ministry of Education, Jinan University, Guangzhou, China, Guangzhou,
Several studies observed that BCR-ABL1 or Wilms’ tumor protein 1 (WT1) antigen China (People’s Republic); 3Institute of Hematology, Jinan University, Guangzhou,
specific cytotoxic T cells can be detected in parts of CML patients and healthy donors China; Department of Hematology, First Affiliated Hospital, Jinan University,
after antigen peptides vaccination. In this study, we aimed to analyze the changes of Guangzhou, China; Department of Clinical Laboratory, First Affiliated Hospital,
the TCR Vb repertoire of T cells from healthy donor (HLA*A0201+) after induction Jinan University, Guangzhou, China; Key Laboratory for Regenerative Medicine of
with WT1 or BCR-ABL1 peptides. PBMCs from a healthy donor was induced with Ministry of Education, Jinan University, Guangzhou, 510632, China, Gaungzhou,
WT1 peptide (RMFPNAPYL, AA 126-134) or mixed BCR-ABL1 peptides and China (People’s Republic)
cultured with IL-2 and IL-7 in vitro for 21 days, then the cells were collected for
high throughput sequencing of TCRb-chain. We found that the top three usage of The distribution of different memory T cell subsets in patients with leukemia remains
Vb subfamilies in the non-treated group were TRBV20-1 (20.3%)、TRBV5-1 unclear. In this study, we investigated the distribution of T memory stem cells
(11.1%) and TRBV29-1 (9.2%), as well as in the cytokines treated group (unspecific (TSCM) which are a unique memory T cell subset with enhanced self-renewal capac-
stimulation). However, the top three Vb subfamilies were changed into TRBV29- ity and the ability to differentiate into potent effectors, T central memory (TCM), T
1(15.4%), TRBV12-3(13.5%) and TRBV20-1 (10.8%) in WT1 group and TRBV7- effector memory (TEM) and T terminal effector (TE) cells in CD4+ and CD8+ T
9 (30.3%), TRBV20-1 (24.8%) and TRBV5-1 (15.4%) in BCR-ABL1 group. As ex- cells from patients with acute myeloid leukemia (AML) by flow cytometry analysis.
pected, the stimulation along with cytokines did not change the selection of Vb sub- There were lower percentage of CD8+ TSCM and CD8+TCM in peripheral blood
families in normal T cells, while specific stimulation with different polypeptides (PB) AML group (n 5 11) than that in healthy control (HI) group (n 5 16), there
could induce the select proliferation of Vb subfamily T cell clones. Based on the was the trend of lower of CD4+ TSCM and CD4+TCM were found, while it was
dominant usage of the V(D)J rearrangements, according to the prediction of CDR3 not statistic different. Significant higher percentage of TEM and TEF in both
amino acid, the high frequency of CDR3AA sequence (CASSSSGTGPNTEAFF CD4+ and CD8+ T cell subsets were showed in AML group. We also analyzed the
(4.7%)) was observed in WT1 group, which was identified as TRBV11-2/TRBD1/ distribution of the memory T cell subsets in samples from AML in complete remis-
TRBJ1-1 rearrangement. Interesting, this T cell clone is similar to the clonally sion (CR) group (n 5 6), the numbers of both CD8+ TSCM (P 5 0.013) and CD4+
expanded Vb21 (different TCR V region named system) T cells from CML patients TSCM, as well as CD8+ TCM and CD4+ TCM were increased in compared with
that we identified previously. In conclusions, by using high throughput sequencing of AML group, while the numbers of TEM and TEF in both CD4+ and CD8+ T cell
S86 Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
subsets were decreased accordingly. We also compared the percentage of the memory 3111 - ROLE OF EARLY B CELL FACTOR 1 IN HSPCS
T cell subsets in samples from both PB and bone marrow (BM) either in AML or in Aurelie Lenaerts, Marta Derecka, and Rudolf Grosschedl
AML-CR, the difference seemed individually. Combining the results that higher Tim- Max Planck Institute of Immunology and Epigenetics, Freiburg, Germany
3 (mucin domain-containing protein 3) and PD-1 (programmed death 1) expression
and exhausted phenotype (CD244 and CD57) were identified in both CD4+ and Early B cell factor 1 (Ebf1) is an essential transcription factor that orchestrates the B
CD8+ T cells in AML, it might conclude that lower TSCM and TCM may be one cell lineage program. Despite its heavily characterised role in the B cell lineage, it
of the crucial reason of immunodeficiency in patients with AML, even there are has been suggested that Ebf1 plays a role in the haematopoietic stem cell and progen-
higher percentage of TEM and TEF in AML patients, their higher expression of T itor compartment (HSPC). To gain further insight into the potential role of EBF1 in
cell inhibitory receptors and exhaustion status resulted their dysfunction, and could HSPCs we analysed the haematopoietic compartment of Ebf1fl/flTie2Cre conditional
not efficiently play the immune reaction ability to AML cells. KO mice. We observed a modest increase in the number of HSCs in these mice and in
order to test their HSC functionality we performed competitive adoptive transfers of
Ebf1fl/flTie2Cre HSCs into WT recipients. Interestingly, we observed a decrease in
chimerism in primary and secondary adoptive transfers from Ebf1fl/flTie2Cre
HSCs compared to Ebf1wt/wtTie2Cre HSCs. Further analysis of Ebf1fl/flTie2Cre
mice revealed a significant increase of myeloid-biased multipotent progenitors
(MPP2/3) as well as mature cells of the myeloid compartment. Moreover, CFU-as-
says of Ebf1fl/flTie2Cre HSCs showed an increase in the number of myeloid colonies
relative to Ebf1wt/wtTie2Cre HSCs. Together, these data demonstrate that loss of
Ebf1 results in myeloid biased haematopoietic output and suggests that Ebf1 contrib-
utes to the regulation of HSC function.
3110 - FOLLOWING THE IN VIVO EXPANSION OF A 3112 - THE ROLE OF RIG-I-LIKE RECEPTORS IN
MYELOPROLIFERATIVE DISEASE DEVELOPMENTAL HEMATOPOIESIS
Daniel Lewandowski1,2,3,4, Vilma Barroca1, Jean-Luc Villeval5, Stylianos Lefkopoulos1, Na Yin2, Pierre Cauchy1,
William Vainchenker5, and Paul-Henri Romeo1 Natalia A. Martagon Calderon1, Thomas Clapes1, Gianluca Dagati2,
1
CEA, Fontenay aux Roses, Fontenay aux Roses, France; 2INSERM U967, Fontenay Christian Mosimann2, and Eirini Trompouki1
aux Roses, Fontenay aux Roses, France; 3University of Paris-Diderot, Paris 7, 1
Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany;
Fontenay aux Roses, France; 4University of Paris-Sud, Paris 11, Fontenay aux Roses, 2
Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland
France; 5IGR, Villejuif, France
Embryonic hematopoiesis is a complex and tightly regulated process. Recent studies hint
Malignant myeloid hemopathies are characterized by a clonal dominance process linked to at endogenous RNAs, like Endogenous Retroviruses, as novel hematopoietic regulators.
transformation of hematopoietic stem cells (HSCs). In non-BCR/ABL myeloproliferative Searching for the cellular element that could transduce the signal initiated by endogenous
neoplasms (MPN), mutated HSCs acquire a hypersensitivity to growth factors leading to un- RNAs, a family of RNA helicases, known as RIG-I-like receptors (RLRs), caught our
regulated production of mature cells. All these MPNs exhibit mutations that activate JAK2/ attention. RLRs, a family of innate immune receptors consisting of RIG-I, MDA5 and
STAT signaling pathway (Klampfl T, 2013). Interestingly, human JAK2V617F mutation can LGP2, are activated by viral RNAs. However, lately, the notion that they also bind to
be found in healthy subjects (Sidon P, 2006; Nielsen C, 2011) and the polyclonal endogenous RNAs has gained acceptance. Thus, we asked whether RLRs can regulate
JAK2V617F murine models show that this mutation is sufficient to generate MPNs when embryonic hematopoiesis alone or in a network. Although primitive hematopoiesis was
transplantation of mutant HSCs is carried out after total body irradiation (TBI) of mice (Lac- not affected, RIG-I and MDA5 morphants displayed decreased expression of the hemo-
out C, 2006; Delhommeau F, 2009). We thus looked for experimental conditions that allow genic endothelial and Hematopoietic Stem and Progenitor Cell (HSPC) marker, runx1,
the development of MPN without TBI from a local engraftment of JAK2V617F HSCs. To at 26 hpf, while LGP2 morphants had the opposite effect. The impact was consistent,
address this question, we have developed a new technique based upon a local irradiation of regarding the expression of c-myb at 48hpf and rag1 in the thymus at 4 dpf. Our pheno-
one mouse leg to create a site from which the engrafted hematopoiesis may spread in non- types were verified in mutants generated by CRISPR/Cas9 technology. Simultaneous defi-
irradiated tissues. We showed that WTand JAK2V617F mouse grafts can implant and partic- ciency in RIG-I and LGP2, but also in MDA5 and LGP2 rescued HSPCs, suggesting an
ipate in hematopoiesis of recipient irradiated mouse leg. Interestingly, in the non-irradiated interplay between them. To test whether RNA binding plays a role in their effect, we at-
leg, a significant increase of chimeric cells was observed after JAK2V617F graft but not WT. tempted to rescue the morphant phenotypes with human RLR mRNA lacking the RNA
We found that an increase of MHCII-low expressed macrophages could precede and/or be helicase domain. Importantly, while the wild type mRNA rescued the phenotype, the heli-
concomitant with engraftment and invasion of JAK2V617F cells in non-irradiated bone case mutant did not, proving that the RNA binding domain is essential. To shed light on the
marrow and spleen. Nonetheless, splenectomy prior to or during the spent phase of Polycy- transcriptional landscape, we performed an RNA-sequencing analysis of endothelial cells,
themia Vera did prevent expansion of red blood cells and appearance of JAK2V617F he- from which HSCs derive. Our analysis revealed a minimal cluster of genes essential for
matopoietic cells in non-irradiated bone marrow, preventing the appearance of the endothelial to hematopoietic transition, positively regulated by LGP2 knock down, but
disease. We are currently analyzing the clonal dissemination of the JAK2V617F cells using negatively by RIG-I or MDA5 knockdown. Upstream regulator analysis of this cluster re-
the bar-code method. Using this new technique, we will be able to study the intrinsic and/or vealed DDIT3, FOXO3 and TNF as concomitant regulators of the cluster. Importantly,
extrinsic factors regulating the evolution of a MPN in a non-irradiated environment. This LGP2 knock down suppressed the negative regulation of this gene network by RIG-I
work opens new research avenues in understanding the progressive invasion of malignant and MDA5 knock down and thus, rescued HSPCs. Our data suggest RLRs as novel reg-
hematological diseases in a homeostatic microenvironment. ulators of embryonic HSPCs that mediate their effect through their helicase domain.
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136 S87
3113 - HETEROGENEITY OF EMERGING demonstrates the importance of using correct control mice for transplantation and expression
HEMATOPOIETIC STEM CELLS analysis to discriminate mouse background-induced artefacts from genuine phenotypes.
Yu Lan1 and Bing Liu2
1
Institute of Hematology, School of Medicine, Jinan University, Guangzhou, China
(People’s Republic); 2Affiliated Hospital, Academy of Military Medical Sciences,
Beijing, China (People’s Republic)
3114 - INHERENT ENGRAFTMENT DIFFERENCES 3115 - INTERACTIONS WITH THE BONE MARROW
BETWEEN CD45.1 AND CD45.2 HSCS ARE CAUSED BY MICROENVIRONMENT AS DETERMINANT OF
DIFFERENTIAL EXPRESSION OF CXCR4 BIOLOGICAL PHENOTYPE AND MYELOID
Luisa Ladel1,2, Simon Renders3, Jasper Panten1,2, DIFFERENTIATION IN IMATINIB-RESISTANT CHRONIC
Katharina Sch€onberger1,2, Pia Sommerkamp1,2, Petra Zeisberger1,2, MYELOID LEUKEMIA
Nina Cabezas-Wallscheid1,2,3, and Andreas Trumpp1,2,4 Rahul Kumar1, Melanie Meister1, Eva Weissenberger1,
1
Division of Stem Cells and Cancer, Deutsches Krebsforschungszentrum (DKFZ), Alexander Azimpour1, Frank Schn€uttgen2, Thomas Oellerich3,
Heidelberg, Germany; 2Heidelberg Institute for Stem Cell Technology and Charles Lin4, Richard Etten5, and Daniela Krause1
Experimental Medicine (HI-STEM gGmbH), Heidelberg, Germany; 3Department of 1
Georg-Speyer Haus, Frankfurt, Germany; 2Universit€atsklinikum der Goethe-
Cellular and Molecular Immunology, Max Planck Institute of Immunobiology and Universit€at, Frankfurt, Germany; 3University Hospital Frankfurt, Frankfurt,
Epigenetics, Freiburg, Germany; 4Germany and German Cancer Consortium Germany; 4MGH-Harvard, Boston, United States; 5Chao Family Comprehensive
(DKTK), Heidelberg, Germany Cancer Center, Irvine, CA, United States
The CD45.1/2 system is commonly used in mouse to discriminate the progeny of trans- Mutations within BCR-ABL1, the oncogene giving rise to chronic myeloid
planted cells from those of the recipient. To distinguish the two genetic alleles, discrimina- leukaemia (CML), can lead to resistance to tyrosine kinase inhibitors and some are
tory antibodies specific for a single lysine to glutamic acid amino acid difference are associated with clinically more aggressive disease and worse outcome (Branford et
available (Mercier et al; Stem Cell Reports, 2016). Since CD45 is expressed on most nucle- al., 2003). This phenomenon was faithfully recapitulated in our murine transduc-
ated cell of the hematopoietic lineage, it is possible to analyse the origin of various stem, pro- tion/transplantation model of BCR-ABL1+ CML, in which recipients of bone
genitor and differentiated cell populations in bone marrow reconstitution experiments by marrow transduced with the imatinib-resistant BCR-ABL1 point mutant T315I (or
FACS and compare their respective contributions under competitive conditions. However, Y253F) had shortened survival, compared to native BCR-ABL1. To test how BCR-
an important concern in the field is the observation, that CD45.2 cells are inherently advan- ABL1-positive leukaemia-initiating cells may interact with the bone marrow micro-
tageous to CD45.1 ones, even after extensive backcrossing probably due to genetic linkage environment (BMM), we used a combination of confocal and 2-photon intravital mi-
(Waterstrat et al; Blood, 2010). This complicates the interpretation of published transgenic croscopy of murine calvarium. We showed that BCR-ABL1T315I + Lin– c-Kit+ Sca-
animal studies and requires carefully chosen controls. To investigate the functional reason for 1+ (LKS) cells, homed closer to osteoblastic cells than LKS cells expressing native
this competitive advantage, we performed RNA-seq of CD45.2 and CD45.1/2 hematopoietic BCR-ABL1 which suggests a differential homing location of BCR-ABL1T315I
stem cells (HSCs) isolated from competitive transplanted chimeras. Interestingly, one of the leukemic stem cells (LSC) and may be due to differences in adhesion or migration.
top differentially expressed genes was Cxcr4, a well known cell surface receptor that has Indeed, immunofluorescence revealed an altered actin cytoskeleton and striking dif-
been shown to be important for HSC maintenance and mobilization. In agreement, we found ferences in focal adhesion kinase in BCR-ABL1T315I+ compared to native BCR-
a gradual decrease of Cxcr4 mRNA and protein expression in CD45.1 compared to CD45.2 ABL1 cells. Additionally, integrin b3, was upregulated in BCR-ABL1T315I cells.
mice obtained from different suppliers. Next, we performed competitive transplantations, Consistent with the situation in humans we found an increase in the number of imma-
while treating recipient mice with a Cxcr4-inhibitor. Strikingly, this treatment led to the alle- ture blast-like cells and alterations of the immunophenotypic composition of cells in
viation of the advantage seen upon transplantation of CD45.2 HSCs. Collectively, this data the bone marrow of murine recipients of BCR-ABL1T315I+. Furthermore, the
S88 Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
deposition of fibronectin by BCR-ABL1T315I + cells was decreased compared to 3117 - DECLINED PRESENTATION
native BCR-ABL1 cells. Intrafemoral or intravenous administration of fibronectin, INHIBITION OF CARBONIC ANHYDRASE IX AS
however, led to a significant prolongation of survival in 70% of mice with BCR- A NOVEL STRATEGY TO TARGET FLT3/ITD MUTATED
ABL1T315I + CML. In summary, these data suggest that interactions with BMM
AML CELLS UNDER HYPOXIC CONDITIONS
via the integrin b3-mediated signaling pathway may regulate myeloid differentiation
and clinical outcome in BCR-ABL1T315I+ imatinib-resistant CML. Targeting the
Heiko Konig, Fangli Chen, Adriana Rogozea, Garrett Kinnebrew,
interaction of BCR-ABL1T315I+ leukemia cells with the BMM in the form of fibro- Mircea Ivan, and Heiko Konig
nectin may offer a beneficial, innovative, adjuvant treatment strategy in patients with Indiana University Simon Cancer Center, Indianapolis, United States
BCR-ABL1T315I+ CML. Background: Recent data suggests that the hypoxic properties of the bone marrow niche
play a key role in the evolution of drug resistance in FLT3/ITD+AML, thus indicating a
role for hypoxia-specific drugs in AML therapy. However, the mechanisms by which low
oxygen (O2) levels distort drug responses of FLT3/ITD+AML cells are mostly unknown.
Here we investigated the cytotoxic activity of classic and novel agents under hypoxic
(1%) and normoxic (21%) conditions against FLT3/ITD+AML. Methods: Molm14
(M14) and primary cells from relapsed/refractory FLT3/ITD+AML patients were treated
with Cytarabine (C), Quizartinib (Q) or FC531 (FC; a selective inhibitor of the hypoxia
responsive gene carbonic anhydrase IX [CA-IX]) under 21% and 1% O2. After 48h, prolif-
eration and apoptosis induction were assessed per MTT and FACS assays. Cells were also
assessed for FLT3 protein and Hypoxia inducible factor (HIF) target gene expression by
western blot and PCR arrays. Results: The growth inhibitory effects of C and Q against
FLT3/ITD+ cells were significantly dampened under 1% compared to 21% O2. Similarly,
hypoxia weakened the pro-apoptotic effects of either drug. In contrast, Q potently inhibited
FLT3 activity under both 1% and 21% O2. We next sought to identify tractable pro-survival
genes induced by hypoxia, and to assess their expression in response to therapy. Our data
shows that the HIF pathway is upregulated in response to 1% O2 and remains widely func-
tional in C or Q treated cells. CA-IX, a druggable HIF target, was significantly upregulated in
FLT3/ITD+ cells under 1% O2. When we exposed M14 cells to FC, we found that cell
growth was significantly inhibited under 1% but not 21% O2. Further, FC was significantly
more effective than C or Q in inducing apoptosis under hypoxia. Conclusions: 1) Hypoxia
blunts the cytotoxic effects of C and Q in FLT3/ITD+AML cells. 2) The HIF signaling pathway
remains largely functional under hypoxic conditions, despite treatment. 3) FC is significantly
more cytotoxic than C or Q under hypoxic conditions. 4) Further investigation of combined
therapies between standard AML agents and FC in FLT3/ITD+AML is warranted.
Signalling pathways influence fate decisions of hematopoietic cells. However, quan- Whole exome sequencing analyses are increasingly performed on patients presenting
tifying signalling dynamics over time and linking these to future fate decisions of the with suspected inherited disease but lacking classical mutations linked to the pre-
same individual cells have been technically infeasible. Here, we establish continuous sented phenotype. However, in case a novel mutation is found, its causal contribution
single-cell time-lapse imaging to quantify the activity of various signalling pathways to the patients’ clinical symptoms is yet unclear and requires further exploration in
over time in living hematopoietic stem and progenitor cells (HSPCs). Live signalling functional studies. Here we explore the utility of the zebrafish model for such studies
biosensors reporting the activity of the ERK, PI3K/AKT, STAT3, WNT and NOTCH by exploring the functional relevance of gene X, which has been newly identified as
signalling pathways are combined to generate a transgenic mouse line simultaneously mutated in a patient with unexplained neutropenia (Carapito et al, unpublished). Spe-
displaying their activity. Isolating known single cells after live observation allows to cifically, we performed loss-of-function experiments using two different antisense
link signalling dynamics to live functional assays or to destructive high-dimensional morpholino oligonucleotides (MO) to inhibit pre-mRNA splicing followed by rescue
RNA or protein quantification. We expect to identify novel HSPC sub-populations experiments with wildytpe or mutated human protein to asses its functional rele-
with specific signalling responses, and a better understanding of the relevance of sig- vance. Indeed, reduced neutrophil numbers were observed in MO versus control in-
nalling pathways in controlling transcriptional programs and HSPC fates, of molec- jected fish when analyzed by whole mount in situ hybridization detecting the
ular HSC control in general. expression of the neutrophil marker mpx and by flow cytometry detecting mpx+ or
respectively lyz+ neutrophils in corresponding transgenic lines at 48 hpf. Interest-
ingly, quantification of results indicated morphants to display not only quantitatively
but also qualitatively impaired neutrophils, which – as shown in tail fin injury assays
– were less able to migrate to the injury site in an emergency situation. Importantly,
co-injection with human wild-type but not mutated mRNA was able to rescue neutro-
phil numbers, suggesting that the observed effects are specific and the mutated allele
most likely a functional null. Supporting our data, in the corresponding Sanger
mutant, homozygous fish display severe neutropenia and are not viable, while hetero-
zygous fish survive, but show significantly suppressed neutrophils in comparison to
wildtype siblings and can be equally rescued by injection with the human mRNA.
Together, these in vivo analyses support the notion that the newly identified mutation
in protein X is responsible for the phenotype observed in patients. This model shall
be used in future drug screening projects to identify drugs able to improve this
phenotype.
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136 S89
3123 - MICROTUBULE PLUS-END TRACKING PROTEIN 3125 - GFI1B – A NOVEL ONCOSUPPRESSOR, WHICH
CLASP2 IS REQUIRED FOR HEMATOPOIETIC STEM RESTRICTS NUMBER OF LEUKEMIC STEM CELLS
CELL GENERATION DURING EMBRYONIC Cyrus Khandanpour1, Aniththa Thivakaran2, Judith Schutte2,
DEVELOPMENT Lothar Vassen2, Ulrich Duhrsen2, and Yahya Almaray2
1
Anna Klaus1, Thomas Clapes2, Laurent Yvernogeau1, Niels Galjart3, and Department of Hematology, University Hospital Essen, Essen, Germany;
2
Catherine Robin1 University Hospital Essen, Essen, Germany
1
Hubrecht Institute, Utrecht, Netherlands; 2Max Planck Institute of Immunobiology
Background: Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are
and Epigenetics, Freiburg, Germany; 3Erasmus MC, Rotterdam, Netherlands
hematopoetic disorders, which affect the myeloid lineages of hematopoiesis. Both are char-
Hematopoietic stem cells (HSCs) are initially generated during embryonic develop- acterized by an accumulation of blast cells in the bone marrow (BM) that have the lost the
ment from hemogenic endothelial cells via an endothelial to hematopoietic transition ability to differentiate to mature cells. The proper differentiation of hematopoietic stem cells
(EHT). After emergence, the first HSCs and their precursors (pre-HSCs) form clus- (HSCs) is regulated by transcription factors. Growth factor independence 1b (Gfi1b) is a re-
ters that remain transiently associated with the aortic endothelium. Pre-HSCs and pressing transcription factor regulating quiescence of HSCs and the proper emergence and
HSCs then migrate to the fetal liver where they mature/amplify before the HSC maturation of erythrocytes and platelets. Aims: Aim of the study was to identify I) do
pool colonizes the bone marrow. The journey of HSCs requires cell polarisation, different level of Gfi1b influence onset and development of MDS and AML in human pa-
cell shape remodelling, cell division, homing and cell attachment. All these processes tients II) how does Gfi1b act in MDS/AML development on a molecular level.
involve extensive cytoskeletal rearrangements. CLASP2 is a microtubule-associated Methods: We correlated Gfi1b expression level in blast cells of patients with MDS and
protein, regulating microtubule dynamics by stabilizing the plus-end of microtubules. AML with the overall disease course. To get a better insight how does different Gfi1b level
Interestingly, Clasp2 knockout mice have an HSC defect in the bone marrow, result- influence MDS/AML development, we used three different murine models of human AML
ing in severe pancytopenia. Our goal is now to investigate the role of CLASP2 in with expression of different oncogenes (NUP98/HOXD13, MLL-AF9 and expression of a
HSCs during embryonic development. Our preliminary data show that the loss of mutated K-Ras). In these models we either downregulated or conditionally knocked out
CLASP2 does not impact the number or type of committed progenitor cells in the Gfi1b expression. Finally, we performed ChIP Seq analysis as well as whole genome
aorta, yolk sac and fetal liver. However, a reduced HSC activity in the aorta and fetal gene expression arrays to study the molecular functions of Gfi1b in AML development.
liver, and the reduced number of cluster cells in the aorta of Clasp2-/- embryos, sug- Results: Low expression or absence of Gfi1b expression was associated with an inferior
gest a role of Clasp2 during EHT and HSC production and maintenance. Zebrafish outcome with regard to overall-survival as well as event-free survival of MDS/AML pa-
CLASP2 knockout lines have been generated to study the EHT process in detail. tients. Using the above murine models of MDS/AML, loss or low expression of Gfi1b
The comparison of single cell RNA sequencing data of cluster and hemogenic endo- accelerated AML development. Additionally we could show that loss of Gfi1b signifi-
thelial cells sorted from the aorta of mouse wild type and Clasp2-/- embryos will also cantly enhanced number of functional leukemic stem cells. It is well known that Gfi1b
help deciphering the mechanism of action of CLASP2 during EHT. Overall, we has a function to recruit histone modifying enzymes to induce among other deacetylation
demonstrate here a critical role for CLASP2 during the first steps of HSC production of H3K9. ChIP seq data of Gfi1b deficient leukemic cells revealed that loss of Gfi1b led to
during embryonic development. a higher H3K9 acetylation of a number of target genes, among them a number of onco-
genes. Among these target genes, we found MAPK as well as Reactive oxygen species
(ROS) signalling, as one of the top hit in our data. Previously it was reported that loss
of Gfi1b enhanced the ROS level in HSCs. In our case we also see an increased expression
of ROS in Gfi1b deficient leukemic cells, a higher activity of the FOXO pathway as well
as reduced p38 activity. The combination of these foundings contributes to the higher
number of leukemic stem cells in Gfi1b deficient leukemic cells. To reduce the high level
of ROS in leukemic stem cells we use with N-Acetylcystein (NAC). Use of NAC impeded
growth of Gfi1b deficient cells in-vitro and in-vivo. Conclusion: Gfi1b act as a tumorsup-
pressor by restricting number of leukemic stem cells and treatment with NAC opens a po-
tential targeted therapy for AML patients with low/absent expression of Gfi1b.
3124 - MICRORNA-22 CONTROLS INTERFERON ALPHA 3126 - GROWTH FACTOR INDEPENDENCE 1 (GFI1)
PRODUCTION AND ERYTHROID MATURATION IN REGULATES THE AML SUPPORTING FUNCTION OF
RESPONSE TO INFECTIOUS STRESS MESENCHYMAL STROMAL CELLS
Katherine King1, Claudine Kadmon2, Cameron Landers2, Haiyan Li3, Cyrus Khandanpour
Stephanie Watowich3, and Antony Rodriguez2 Department of Hematology, University Hospital Essen, Essen, Germany
1
Baylor College of Medicine, Houston, United States; 2BCM, Houston, United
Mesenchymal stromal cells (MSCs) harbor and support the function of normal hemato-
States; 3MD Anderson Cancer Center, Houston, United States
poietic stem cells. Less is known about their interaction with leukemic cells, e.g. in acute
MicroRNA-22 (miR-22) is a highly conserved microRNA that plays a role in diverse myeloid leukemia (AML). The prognosis of AML, a clonal malignant disease of the bone
cellular functions including cell proliferation, oncogenesis, and cell maturation, particu- marrow (BM), is still poor with only 25% of patients living longer than 5 years. We there-
larly in response to stress. Recent work demonstrates that miR-22 participates in regulation fore investigated the interaction between MSC and AML cells. MSCs from AML patients
of the interferon response, and expression profiling studies suggest that it is variably ex- called AML-associated MSCs (AMSCs) or from murine models of human leukemia
pressed at different stages in erythroid differentiation. We thus hypothesized that miR- enhance significantly in vitro the growth of leukemic cells compared to AML cells
22 regulates maturation of erythroid progenitors during stress hematopoiesis. We growing without MSCs or in presence of MSCs from non-leukemic patients or mice.
compared the blood and bone marrow of wild type (WT) and miR-22-deficient mice at Among other, AMSCs increased entry of leukemic cells into the cell cycle, and at the
baseline and upon infectious challenge with systemic lymphochoriomeningitis (LCMV) same time protected the leukemia cells against exogenous toxic events such as chemo-
virus. MiR-22-deficient mice maintained platelet counts better than WT mice during infec- therapy or irradiation. The interaction between AMSCs and leukemia cells is dependent
tion, but they showed significantly reduced red blood cells (RBC) and hematocrit. Analysis on cell-to-cell contact. In vivo, absolute and relative numbers of AMSCs and other stro-
of bone marrow progenitors demonstrated increased colony forming units and improved mal cells, i.e. endothelial cells and osteoblast lineage cells were highly expanded in the
hematopoietic stem cell homeostasis in infected miR-22-null mice compared to WT. BM of mice modeling of human AML. AMSCs showed a higher efficiency of capillary
Absence of miR-22 blunted the interferon response to LCMV challenge, which may ac- tube formation in the matrigel assay than normal MSCs which gives an additional indi-
count for these results since interferon responses are known to impair HSC function. Addi- cation that AMSCs were polarized by leukemia cells towards a tumor-supporting state.
tionally, we found that miR-22 was exclusively expressed in Stage 2 erythroid precursors On a molecular level, the polarization of MSCs towards an AML-supporting state de-
and was downregulated upon infection in WT mice. The effects of infection on the distri- pends on upregulated expression of the transcription factor Growth factor independence
bution of erythroid precursors were exaggerated in miR-22 null mice. Collectively, our re- 1 (Gfi1). Loss of Gfi1 diminished the tumor-supporting state of AML-associated MSCs.
sults indicate that miR-22 functions at baseline as a brake to slow erythroid differentiation We conclude that leukemia cells polarize AMSCs towards a leukemia-supporting state in
and maintain adequate erythroid potential, and furthermore shows that impaired regulation a Gfi1-dependent manner, which could open the way to new therapeutic approaches.
of erythrogenesis in the absence of miR-22 can lead to anemia during infection.
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136 S91
3127 - EXPLORING THE ROLE OF JAGN1 IN NEUTROPHIL observed initially delayed repopulation kinetics as a common phenotype: gene-
EXTRACELLULAR TRAP (NET) PRODUCTION AND marking (%BFP+) of human CD45+ and lin-CD34+ cells in bone marrow was
FUNCTION: IMPLICATIONS FOR SEVERE CONGENITAL reduced relative to input and control at 4w post transplantation. Whereas by 20w
engraftment reached or exceeded input levels and secondary transplants showed
NEUTROPENIA
that HSC frequency was maintained or increased. This particular pattern of repopu-
Avinash Khandagale1, Beatrice Lazzaretto2, G€oran Carlsson3, lation combined with the observation of transcriptional upregulation of these genes in
Sulman Shafeeq4, Ute R€omling4, and Bengt Fadeel2 subsets of paired diagnosis-relapse samples from AML patients (Shlush et al, in revi-
1
Karolinska Institutet, Stockholm, Sweden; 2Division of Molecular Toxicology, sion) is consistent with the hypothesis that latent LSC can survive chemotherapy and
Institute of Environmental Medicine-IMM, Karolinska Institutet, Stockholm, initiate relapse. Our studies implicate these candidates as regulators of LSC self-
Sweden; 3Department of Women’s and Children’s Health, Karolinska Institutet, renewal and persistence and functional studies of the relevant pathways involved
Karolinska University Hospital Solna, Stockholm, Sweden; 4Department of may uncover new therapeutic targets for LSC eradication in AML.
Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden
3130 - EXPANSION AND MAINTENANCE OF mast cells, we generated bone marrow derived mast cells (BMMCs). We observed
HEMATOPOIETIC STEM AND PROGENITOR CELLS IN that bone marrow from Cebpg KO mice produced reduced number of BMMCs in
COURSE OF LONG-TERM INHIBITION OF CXCR4/CXCL12 comparison to WT controls. Functionally, we demonstrated that deletion of Cebpg
reduced mast cell migration towards antigen, SCF or PGE, and impaired degranula-
SIGNALING
tion upon FcεRI-mediated activation. Next, we aimed to investigate the mechanisms
Darja Karpova1, Julie Ritchey1, Matthew Holt1, Grazia Abou-Ezzi1, by which C/EBPg is controlling these processes. Our data showed that BMMCs
Darlene Monlish1, Laura Schuettpelz1, Wei Yang2, Allison Pettit3, exhibit increased C/EBPa levels in the absence of C/EBPg, probably contributing
Michael Rettig1, Halvard Boenig4, and John DiPersio1 to the observed defects. In summary, we revealed C/EBPg as a new important
1
Washington University School of Medicine, St. Louis, United States; 2Genome Technology component of the mast cell transcriptional network which suppresses C/EBPa
Access Center, Washington University, St. Louis, United States; 3Mater Research Institute- expression, thereby favoring mast cell development and function.
The University of Queensland, Faculty of Medicine, Translational Research Institute,
Woolloongabba, Australia; 4German Red Cross Blood Service and Institute for Transfusion
Medicine and Immunohematology of the Goethe University, Frankfurt, Germany
The critical role of the interaction between the chemokine receptor CXCR4 and its chief ligand
CXCL12 for retention and migration of hematopoietic stem and progenitor cells (HSPC) is
well established. Interference with CXCR4/CXCL12 signalling is currently being exploited
as a strategy to mobilize HSPC indirectly with G-CSF as well as directly with the bicyclam
CXCR4 antagonist Plerixafor (AMD3100). In this study, qualitative and quantitative effects
of long-term pharmacologic inhibition of CXCR4/CXCL12 axis within the HSPC compart-
ment were investigated in mice using the non-peptidic small molecule CXCR4 antagonists
Plerixafor and ALT1188 along with the Protein-Epitope Mimetics Inhibitor POL5551. Up
to 12-14 fold higher mobilization efficiency was achieved by applying the antagonists via
continuous infusion (up to 8-10x104 CFU-C and LSK/ml) as compared to bolus treatment
(4-6x103 CFU-C and LSK/ml) or 5-day course of G-CSF (3-6x103 CFU-C/ml). Despite dra-
matic increase in numbers of circulating HSPC, the BM HSPC pool dis not decrease; in fact it
expanded up to 2-4-fold compared to steady state reservoir. Cell cycle analysis showed a 2-3–
fold increase in cycling activity of BM HSPC: only 10-20% of LSK and 30-40 % of LSK
SLAM cells were found to be quiescent (in G0 phase of the cell cycle) after two weeks of
CXCR4 antagonist infusion versus 50-60 % of LSK and 70 % of LSK SLAM found in G0
under homeostatic conditions. Profiling of differentially treated BM HSC via microarray anal-
ysis did not reveal substantial effects of CXCR4 inhibitor infusion on the expression signature.
No major cytological changes were detected in continuous infusion of POL5551 exposed BM
suggesting limited effects within the BM niche compartment. Moreover analysis of the BM
HSPC after different washout periods at the end of continuous infusion treatment revealed a
rapid reestablishment of steady state HSPC numbers in the BM. Our data suggest that pro-
longed pharmacologic blockade of the CXCR4/CXCL12 axis using multiple small molecule
inhibitors represents an approach superior to any other known HSPC mobilization strategy but
also may serve as an effective method to expand BM HSPCs.
3131 - THE TRANSCRIPTION FACTOR C/EBPg 3132 - TET1 PROMOTES LEUKEMIC GROWTH OF
REGULATES AML1-ETO+ AML VIA 5-HYDROXYMETHYLCYTOSINE
MAST CELL DEVELOPMENT AND FUNCTION MARKS AND ITS ONCOGENIC ROLE AND HIGH
Miroslava Kardosova1, Lucie Potuckova2, Ivana Halova2, EXPRESSION CAN BE ANTAGONIZED USING OLAPARIB,
Polina Zjablovskaja2, Lubica Draberova2, Petr Draber2, Daniel Tenen3, AN INHIBITOR OF ITS BINDING PARTNER PARP1
and Meritxell Alberich-Jorda2 Jing He1, Shiva Bamezai1, Deniz Sahin1, Fabian Mohr1, Naidu Vegi1,
1
Laboratory of Hematooncology, Institute of Molecular Genetics of the Fabio Ciccarone2, Alex Jose1, Medhanie Mulaw1, Paola Caiafa2,
ASCRPrague, Czech Republic; 2Institute of Molecular Genetics of the ASCR, Konstanze D€ohner3, Hartmut D€ohner3, Michaela Buske3,
Prague, Czech Republic; 3Cancer Science Institute, National University of Singapore, Christian Buske1, and Vijay Rawat1
Singapore, Singapore 1
Institute of Experimental Cancer Research, CCC and University Hospital of Ulm,
Mast cells represent an important part of the immune system. They play a key role in Ulm, Germany; 2Department of Cellular Biotechnology and Haematology, Medicine
multiple pathologies including allergies and autoimmune disorders, and conse- 2, University of Rome ‘La Sapienza’, Rome, Italy; 3Department of Internal Medicine
quently, it is critical to fully understand the molecular mechanisms that control their III, University Hospital Ulm, Ulm, Germany
generation and activity. Development of hematopoietic stem cell through progenitor AML1-ETO (AE) is the most commonly occurring fusion gene in AML and shows a
stages towards functionally mature mast cell is orchestrated by networks of interact- distinct methylation pattern, the underlying mechanisms for which is poorly under-
ing transcription factors. Expression of several transcription factors, such as GATA1, stood. TET1 dioxygenase regulates methylation patterns via conversion of 5-methyl-
GATA2, STAT5, and MITF, and downregulation of C/EBPa are already known to be cytosine (5mC) to 5-hydroxymethylcytosine (5hmC), thereby regulating a variety of
important determinants of mast cell identity. Recently, we described C/EBPg as a C/ biological processes. However, the role of TET1 in AE+ AML cases is yet unex-
EBPa target gene, and observed that C/EBPg is abundantly expressed in mast cells. plored. Using qRT- PCR we observed that TET1 was significantly higher expressed
In order to investigate the role of C/EBPg in mast cell development and function, we in the majority of AE+ patients compared to other AML subtypes and CD34+ normal
used Cebpg conditional knockout mice, which allow specific excision of Cebpg in the BM cells. This observation was consistent with published cDNA microarray and tran-
hematopoietic system from early embryogenesis. Cebpg flox/flox Vav-1Cre- and scriptome data. Knockdown (KD) of TET1 using two shRNAs in AE+ AML cell lines
Cebpg flox/flox Vav-1Cre+ mice, referred here as WT and Cebpg KO respectively, impaired their cell growth and clonogenicity in vitro. KD of Tet1 in AE9a+ murine
showed similar numbers of peritoneal mast cells in steady state conditions. Neverthe- leukemic cell line inhibited its clonogenicity in vitro and delayed onset of leukemia
less, mice lacking Cebpg presented defective peritoneal mast cell repopulation after in vivo. Tet1-ko mouse derived HSPCs transduced with AE9a showed impaired serial
intraperitoneal injection of distilled water, which specifically abolishes the presence replating capacity compared to AE9a transduced Tet1-wt HSPCs in vitro. hMeDIP
of mast cells in the peritoneum. To further explore the effects of Cebpg ablation in
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136 S93
and MeDIP-seq performed on TET1 depleted KASUMI1 cells revealed 8,562 5hmC- tion, MAPkinase activity and cell migration as determined by microarray gene
enriched promoters (-5kbTSS) in the scrambled arm and a 50% decrease in 5hmC- expression analysis. Overexpression of TET3 in AML cell lines further enhanced
enriched promoters in KD arm. In RNA-seq the genes associated with AML, Pleur- their clonogenicity compared to control. Overexpression of wild-type TET3 or the
ipotency and WNT signaling were downregulated upon TET1 KD and over 200 of TET3-CD, construct containing only the catalytic domain, in human CD34+ CB cells
these genes exhibited loss of 5hmC on their promoters. MeDIPseq and RNA-seq impaired their myeloid colony formation in the CFC assays (n54, p ! 0.002)
data revealed a global decrease of promoter methylation and increased expression whereas erythroid colony formation was not affected. In contrast KD of TET3 in
of myeloid differentiation associated genes. IP analysis confirmed that TET1 physi- CD34+ CB cells showed reduction in erythroid antigen positive cells and increase
cally interacted with PARP1 in AE+ cell line. Oncogenic TET1 expression was antag- in myeloid antigen cells in proliferation assay (n53). In conclusion, our data indi-
onized by PARP inhibitor Olaparib in AE+ human leukemic cell lines, which induced cates that ordered expression levels of TET3 are necessary for normal human HSC
reduction of H3K4me3 marks on the TET1 promoter and 5hmC levels in AML cell lineage differentiation. The growth and engraftment potential of AML cells are de-
lines. Furthermore, Olaparib treatment decreased cell growth and clonogenicity of pending on high TET3 expression levels.
human and murine AE+ cell lines. In conclusion, our data indicate that aberrant
TET1 expression contributes to the growth of AE+ AML by maintain 5hmC marks
and that the PARP inhibitor olaparib can at least partially antagonize the oncogenic
effect of TET in AML.
3133 - THE DIOXYGENASE TET3 IS ABERRANTLY HIGH 3134 - CLONAL DIFFERENCES IN DRUG SENSITIVITY
EXPRESSED IN ACUTE MYELOID LEUKEMIA, AND GROWTH BEHAVIOR IN PATIENTS ALL CELLS
PROMOTES LEUKEMIC GROWTH AND IMPAIRS GROWING IN MICE
MYELOID DIFFERENTIATION OF NORMAL Irmela Jeremias1,2 and Cornelia Finkenzeller3
HEMATOPOIETIC STEM PROGENITORS
1
Helmholtz Center Munich, Munich, Germany; 2Dr. von Haunersches Kinderspital,
Alex Jose1, Fabian Mohr2, Shiva Bamezai2, Karlheinz Holzmann3, LMU Munich, Munich, Germany; 3layer, Munich, Germany
Ursula Kohlhofer4, Leticia Quintanilla Martinez Fend4, Tadafumi Iino5, Acute lymphoblastic leukemia (ALL) consists of genetically heterogeneous cell sub-
Koichi Akashi5, Konstanze D€ohner6, Hartmut D€ohner7, populations, but little is known about how genetic differences lead to functional dif-
Michaela Feuring-Buske6, Christian Buske2, and Vijay Rawat2 ferences between the clones. Aggressive subpopulations determine the prognosis of
1
Institute of Experimental Cancer Research, Ulm, Germany; 2Institute of patients and require eradication by treatment. We aimed at characterizing functional
Experimental Cancer Research, University Hospital of Ulm, Ulm, Germany; and genetic characteristics of single stem cell clones in order to identify new treat-
3
Genomics Core Facility, University Hospital of Ulm, Ulm, Germany; 4Institute of ment options for adverse subclones. Primary tumor cells from a 5-year old girl at first
Pathology, University Hospital of T€ubingen, T€ubingen, Germany; 5Department of relapse ALL with chromosomal hyperdiploidy involving a trisomic X chromosome
Medicine and Biosystemic Science, Kyushu University, Fukuoka, Japan; were transplanted into severely immune-compromised mice and lentivirally modified
6
Department of Internal Medicine III, University Hospital of Ulm, Ulm, Germany; to express the fluorochromes red, blue and green at different amounts and combina-
7
Department of Internal Medicine III, Ulm, Germany tions (RGB marking, Weber et al., Nat. Protoc. 2012). Eight single stem cell clones
were generated by limiting dilution transplantation and their uniqueness verified by
The epigenetic regulator TET3 catalyzes the oxidation of 5-methylcytosine (5mC)
ligation-mediated (LM) PCR. Several single cell clones were mixed in a single
into 5-hydroxymethylcytosine (5hmC) and plays an important role in gene regula-
mouse for in vivo assays and distinguished from each other by flow cytometry due
tion. Epigenetic aberrations play a key role in the pathophysiology of acute myeloid
to their unique color staining. We found that clones differed substantially in growth
leukemia (AML), the reasons for the highly distorted epigenome in AML are poorly
rate and that faster and slower growing clones co-existed in the sample. Slow growth
known. However, the role of TET3 in human myeloid leukemia is still unknown. In
was associated with marked resistance against chemotherapy treatment of mice. The
this study, by performing qRT-PCR we could demonstrate that TET3 is higher ex-
slowest subclone was most resistant to in vivo treatment with Glucocorticoids. The
pressed in human CD34+ BM cells and myeloid cells compared to total BM cells
slowly proliferating, Glucocorticoid-resistant clone had lost the additional X chromo-
and lymphoid cells respectively. In AML patients, TET3 showed a broad range of
some, which was present in all other clones and the bulk. Taken together, our studies
expression (n580). qRT-PCR analysis on primary AML leukemic cells shows that
allow identifying and characterizing single cell clones in order to develop efficient
TET3 was higher expressed in 30% of the cases in CN-AML (n550) and in 95%
novel treatment approaches to eliminate aggressive stem cell clones in ALL.
of cases in PML-RARa+ AML (n521) compared to total BM and CD33+ myeloid
cells respectively. The AML cell lines OCI AML-3, NB-4 and KASUMI-1 showed
higher expression of TET3 compared to other AML cell lines. shRNA mediated
knockdown of TET3 in these human AML cell lines significantly reduced the cell
growth, clonogenicity in vitro and the leukemic engraftment in NSG mice (shRNA
n512; scr n511). TET3 depletion decreased total 5hmC level in the AML cell lines
and in parallel changed expression of genes involved in regulation of cell prolifera-
S94 Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
3135 - SINGLE CELL CENSUS OF EARLY MURINE found in myelodysplastic patients (MDS). Even without stress, high aTGFb1 was suf-
ORGANOGENESIS REVEALS A NOVEL PATHWAY ficient to induce mild cytopenia and myeloid dysplasia. TGFb signaling is overacti-
REGULATING GENERATION OF BLOOD PROGENITORS vated in MDS (Bhagat et al., Blood, 2013). Thus, we propose that aberrant TGFb
signaling is sufficient to induce bone marrow failure and some phenotypes associated
Wajid Jawaid1,2,3, Ximena Ibarra - Soria4, Blanca Pijuan - Sala5,
with MDS, perhaps by inhibiting DNA repair.
Vasileios Ladopoulo5, Fernando Calero-Nieto5, Antonio Scialdone6,
David Jorg5, Ludovic Vallier5, Benjamin Simons5, Berthold Gottgens5, and
John Marioni4
1
Cambridge Stem Cell Institute, University of Cambridge, Cambridge, United
Kingdom; 2Department of Haematology, University of Cambridge, Cambridge, United
Kingdom; 3Depatment of Paediatric Surgery, Addenbrookes Hospital, Cambridge, United
Kingdom; 4CRUK CI, Cambridge, United Kingdom; 5University of Cambridge,
Cambridge, United Kingdom; 6EMBL-EBI, Cambridge, United Kingdom
Post gastrulation cells within the three definitive germ layers continue to re-arrange and orga-
nise themselves thorugh gross morphological changes, anatamical localisation and integra-
tion. The first recognisable functional regions thus formed, give a semblance of early
rudimentary organ units. Harnessing the utility of unbiased single-cell transcriptome analysis
we sampled 3 murine embryos (E8.25, Theiler stage 12) after removing the ectoplacental
cone. This resulted in 6882 cells that passed quality control with essentially equivalent con-
tributions from each embryo. Unsupervised clustering revealed 18 major cell groups with
balanced contributions from each of the 3 embryos excepting one embryo in which the
yolk sac was removed. Examples of clusters include several neural clusters, definitive endo-
derm, cardiac, endothelial, embryonic blood and multiple other mesodermal clusters. Prior
knowledge of prototypal genes characteristic of the designated clusters revealed readily
appreciable further diversity and substructure. For example the foregut endoderm cluster
was shown to unambigiously consist of three separate populations matching regionalisation
patterns demonstrated by in-situ hybridisation at the corresponding developmental stage. Ex-
ploiting the well described Fgf8 mesodermal gradient in the caudal mouse embryo we
computationally arranged somitic progenitors along a pseudospace identifying dynamic
waves of transcription and new putative candidate regulators. Focusing on the endothelial
cluster we identify a rare population of cells displaying a signature consistent with activation
of an erythroid-myeloid progenitor (EMP) transciptional programme. Gene expression anal-
ysis revealed a previously unidentified druggable biochemical pathway upregulated in these
early EMPs. Functional assays confirm this pathway promotes the generation of haemato-
poietic precursors. This comprehensive single cell map therefore for the first reports the
whole spectrum of cell types in the post-gastrulation mouse embryo and can be exploited
to reveal previously unrecognised pathways contributing to tissue development.
3136 - HIGH TGFB SIGNALING ALTERS DNA REPAIR AND 3137 - COMPREHENSIVE PROTEOMIC DESCRIPTION
ACCELERATES BONE MARROW FAILURE DURING OF ONTOGENIC CHANGES IN HEMATOPOIETIC STEM
STRESS HEMATOPOIESIS AND PROGENITOR CELLS
Jose Javier1,2, Ashwini Hinge3, Juying Xu3, and Marie-Dominique Filippi3
Maria Jassinskaja, Trine Kristiansen, Hugo Akerstrand, Karin Olsson,
1
University of Cincinnati College of Medicine Cancer and Cell Biology Graduate Kristoffer Sj€oholm, Simon Hauri, Johan Malmstr€om, Joan Yuan, and
Program, Cincinnati, United States; 2Division of Experimental Hematology and Jenny Hansson
Cancer Biology, Cincinnati Children’s Hospital Research Foundation, Cincinnati, Lund University, Lund, Sweden
United States; 3Cincinnati Children’s Hospital Research Foundation, Cincinnati,
United States Hematopoiesis in the fetus and adult is characterized by distinct features that reflect
the vastly different needs of the organism during different stages of ontogeny.
Hematopoietic stem cells (HSCs) are able to reconstitute all blood lineages and self- Although the functional differences between fetal and adult hematopoietic stem
renew, and are used to treat hematopoietic diseases via bone marrow transplantation and progenitor cells (HSPCs) are well-established, the molecular programs that
(BMT). However, BMT and chemotherapy induce a variety of stresses in HSCs, govern these differences remain elusive. In this study, we have utilized an in-depth
which impair their long-term regenerative potential. The mechanisms that control mass spectrometry-based quantitative proteomics approach to compare the full pro-
HSC function during stress hematopoiesis are still ill-defined. We recently reported teomes of ex vivo isolated and FACS-sorted mouse fetal liver and adult bone marrow
that an autocrine TGFb/p38 MAPK pathway increases in HSCs during stress hema- HSPCs. Close to 7000 proteins were identified, allowing us to comprehensively
topoiesis and drives HSC differentiation at the expense of self-renewal (Hinge et al. describe the proteomic differences between these cells. 454 proteins showed differ-
Nat Comm. 2017). To explore the role of TGFb signaling further, we used a trans- ential expression, indicative of the divergent nature of fetal and adult hematopoiesis.
genic mouse model that conditionally overexpresses active TGFb1 (aTGFb1) in he- While the proteome of fetal HSPCs is fairly simple, with main features being cell cy-
matopoietic cells. After 5-fluorouracil-mediated myeloablation, aTGFb1- cle and cell proliferation, their adult counterpart has a more advanced proteome,
overexpressing mice (Tg-Cre+) had impaired reconstitution of peripheral white blood including an arsenal of proteins important for viral and bacterial defense, as well
cells, neutrophils and platelets, and reduced frequencies of long-term HSCs, short- as protection against ROS-induced protein oxidation. Our further analyses of Type
term HSC and multipotent progenitors, indicating that high TGFb impairs HSC I interferon signaling showed that fetal HSPCs are sensitive to Interferon alpha
regenerative capacity. Interestingly, Tg-Cre+ neutrophils had higher frequency of mi- (IFNa) in vitro, although their exposure and response to IFNa in their native environ-
cronuclei formation than control, suggesting abnormal DNA repair during replicative ment is mild. Our results provide new and important insights into the molecular land-
stress. Likewise, intracellular phospho-flow staining showed elevated levels of scape of fetal and adult hematopoiesis that advance our understanding of normal and
gH2AX, a DNA double strand break marker, in Tg-Cre+ cells. Cell cycle analysis malignant hematopoiesis during fetal and adult life.
also showed fewer quiescent HSCs in Tg-Cre+ mice 7 days after stress. Using poly-
inosinic:polycytidylic acid challenge to induce replicative stress, we noted that Tg-
Cre+ mice had persistent cytopenias up to 5 months after stress, and unexpectedly
had dysplastic bone marrow erythroid and myeloid progenitors, similar to those
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136 S95
3139 - ADHESION TO STROMAL CELLS MEDIATES 3141 - IN VIVO CRISPR EDITING OF DNMT3A IN
IMATINIB RESISTANCE IN CHRONIC MYELOID JAK2V617F HEMATOPOIETIC STEM CELLS INDUCES
LEUKEMIA MYELOFIBROSIS
Bithiah Grace Jaganathan Sebastien Jacquelin1, Jasmin Straube1, Axia Song1, Leanne Cooper1,
Associate Professor, Guwahati, India Therese Vu1, Matthew Heidecker1, John Pimanda2, Luke Hesson3,
Chronic Myeloid Leukemia (CML) is a myeloproliferative disorder initiated by
Geff Hill1, Nicole Cloonan1, Dirk Heckl4, and Steven Lane1
1
QIMR, Brisbane, Australia; 2Lowy Cancer Research Centre, University of New
BCR-ABL fusion in which the leukemic stem cells give rise to abnormally high num-
South Wales Australia, Sydney, Australia; 3Faculty of Medicine, UNSW Sydney,
ber of myeloid cells. Tyrosine kinase inhibitor, Imatinib mesylate (IM) is the main-
Australia, Kensigton, Australia; 4Hannover Medical School, Hannover, Germany
line drug used for the treatment of CML. CML responds to IM treatment efficiently.
However, in several patients, CML relapses even after few years of remission, upon JAK2V617F is present in the majority of patients with myeloproliferative neoplasm (MPN),
discontinuation of IM. Persistent leukemic cells in the BM during IM treatment play however, it is unclear how co-occurring mutations in epigenetic regulators (e.g. DNMT3A)
a major role in causing relapse in CML patients. In the present work, MAPK path- in MPN impact disease biology. DNMT3A methylates DNA at cytosine residues in enriched
ways (ERK, P38), NFkB, STAT pathways (STAT3/5), BMP signaling pathway CpG region, and drives promoter hypermethylation. DNMT3A is mutated in advanced MPN
through SMAD, CXCL12-CXCR-4 signaling axis, actin cytoskeleton and RhoAGT- (15% of MF and 17% of AML). These mutations sit in the methyltransferase domain and result
Pase were examined for their role in CML-stromal interactions and CML chemopro- in reduced activity. To determine the functional effects of Dnmt3a loss in MPN, we used
tection. We found that actin cytoskeleton, RhoA GTPase and BMP-SMAD signaling CRISPR-Cas9 technology to edit Dnmt3a function in Jak2V617F murine hematopoietic
play an important role in establishing direct cell-cell interaction between CML and stem and progenitor cells (HSPC). Jak2V617F LKS+ (Lin-Sca-1highKithigh) HSPCs were in-
stromal cells. This effect could be abrogated by use of small molecule inhibitors fected with a lentivirus encoding Cas9 and sgRNA targeting Dnmt3a (Jak2V617F/DDnmt3a)
for ERK-MAPK and BMP-SMAD signaling as well as actin cytoskeleton disruptions or non-targeting controls (Jak2V617F). In vitro CFU from Jak2V617F/DDnmt3a had
in combination with IM. Moreover, it was observed that as a result of stroma medi- enhanced serial replating and increased expression of markers Kit and Cd34. CFU RNAseq
ated chemoprotection, CML cell line K562 cells could persist long-term in the pres- confirmed Dnmt3a deletion in Jak2V617F/DDnmt3a, as well as transcriptional upregulation
ence of IM when attached to stromal cells. These persistent K562 cells acquired of key stemness genes, and de novo expression of imprinted genes Igf2 and H19, together
stroma-independent chemoresistance and could be maintained in stroma-independent with decreased H19 promoter methylation in Jak2V617F/DDnmt3a compared to controls.
suspension culture in the presence of IM, which resembles a relapse status in the pa- To assess the functional effect of Dnmt3a loss on Jak2V617F driven MPN, we transplanted
tient. The resulting chemoresistant cells showed activation of BCR-ABL independent Jak2V617F/DDnmt3a or Jak2V617F LKS+ into irradiated recipients. Jak2V617F/DDnmt3a
signaling pathways such as ERK and BMP/SMAD pathways and the cells could be became severely pancytopenic with bone marrow failure, fibrosis and the accumulation of
induced to undergo apoptosis by use of small molecule inhibitors against these path- myeloid cells. Jak2V617F/DDnmt3a bone marrow showed osteosclerosis, disorganized archi-
ways. In conclusion, the present study showed that a reciprocal interaction exists be- tecture with dense fibrocellular infiltrate. RNAseq on Jak2V617F/DDnmt3a LKS showed
tween the CML cells and the stromal cells in their microenvironment. The stromal strong enrichment in gene sets previously annotated by Dnmt3a-/- in normal HSPC (Challen,
cells, modified by the leukemic cells, chemoprotected CML cells from IM induced Nat Gen 2011) and oncogenic Dnmt3a R878H (Guryanova,Nat Med 2016), together with LT-
cell death, which further developed into stroma independent chemoresistance which HSC signatures and PRC2 deregulation. This novel model of mutant Dnmt3a, achieved
can be effectively targeted by inhibiting oncogene-independent signaling pathways. through in vivo CRISPR-Cas9 editing, links loss of Dnmt3a with acquisition of self-renewal
and disease progression in Jak2V617F HSPC. Such knowledge has the potential to inform
the development of targeted therapeutic approaches in transformed MPN, a highly chemore-
fractory disease associated with poor prognosis.
S96 Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
3142 - POSTER NUMBER HAS CHANGED TO 3263 3144 - SFRP2 FROM THE NICHE IS REQUIRED TO
MAINTAIN THE REGENERATION OF THE
HEMATOPOIETIC STEM CELL POOL
Rouzanna Istvanffy, Franziska Ruf, Christina Schreck, Sandra Romero,
Franziska Hettler, Christian Peschel, and Robert Oostendorp
3rd Department of Internal Medicine, Klinikum Rechts der Isar der Technischen
Universit€at M€unchen, Munich, Germany
3143 - NICHE WNT5A REGULATES THE ACTIN 3145 - ROLE OF KDM2B, A COMPONENT OF NON-
CYTOSKELETON DURING REGENERATION OF CANONICAL PRC1.1, IN HEMATOPOIESIS
HEMATOPOIETIC STEM CELLS Yusuke Isshiki1, Yaeko Nakajima-Takagi1, Motohiko Oshima1,
Rouzanna Istvanffy1, Christina Schreck1, Christoph Ziegenhein2, Kazumasa Aoyama1, Haruhiko Koseki2, and Atsushi Iwama1
1
Theresa Sippenauer1, Sandra Romero1, Franziska Hettler1, Department of Cellular and Molecular Medicine, Chiba University, Chiba, Japan;
2
Carolina Florian3, Claudia Waskow4, Mareike Essers5, Christian Peschel1, Labolatory for Developmental Genetics, RIKEN Research Center for Integrative
Hartmut Geiger6, Wolfgang Enard2, and Robert Oostendorp1 Medical Science, Yokohama, Japan
1
3rd Department of Internal Medicine, Klinikum Rechts der Isar der Technischen Kdm2b is a component of non-canonical polycomb repressive complex (PRC) 1.1 and re-
Universit€at M€unchen, Munich, Germany; 2Anthropology and Human Genomics, cruits non-canonical PRC1.1 to unmethylated CGI. In contrast to canonical PRCs, the
Department of Biology II, Ludwig-Maximilian-Universit€at, Munich, Germany; function of non-canonical PRCs are still unknown. In order to clarify the role of
3
Institute of Molecular Medicine, University of Ulm, Ulm, Germany; 4Regeneration Kdm2b in hematopoiesis, we performed detailed analysis of mice deficient for Kdm2b
in Hematopoiesis and Animal Models in Hematopoiesis, Institute for Immunology, specifically in hematopoietic cells. We transplanted bone marrow (BM) cells from Cre-
TU Dresden, Dresden, Germany; 5Heidelberg Institute for Stem Cell Technology and ERT;Kdm2bfl/fl mice into lethally irradiated recipient mice. After engraftment, we
Experimental Medicine, Deutsches Krebsforschungszentrum, Heidelberg, Germany, deleted Kdm2b by intraperitoneal injection of tamoxifen 4 weeks after transplantation. Pe-
Heidelberg, Germany; 6Institute of Molecular Medicine and Aging Research Center ripheral blood (PB) analysis revealed that Kdm2b KO mice maintained normal myelopoi-
Ulm, University of Ulm, Munich, Germany esis but showed severe lymphocytopenia of both T and B cell lineages. Kdm2b KO mice
Here, we show that the Wnt5a-haploinsufficient niche regenerates dysfunctional had significantly decreased numbers of hematopoietic stem cells (HSCs), multipotent pro-
HSCs, which do not successfully engraft in secondary recipients. RNA sequencing genitors (MPPs), lymphoid progenitors in the BM compared to wild-type mice. In
of the regenerated donor Lin SCA-1+ KIT+ (LSK) cells shows dysregulated contrast, granulocyte and macrophage progenitors (GMPs) were moderately increased
expression of ZEB1-associated genes involved in the small GTPase-dependent actin in KO mice, suggestive of myeloid-biased hematopoiesis. RNA sequence analysis re-
polymerization pathway. Misexpression of DOCK2, WAVE2, and activation of vealed activation of myeloid gene sets in Kdm2b KO hematopoietic stem and progenitor
CDC42 results in apolar F-actin localization, leading to defects in adhesion, migra- cells (HSPCs) including Cebpa and Cebpε, encoding key myeloid regulators C/EBPa and
tion and homing of HSCs regenerated in a Wnt5a-haploinsufficient microenviron- C/EBPε, respectively, and their downstream target genes. ChIP sequence analysis in an
ment. Moreover, these cells show increased differentiation in vitro, with rapid loss immature hematopoietic cell line EML confirmed the binding of Kdm2b to the promotor
of HSC-enriched LSK cells. Our study further shows that the Wnt5a-haploinsuffi- of Cebpa and Cebpε. These data suggested that Kdm2b restrains premature activation of
cient environment similarly affects BCR-ABLp185 leukemia-initiating cells, which myeloid program in HSPCs to maintain their multi-lineage differentiation potential.
fail to generate leukemia in 42% of the studied recipients, or to transfer leukemia
to secondary hosts. Thus, we show that WNT5A in the bone marrow niche is required
to regenerate HSCs and leukemic cells with functional ability to rearrange the actin
cytoskeleton and engraft successfully.
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136 S97
3146 - CHRONIC VIRAL INFECTIONS INDUCE MAJOR From 800 miRNAs evaluated in this study, there were 37 miRNAs with significantly
DISRUPTION OF BONE MARROW STROMAL CELL differentially expressed in APL patients. 26 miRNAs were found to be up regulated
NETWORKS AND PERSISTENT LOSS OF and 11 miRNAs were identified as down regulated. The miR-181 family, which in-
cludes miR-181a, miR-181b and miR-181c had been found significantly up regulated
HEMATOPOIETIC STEM CELL FUNCTION
in APL patients. More interestingly, its expression sharply decreased after ATRA
Stephan Isringhausen1, Nike Kr€autler2, Ute Suessbier1, Patrick Helbling1, treatment. mRNAs expression profiles revealed the most differentially expressed
Alvaro Gomariz Carillo1, Larisa Kovtonyuk1, Wong Hui Chyn1, genes in APL patients were found in Notch signalling pathway, followed by tran-
Markus Manz1, Annette Oxenius2, and Cesar Nombela-Arrieta1 scriptional regulation, MAPK, apoptosis, and RAS signalling pathways. The heat
1
University of Zurich, Zurich, Switzerland; 2ETH Zurich, Zurich, Switzerland map of directed global significance score showed that most genes from 13 evaluated
canonical pathways were under expressed in APL patients compared to the normal
Hematopoiesis is a highly demand-adapted and tightly regulated process that is sus-
samples and post treatment patients, particularly in Notch, Apoptosis, Hedgehog
tained by a rare population of self-renewing, multipotent hematopoietic stem and pro-
and MAPK signalling pathways. The most significantly down regulated gene in
genitor cells (HSPCs) residing in specialized microenvironments within bone marrow
APL patients was TIAM1 gene (p ! 0.001). In conclusion, our findings provide
(BM) cavities. The basic tissue infrastructure of the BM is provided by stromal
some new and meaningful candidate miRNAs and mRNAs to highlight important
cellular networks of mesenchymal, neural and vascular origin, which are critically
molecular markers for the therapeutic targets of APL.
involved in the fine regulation of different stages of hematopoiesis. Viral infections
act as major stressors to the hematopoietic system, inducing massive and adaptive re-
sponses in cellular output. Albeit the effects of viral challenge and ensuing inflamma-
tory responses on hematopoietic cells have been studied, whether viral infections
alter BM stromal scaffolds and thus shape hematopoietic responses remains poorly
defined. By combining conventional in vitro and in vivo assays with 3D confocal im-
aging, we herein investigated the structural and functional alterations imposed on the
BM after a chronic infection with Lymphocytic Choriomeningitis Virus (LCMV).
Our data shows that chronic LCMV infections trigger a substantial loss of BM endo-
thelial and mesenchymal CXCL12-abundant reticular cells. Upon viral challenge,
conspicuous vasodilation of BM sinusoidal vessels is induced which was followed
by intense vascular remodeling and substantial disruption of extracellular matrix net-
works throughout the BM cavity. Major damage to BM stromal integrity is accom-
panied by a profound and sustained reduction in the number of both HSPCs as
well as hematopoietic stem cells by phenotype. Competitive repopulation assays re-
vealed that the remaining HSCs are additionally impaired in their repopulation capac-
ity for prolonged times after LCMV infection. Finally, our results indicate that the
observed alterations in the composition and functionality of cells in the BM are medi-
ated by virus-specific CD8 T cells and partly dependent on the production of specific
cytokines. Our observations thus indicate that chronic infections result in persistent
damage to HSPC function, which might be partially explained by an impaired regu-
latory function of the stromal compartment of the BM.
upregulated in CD9+ HSCs upon Thpo signaling. Thpo stimulated CD9+ HSCs ex- 3150 - FUBP1 PROMOTES LEUKEMIA PROGRESSION BY
hibited increase of phosphorylated-STAT3 (S727) which is known to translocate to REGULATION OF CELL CYCLE AND APOPTOSIS
and activate mitochondria metabolism. Our data indicate that Thpo-mediated exit Van T. Hoang1, Katharina Gerlach1, Uta Kuller-M€uller2,
from cell cycle quiescence is coupled with emergence of Mk-biased HSCs. While
Eva Weissenberger1, Daniela Krause1, and Martin Z€ornig1
facilitating cell cycle proliferation, Thpo signaling metabolically fortifies Mk-biased 1
Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy,
HSCs through mitochondrial activation, enabling them fit for Mk production. This
Frankfurt, Germany; 2German Red Cross Blood Donor Service Baden-Wuerttemberg –
suggests clonal expansion of HSCs which are metabolically adjusted for Mk-differ-
Hessen, Frankfurt, Germany
entiation during stress hematopoietic states.
We have shown the important role of FUBP1 (Far Upstream Element Binding Protein 1)
in the maintenance of hematopoietic stem cells (HSCs) (Rabenhorst, Thalheimer et al.
2015). Fubp1-/- gene trap mice died in utero at day E15.5 with reduced numbers of total
fetal liver cells, and their HSCs failed to engraft long-term. In adult mice, FUBP1 is also
required for HSC self-renewal, and it promotes cell proliferation and inhibits apoptosis
through regulation of target gene expression (e.g. c-myc, cyclin D2, p21, p27). We
then decided to investigate a potential function of the molecule in chronic (CML) and
acute myeloid leukemia (AML). Eppert et al. demonstrated an increased expression of
FUBP1 in CD34+ CD38- cells compared to total bone marrow (BM) derived from human
primary AML in their microarray analysis (Eppert, Takenaka et al. 2011). To study its
role in leukemia development and leukemia initiating cells, and to test a potential treat-
ment of leukemia with FUBP1 inhibitors, we employed the retroviral transduction/trans-
plantation mouse model of CML and AML induced by BCR-ABL1 and MLL-AF9,
respectively. Mice that received BM transduced with the oncogene plus Fubp1 shRNA
survived longer than the control mice. BCR-ABL+ Fubp1 shRNA+ CML cell populations
including total BM, Lin- and LSK cells showed more apoptosis and less proliferation.
Furthermore, pharmacological inhibition of FUBP1 improved the survival of AML
mice significantly, even to a larger extent than Ara-C treatment. Next, we analyzed
FUBP1 expression in biopsies derived from CML (n511) and AML patients (n572)
by immunohistochemistry and compared with healthy BM (n58). Of note, no enhanced
FUBP1 expression was observed in leukemic samples. However, we noticed a shorter
overall survival in AML patients with strong FUBP1 expression compared to those
with no or moderate expression, suggesting that FUBP1 levels might correlate with
the aggressiveness of leukemia. In summary, our data suggest that FUBP1 acts as a
cell cycle regulator and anti-apoptotic factor in leukemia, similar to its oncogenic role
in solid tumors, and the protein can be a potential target for therapeutic purpose.
3149 - FOXP2 IS ESSENTIAL FOR THE QUIESCENT STATE 3151 - THE DUAL FUNCTION OF LMO2 IN DRIVING
AND SELF-RENEWAL CAPACITY OF HEMATOPOIETIC ERYTHROID CELL FATE
STEM CELLS Trang Hoang1,2, Marie-Claude Sincennes3, Veronique Lisi4,
Kentaro Hosokawa, Saki Morimoto, Yuya Kunisaki, Tomoko Hyoda, Diogo Veiga5, Magali Humbert6, Francois Major7, Bachir Affar8, and
Yasufumi Uehara, Haruka Imanishi, and Fumio Arai Alain Verreault7
1
Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan Institute for Research in Immunology and Cancer, Montreal, Canada; 2Montreal,
Canada; 3University of Ottawa, Ottawa, Canada; 4University of California, Santa
Cell cycle quiescence is crucial for maintenance of the hematopoietic stem cells Barbara, Santa Barbara, United States; 5Jackson Genomics Institute, Farmington,
(HSCs) activity. Activation of the cell cycle and continued proliferation reduced United States; 6Bern University, Bern, Switzerland; 7IRIC, University of Montreal,
the self-renewal capacity and eventually resulted in the reduction of HSC pool. In Montreal, Canada; 8University of Montreal, Montreal, Canada
this study, we found that Foxp2 – a forkhead transcription factor which tightly asso-
ciated with the speech and language development – specifically expressed in the Erythroid precursors and pro-erythroblasts are among the highest proliferative cells in the
quiescent long-term (LT)-HSCs. It is reported that Foxp2 is the responsible gene bone marrow. In the erythroid lineage, LMO2 protein levels are highest in pro-erythroblasts
of the inherited speech and language disorder while the role of Foxp2 in the regula- and progressively decrease with maturation through four stages of differentiation to ortho-
tion of bone marrow (BM) HSCs is poorly understood. We then examined the func- chromatic erythroid cells. Interestingly, this correlates with decreased proportion of cells
tion of Foxp2 in the maintenance of HSCs. First, we analyzed the function of Foxp2 in S/G2/M from 52% to 25%. Moreover, decreasing LMO2 levels in pro-erythroblasts
in the maintenance of colony formation capacity of HSCs. Knockdown of Foxp2 in was sufficient to lower the proportion of cycling cells to 25%, indicating that LMO2 levels
LT-HSCs decreased the number of colony forming unit in culture (CFU-C) while control their high cycling status. LMO2, a LIM only domain protein, does not bind DNA but
overexpression of Foxp2 increased CFU-C formation. Notably, overexpression of interacts with the SCL and GATA1 transcription factors to drive erythroid gene expression
Foxp2 in HSCs increased the number of immature colonies (CFU-GEMM) compared program. The proteomics of the SCL-LMO2 pentameric complex formed on DNA indicate
with the control. We next examined the effect of the overexpression and knockdown that LMO2 is stoichiometric with SCL tethered on erythroid gene promoters. Nonetheless,
of Foxp2 in LT-HSCs in the long-term reconstitution (LTR) ability of HSCs. We 25% of the nuclear pool of LMO2 was not associated with SCL, indicating that LMO2 has
found that the knockdown of Foxp2 reduced the engraftment of donor HSCs in pe- SCL-independent functions. Through a proteome-wide screen for novel LMO2 interaction
ripheral blood and reduced the number of quiescent HSCs. Conversely, the overex- partners in Kit+Lin- hematopoietic progenitors, we uncovered un unexpected role for LMO2
pression of Foxp2 maintained LTR ability of donor HSCs. To clarify the molecular in directly controlling DNA replication, via interaction with three essential replication en-
mechanism of Foxp2 in the maintenance of HSC function, we analyzed the effect zymes: POLD1, PRIM1 and MCM6. In sharp contrast, SCL does not interact with these en-
of exogenous Foxp2 on the expression of cell cycle-related genes and found that zymes. Moreover, tethering LMO2 to DNA is sufficient to recruit these replication enzymes
Foxp2 overexpression upregulated the expression of Cdkn1a (p21) in HSCs. These to DNA and converts LMO2-bound sequences into origins of replication. Interestingly,
data suggest that Foxp2 contributes the maintenance of the quiescent state of ectopic LMO2 expression interfered with the production of TER119+ cells in the bone
HSCs through the induction of p21. marrow. In contrast, decreasing LMO2 protein levels, either via short hairpin RNA or ectopic
expression of miR-223, was sufficient to decrease the proliferative status of proerythroblasts
and favor their differentiation. We conclude that LMO2 controls cell fate in the erythroid
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136 S99
lineage via two mechanisms. First, LMO2 controls the expression of erythroid genes through 3153 - METABOLIC PROFILE OF LEUKEMIC CELLS
a well characterized interaction with the SCL complex. Second, LMO2 interacts with the INFLUENCES RESPONSE TO CURRENT THERAPY
DNA replication complex that drives entry into S phase in erythroid precursors and prevents Katerina Hlozkova1, Alena Pecinova2, David Pajuelo Reguera2,
commitment to terminal erythroid differentiation.
Marketa Simcikova1, Marketa Zaliova1, Natividad Alquezar Artieda1,
Alena Musilova1, Tomas Mracek2, Jan Trka1, and Julia Starkova1
1
CLIP - Childhood Leukemia Investigation Prague, Department of Pediatric Hematology
and Oncology, Second Faculty of Medicine, Charles University, Prague, Czech Republic;
2
Institute of Physiology, the Czech Academy of Sciences, Prague, Czech Republic
Bmi1 has been reported as a master regulator of several types of stem cells, including
hematopoietic stem cells (HSCs). In addition, Bmi1 has been found overexpressed in
several hematologic tumours such as lymphomas, leukaemia or myelodysplastic syn-
dromes, where its high expression also confers worse prognosis, although the causal-
ity of these observations is to a large extent lacking. To date, most studies exploring
the normal role of Bmi-1 have utilized loss-of-function models. Here, we developed a
novel and inducible gain-of-function Bmi1 model. While Bmi1 overexpression alone
does not appear to cause any hematopoietic abnormalities, long-term analysis of seri-
ally transplanted HSCs suggests intrinsic effects of prolonged Bmi-1 expression on
hematopoietic output. Continuous work aims to unravel the effect of Bmi1 overex-
pressing HSC in the settings of additional/secondary stressors.
S100 Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
3155 - DECLINED PRESENTATION component of the hematopoietic stem cell (HSC) niche. We have extended these ob-
LOSS OF TRNA-METHYLATION AT CYTOSINE 38 BY servations demonstrating CD41/CD61bright and CD41/CD61dim megakaryocytes
DNMT2 INDUCES A HEMATOPOIETIC STEM CELL differentially regulate HSC proliferation in vitro. With the essential physiological
roles megakaryocytes and platelets play, better understanding the regulation of meg-
AGING-LIKE PHENOTYPE
akaryocytopoiesis, as well as how different subfractions of megakaryocytes regulate
Friederike Herbst1,2, Francesca Tuorto1, Tim Kindinger1,2, Frank Lyko1, HSCs will lead to a better understanding of how HSCs can be manipulated for ther-
and Hanno Glimm1,2 apeutic benefit.
1
German Cancer Research Center (DKFZ), Heidelberg, Germany; 2National Center
for Tumour Diseases (NCT), Heidelberg, Germany
During hematopoietic aging, stem cells undergo a variety of molecular changes leading
to genomic instability, epigenetic alterations and stem cell exhaustion. Within the last
years, it has been reported that the loss of proteostasis also contributes to the develop-
ment of age-related diseases. We have shown previously that the t-RNA methyltransfer-
ase Dnmt2 modifies several tRNAs at cytosine 38 and that Dnmt2-deficiency leads to a
reduced bone marrow cellularity in newborn mice, which is accompanied by a
decreased LSK cell population and a cell-autonomous differentiation defect compared
to wildtype (wt) littermates. As serial BM transplantation experiments revealed reduced
engraftment efficiency of young Dnmt2-/- BM in 3 recipients with reduced lymphoid
lineage output, we hypothesized that Dnmt2 plays an important role during hematopoi-
etic aging. To subsequently investigate the function of Dnmt2 in aged HSCs, we
analyzed PB and BM samples of 2 year old Dnmt2-/- (n512) and wt mice (n59).
RBC and PLT counts were slightly increased (1.23-fold and 1.65-fold, respectively)
as well as the proportion of LT-HSCs upon Dnmt2 deficiency (1.22-fold). Interestingly,
the ratio of Dnmt2-/- MPP4 cells, reported to give rise to lymphoid cells, is less
(p50.041) compared to wt, which is in line with a reduced lymphoid (D2-/-: 57.4 6
9.3 [col. 6 SEM]; n55 vs. wt: 97.9 6 22.1; n56) but similar myeloid colony forming
capacity (D2-/-: 82.3 6 5.6 [col. 6 SEM]; n58 vs. wt: 82.8 6 7.5; n59) of old Dnmt2-/-
BM assessed in differentiation assays in vitro. Moreover, aged Dnmt2-/- BM cells
engraft 1.8-fold (p50.016) less in primary recipients (n520) with reduced neutrophil
and lymphoid blood cell counts as well as a delayed RBC and PLT repopulation within
the first eight weeks. Our results suggest that Dnmt2 may play a role during HSC aging
and further research is needed to investigate mistranslation of specific codons by tRNAs
lacking Dnmt2-dependent methylation to elucidate its role in age-related processes.
autologous stem-cell transplantation (Auto-SCT). Therefore, we next analyzed 3159 - EXPLORING ACUTE MYELOID LEUKAEMIA
CD34+ cells mobilized by G-CSF for Auto-SCT in patients with cancers, and de- BIOENERGETICS AND DRUG SENSITIVITY BY
tected rearrangement bands. These results may explain the mechanism of donor-orig- RENDERING ACUTE MYELOID LEUKAEMIA CELL LINES
inated leukemia after SCT or infant MLL-leukemia, both of which are under high
IMPAIRED FOR EITHER GLYCOLYSIS OR OXIDATIVE
cytokine stimulation circumstances. It is a warning against stem cell expansion in vi-
tro and in vivo.
PHOSPHORYLATION
Minhee Halemba
Monash University, Melbourne, Australia
3158 - INVESTIGATING THE ROLE OF BONE MARROW 3160 - HUMAN HAEMATOPOIETIC STEM CELL
STROMA IN THE RESPONSE OF HAEMATOPOIETIC DIFFERENTIATION FOLLOWS A CONTINUOUS
STEM CELLS TO PLASMODIUM BERGHEI INFECTION WADDINGTON-LIKE LANDSCAPE
Myriam Haltalli, Kira Glatzel, Nicola Ruivo, Andrew Blagborough, and Simon Haas1, Lars Velten2, Simon Raffel3, Andreas Trumpp1,3,
Cristina Lo Celso Marieke Essers4, and Lars Steinmetz4
1
Imperial College London, London, United Kingdom HI-STEM / German Cancer Research Center, Heidelberg, Germany; 2EMBL,
Heidelberg, Germany; 3DKFZ, Heidelberg, Germany; 4Stanford / EMBL, Palo Alto,
Haematopoietic stem cells (HSCs) maintain the turnover of all blood cell lineages United States
and reside within the bone marrow (BM), where they are critically dependant on in-
teractions with complex and specialised niches. Infection can have profound effects Blood formation is believed to occur through step-wise progression of haemato-
on haematopoiesis. HSCs tend to adapt their progeny output to promptly cope with poietic stem cells (HSCs) following a tree-like hierarchy of oligo-, bi- and unipotent
the increased need for immune cells, which often leads to exhaustion of the stem progenitors. However, this model is based on the analysis of predefined flow-sorted
cell pool. It has been shown that chronic infections result in a loss of HSC function cell populations. Here, we mapped human bone marrow hematopoiesis by quantita-
(1), whereas acute infections induce more transient perturbations (2). Questions tively integrating flow cytometric, transcriptomic and functional lineage fate data at
remain about how the HSC response contributes to stressed haematopoiesis and the single-cell level to map early differentiation of human HSCs towards lineage
whether it may affect HSC function in the long-term; how the transition from stem commitment. We found that individual HSCs do not pass through discrete intermedi-
cell to progenitor cell changes in an infected setting; and whether the changes ate progenitor cell stages. In contrast, HSC lineage commitment occurs in a gradual
observed are mediated by alterations in the BM niche. Using a mouse model of ma- manner best described by a continuous Waddington landscape with initially flat but
laria, based on the natural route of sporozoite inoculation by the bites of mosquitos progressively deepening valleys. Our data determine a detailed model of develop-
infected with Plasmodium berghei, we are able to investigate these questions and gain mental trajectories within this landscape and demonstrate that distinct gene expres-
an insight into haematopoietic perturbations as infection progresses. We have previ- sion modules operate in a combinatorial manner to control stemness, early lineage
ously shown that P. berghei infection has significant effects on progenitor cells, while priming and the subsequent progression into all major branches of haematopoiesis.
HSC numbers remain largely unchanged (3). Here I demonstrate that HSC function is These results establish the concept of a developmental continuum, which can replace
maintained through infection, using BM transplantation assays, and this is despite the the ‘differentiation tree’ as a comprehensive model of hematopoiesis.
fact that their proliferation is dramatically altered - as early as the liver stage of infec-
tion, before parasitemia is detectable in the blood. I hypothesise that this may be
mediated by profound alterations of the BM microenvironment and use intravital
confocal 2-photon imaging of calvarium BM in control and infected mice to visualise
the effects of P. berghei on various niche components.
S102 Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
3161 - CELL CYCLE PROGRESSION AND FATE DECISIONS human AML in the TCGA dataset, suggesting that TNF signaling may similarly
IN HEMATOPOIETIC STEM CELLS disrupt HSC function in miR-146a-depleted MDS and AML.
Tatyana Grinenko1, Anne Eugster2, Lars Thielecke3, Ingmar Glauche3,
Onur Basak4, Hans Clever4, Triantafyllos Chavakis1, and Ben Wielockx1
1
TU Dresden, Dresden, Germany; 2CRTD Dresden, Dresden, Germany; 3IMB TU
Dresden, Dresden, Germany; 4Hubrecht Institute, Utrecht, The Netherlands
Hematopoietic stem cells (HSCs) continuously replenish all blood cell types through
a series of differentiation steps that involve the generation of lineage-committed pro-
genitors and necessary expansion due to repeated cell divisions. However, it is un-
clear whether cell division in HSCs precedes differentiation. To address this, we
used an HSC cell tracing approach and Ki67RFP knock-in mice to simultaneously
assess the divisional history, cell cycle progression, and differentiation of adult
HSCs in vivo. Our results reveal that HSCs are able to differentiate into restricted pro-
genitors, especially restricted megakaryocyte-erythroid progenitors (PreMEs) and
pre-megakaryocyte progenitors (PreMegs), without undergoing cell division and
even before entering the S phase of the cell cycle. Additionally, the phenotype of
the undivided but differentiated progenitors correlated with the expression of line-
age-specific genes that manifested as functional differences between HSCs and Pre-
Megs. Thus, HSC fate decisions appear to be uncoupled from physical cell division.
These findings, together with those made in embryonic stem cells, strongly implicate
the presence of a fundamental and conserved mechanism shared by pluripotent and
multipotent stem cells. Our results will facilitate a better understanding of the mech-
anisms that control fate decisions in hematopoietic cells.
3164 - INVOLVEMENT OF THE VASCULAR NICHE IN THE ing us to speculate about the plausibility of the particular assumptions. Our results
REGULATION OF MURINE CHRONIC MYELOID highlight the necessity to understand the mechanisms of immune control in leukemia
LEUKEMIA therapy to employ this effect for optimal treatment results and to furthermore identify
the clinically accessible measures that allow to derive better predictions about the
Sonika Godavarthy1, Stefanie Herkt2, Eva Weissenberger1,
outcome of treatment cessation.
Djamel Aggoune1, Kuan-Ting Pan3, Thomas Oellerich4, Vivian Oehler5,
Richard Van Etten6, Joern Lausen7, and Daniela Krause1
1
Georg-Speyer-Haus, Frankfurt, Germany; 2Institute for Transfusion Medicine DRK-
Blutspendedienst Baden-Wurttemburg-Hessen, Frankfurt, Germany; 3Bioanalytical
mass spectrometry group, Max Planck Institute for Biophysical Chemistry, Frankfurt,
Germany; 4Department of Internal Medicine, Haematology/Oncology, University
Hospital Frankfurt, Frankfurt, Germany; 5Fred Hutchinson Cancer Research Centre,
Seattle, WA, USA, Washington, United States; 6Fred Hutchinson Cancer Research
Centre, Seattle, WA, USA, California, United States; 7Institute for Transfusion
Medicine, DRK- Blutspendedienst Baden-W€urttemberg-Hessen, Frankfurt, Germany
The endosteal bone marrow niche is known to protect leukemic stem cells (LSC)
from chemotherapy in acute myeloid leukemia, but the knowledge about the role
of the vascular niche in the bone marrow microenvironment in leukemia is limited.
E-selectin, which is expressed on bone marrow endothelium, has been described to
regulate the dormancy of normal hematopoietic stem cells. In chronic myeloid leuke-
mia (CML) E-selectin and the adhesion molecule CD44, a ligand for E-selectin,
which is overexpressed on CML-initiating cells, was shown to be responsible for
homing and engraftment of LSC in a murine model of CML. However, exact mech-
anisms of the interactions between BCR-ABL1, the causative oncogene in CML,
CD44 expression and interactions with the vascular niche are unclear. Hypothesizing
that E-selectin influences the LSC cell cycle similar to HSC we treated murine recip-
ients of CML-initiating cells in the retroviral transduction/transplantation model with
the E -selectin inhibitor GMI-1271 and imatinib, considered standard of care in
CML. This increased the survival of mice compared to animals treated with imatinib
alone and decreased the engraftment of CML-initiating cells in this model, as well as
in a xenotransplantation model of NOD SCID IL2 receptor knockout (NSG) mice in-
jected with human CML cells. Further, treatment with GMI-1271 in vitro increased
the cell cycle of LSC, with a concomitant increase in expression of the SCL/TAL1
gene, a transcriptional regulator. Tal1 was shown to negatively regulate CD44 expres-
sion, which was potentiated by imatinib. Downstream, imatinib reduced the AKT-
mediated phosphorylation of Tal1 (T90 and S122), which are known negative regu-
latory phosphorylation sites of Tal1 activity. In summary, inhibition of E-selectin in
murine CML may lead to ‘non-adherence’ of LSC to the vascular niche and improved
eradication by imatinib, as well as reduced engraftment of LSC, likely via an increase
in cell cycle and an increase of Tal1 expression, which is shown to be regulated by
BCR-ABL1. These data connect the role of CD44 for engraftment of LSC in CML
with its regulation by Tal1 and BCR-ABL1 and the biology of the vascular niche
in CML.
hierarchical decision trees in real-world settings. Our analysis is a necessary and 3168 - TRACING THE EMERGENCE OF HEMATOPOIETIC
complementary prerequisite to support experimental approaches for the reconstruc- STEM CELLS FROM THE EXTRA-EMBRYONIC YOLK
tion of hierarchical, hematopoietic decision trees. In particular, the modeling SAC
approach allows, based on the available data, to estimate whether certain decision
Yasamine Ghorbanian1, Lydia Lee2, Hanna Mikkola2, and Matthew Inlay1
processes can be uniquely reconstructed or not. 1
University of California, Irvine, Irvine, United States; 2University of California, Los
Angeles, Los Angeles, United States
While iPSCs offer the potential for an unlimited source of patient HSCs, the gener-
ation of transplantable HSCs from iPSCs has yet to be achieved. This suggests that a
better understanding of how HSCs arise naturally in the course of embryonic devel-
opment is needed. The first primitive waves of hematopoiesis arise from the embry-
onic yolk sac (YS), but definitive waves arise later and produce the first HSCs.
Which tissues produce definitive hematopoiesis, and thereby HSCs, remains unclear.
The aorta-gonad-mesonephros (AGM), placenta, YS, and vitelline vessels are all po-
tential sources. Using a Lyve1-Cre lineage tracing reporter, we previously published
with Dr. Hanna Mikkola’s lab that Lyve1-Cre marks definitive hematopoietic cells
that arise primarily in the YS, with some labeling in the vitelline vessels, and
show that about one-third of adult HSCs are derived from Lyve1-expressing precur-
sors. Our central hypothesis is that the extra-embryonic YS is a significant source of
adult HSCs. We have found that Lyve1-derived cells contribute to each definitive
wave of embryonic hematopoiesis. Lyve1-marked pre-HSCs from the early embryo
are transplantable and give rise to adult HSCs. Lyve1-derived adult HSCs contribute
to cells in all of the major blood lineages. Interestingly, there seems to be a bias in
the development of lymphoid cells, suggesting that Lyve1-derived HSCs have func-
tional properties distinct from HSCs that arise from other tissues. Taken together,
this data suggests that Lyve1-derived cells give rise to engraftable, multipotent,
and long term self-renewing HSCs. We interpret these results to support the notion
that the extra-embryonic YS is an HSC nice in the embryo and contributes to adult
hematopoiesis.
3167 - THE ROLE OF THE GATA2 TRANSCRIPTIONAL 3169 - THE ROLE OF FUBP1 IN HEMATOPOIETIC STEM
PROGRAM IN THE DEVELOPMENT OF ACUTE MYELOID CELLS AND DURING ERYTHROID MATURATION
LEUKEMIA Katharina Gerlach1, Josephine Wesely2, Marlene Steiner1,
Emanuele Gioacchino1, Emma de Pater2, Ivo Touw1, Hans de Looper1, Michael Rieger3, and Martin Z€ornig1
1
Paulette Strien1, Remco Hoogenboezem1, Eric Bindels1, Elaine Dzierzak3, Georg-Speyer-Haus, Frankfurt, Germany; 2Icahn School of Medicine at Mount
and Polynikis Kaimakis4 Sinai, New York, United States; 3University Hospital Frankfurt, Frankfurt, Germany
1
Erasmus MC, Rotterdam (NL), The Netherlands; 2Erasmus MC, Rotterdam,
We previously found out that the transcriptional regulator and established activator of
Netherlands; 3Edinburgh Medical School, Edinburgh, United Kingdom; 4Centro de
c-myc gene transcription, FUSE-binding protein 1 (FUBP1), functions as an impor-
Biologıa Molecular Severo Ochoa, Madrid, Spain
tant oncoprotein in hepatocellular carcinoma (HCC). In addition, two different
Myeloid dysplastic syndrome (MDS) and acute myeloid leukemia (AML) are aggres- gene trap mouse models with impaired FUBP1 function and prospectively isolated
sive forms of blood cancer that are still fatal in more than 50% of patients. Familial murine Fubp1 knockdown hematopoietic stem cells (HSCs) were analyzed to unravel
forms of MDS/AML have very poor cure rates. Germline mutations in GATA2, re- the physiological function of FUBP1. The results of serial competitive transplantation
sulting in haploinsufficiency of the transcription factor GATA2 (Gata2 in animals) experiments, ex vivo single cell tracking and quantitative real-time PCR analyses led
is the most commonly mutated gene in childhood MDS/AML. Between 36-71% of us to conclude that FUBP1 fulfills an essential role in LT-HSC self-renewal by pro-
patients with germline mutations in GATA2 develop AML. Although the disease moting survival and proliferation of HSCs via the regulation of gene expression. We
initiating mutation is known, it is unclear how GATA2 haploinsufficiency predisposes then addressed the question whether FUBP1 also influences the differentiation capac-
to leukemia and contributes to the progression to MDS/AML. Because GATA2 is ity of hematopoietic progenitor cells. Indeed, flow cytometric analyses of FUBP1-
pivotal for hematopoietic stem cell (HSC) generation in the embryo, the disease- deficient fetal liver cells and Fubp1 knockout embryonic stem cell clones revealed
driving defects likely already affect the hematopoietic system during fetal develop- an impaired terminal erythroid maturation. This finding corresponds with the severe
ment. Therefore, we hypothesize that the defect in the hematopoietic system of pa- anemia observed in mouse embryos lacking functional FUBP1 and might explain
tients with a GATA2 haploinsufficiency in newly generated HSCs or in amplifying their lethality at day E15.5 of embryonic development. Our findings suggest at least
HSCs contributes to leukemia predisposition. We have performed RNA sequencing two functions for FUBP1 in the hematopoietic system as an HSC self-renewal factor
on WT and Gata2+/- mouse embryonic and adult phenotypic HSCs and found that and as a transcriptional regulator of erythroid maturation. We continue to analyze the
Gata2 transcriptional levels normalize during HSC development. Despite this regulatory transcriptional network that is controlled by FUBP1 in HSCs and during
normalization, the transcriptome of WT and Gata2+/- adult bone marrow HSCs is erythroid maturation. The ongoing establishment of a conditional Fubp1 knockout
still transcriptionally different. This points to an underlying epigenetic reprogram- mouse line will provide a valuable tool for our current studies.
ming during the embryonic stages. We have found that cell cycle and metabolic sig-
natures are different in embryonic HSCs compared to WT. We will further dissect
these phenotypes in the zebrafish model in which we have generated a knockout of
gata2b and several reporter lines to study these processes in vivo.
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136 S105
3170 - THE BULK OF THE HEMATOPOIETIC STEM CELL suggesting HE precursors with distinct functional output or a continuum of maturing
POPULATION IS DISPENSABLE FOR MURINE STEADY- HE. In sum, HE is recruited from the endothelium beginning at E8.5 and abruptly
STATE AND STRESS HEMATOPOIESIS ceases by E10.5. Whether this reflects an active suppression of HE recruitment or
the passive loss of a specification niche as the embryo develops remains to be
Alexander Gerbaulet1, Kristina Schoedel2, Mina Morcos2,
determined.
Thomas Zerjatke3, Ingo Roeder3, Tatyana Grinenko4, David Voehringer5,
othert6, Claudia Waskow7, and Axel Roers2
Joachim G€
1
Institute for Immunology, Dresden, Germany; 2Institute for Immunology, Medical
Faculty TU Dresden, Dresden, Germany; 3Institute for Medical Informatics and
Biometry, Medical Faculty TU Dresden, Dresden, Germany; 4Institute of Clinical
Chemistry and Laboratory Medicine, Medical Faculty TU Dresden, Dresden,
Germany; 5Department of Infection Biology, University Hospital Erlangen, Erlangen,
Germany; 6Department of Hematology, University Hospital of Essen, Germany,
Essen, Germany; 7Regeneration in Hematopoiesis, Institute for Immunology,
Medical Faculty TU Dresden, Dresden, Germany
Long term repopulating (LT-) hematopoietic stem cells (HSCs) are the most potent
cells at the top of the hematopoietic hierarchy. The regulation of HSC pool size
and its contribution to hematopoiesis is still under debate. We depleted hematopoietic
stem and progenitor cells (HSPCs) in adult mice in situ and found that LT-HSCs
recovered from initially very low levels (! 1%) to below 10% of normal numbers
but not more, while progenitor cells substantially recovered shortly after depletion.
In spite of the persistent and massive reduction of LT-HSCs, steady-state hematopoi-
esis was unaffected for at least 2 years and residual HSCs remained quiescent. He-
matopoietic stress, although reported to recruit quiescent HSCs into cycle, was
well tolerated by HSPC-depleted mice and did not induce expansion of the small
LT-HSC compartment. Only upon 5-Fluorouracil treatment, HSPC-depleted bone
marrow was compromised in re-constituting hematopoiesis, demonstrating that
HSCs and early progenitors are crucial to compensate myeloablation. Hence, a con-
tracted HSC compartment cannot recover in situ to its original size and normal
steady-state blood cell generation is sustained with less than 10% of normal LT-
HSC numbers without increased contribution of the few residual cells.
correlation coefficient R 5 0.98 across all cell types and a mean absolute deviation 3174 - INFLAMMATORY REGULATION OF
(MAD) of only 3.1%. Furthermore, we describe a new method for absolute quantifi- HEMATOPOIESIS IN THE NORMAL AND
cation of cell numbers that is based on a non-methylated reference DNA. Our ‘‘Epi- HYPERGLYCEMIC VERTEBRATE EMBRYO
Blood-Count’’ is applicable to frozen samples, it yields robust results even after long-
Jenna Frame, Virginie Esain, Sung-Eun Lim, and Trista North
term storage, and analysis requires only very small volumes of blood. This approach
Beth Israel Deaconess Medical Center, Boston, United States
may revolutionize leukocyte differential counts and facilitate more standardized and
cost effective analysis of blood counts in clinical application. Hematopoietic stem cells (HSCs) arise in the embryonic aorta through a Runx1-dependent
process of endothelial-to-hematopoietic transition. Once formed, HSCs colonize secondary
niches, where they proliferate and differentiate to maintain lifelong hematopoiesis. Although
many intrinsic factors regulating HSCs have been revealed, there is an emerging role for cell-
extrinsic regulation of HSC formation and long-term function. We previously reported that
acute exposure to excess glucose accelerated the onset and magnitude of HSC formation in
the zebrafish embryo. Additionally, we, and others, recently demonstrated that pro-inflam-
matory cytokines stimulate HSC production across vertebrates. Here, we explored the hy-
pothesis that hyperglycemia augments inflammatory signaling during HSC formation to
influence output and cell fate. In support of this possibility, exposure to elevated glucose
levels during HSC specification (12-36 hours post fertilization (hpf)) increased expression
of several pro-inflammatory cytokines and receptors; morpholino-mediated knockdown of
ifng and il1b each blunted the inductive effects of glucose on runx1 expression. As Il1b pro-
cessing is regulated by inflammasome activity, we treated embryos with inflammasome ac-
tivators, and found that they also increased runx1/cmyb expression and numbers of
functional HSCs, while loss of the inflammasome adapter protein pycard reduced HSC
numbers. We next examined the effects of prolonged glucose exposure, and found that treat-
ment from 24-120hpf robustly increased expression of pro-inflammatory cytokines. Interest-
ingly, glucose exposure during maturation and seeding of secondary organs (72-120hpf)
increased mpo expression and Mpo+ myeloid cells enumerated by flow cytometry;
conversely, rag1 expression was reduced in the thymus, suggesting altered HSC function
or maintenance. Investigation of the mechanisms linking prolonged hyperglycemia and
inflammation implicates both inflammasome activation and insulin signaling in promoting
lineage skewing. As children of diabetic mothers exhibit a higher risk of leukemia, under-
standing the consequences of metabolic alterations on HSCs is of clinical interest.
3173 - REDUCED EXPRESSION OF GFI1 CAUSES A FATAL 3175 - ESTABLISHMENT AND UTILIZATION OF A
MYELOPROLIFERATIVE DISEASE BY SIMULTANEOUSLY PLURIPOTENT CD34 GFP REPORTER STEM CELL LINE
BLOCKING MYELOID DIFFERENTIATION AND P53 Klaus Fortschegger1, Anna-Maria Husa1, Maria Strobl1, Regina Grillari2,
MEDIATED APOPTOSIS and Sabine Strehl3
1
Jennifer Fraszczak1, Riyan Chen2, Marissa Rashkovan2, CCRI, Children’s Cancer Research Institute, St. Anna Kinderkrebsforschung,
Charles Vadnais2, Cyrus Khandanpour3, and Tarik Moroy2 Vienna, Austria; 2Evercyte GmbH, Vienna, Austria; 3Children’s Cancer Research
1
Institut de recherches cliniques de Montreal, Montreal, Canada; 2IRCM, Montreal, Institute, St. Anna Kinderkrebsforschung, Vienna, Austria
Canada; 3Universit€atsklinikum Essen, Essen, Germany
Directed in vitro differentiation of human induced pluripotent stem cells (hiPSCs) to-
Growth Factor Independent 1 (Gfi1) is a transcriptional repressor that regulates he- wards hematopoietic stem cells and their progeny holds great promise for disease
matopoietic stem cells (HSC) and both early lymphoid and myeloid development. modeling, drug screening, and, eventually, cell therapy approaches. In order to allow
Gfi1 knockout (Gfi1 KO) mice have defects in myelopoiesis in particular lack of neu- for monitoring of early steps of hemato-endothelial development, we established a fluo-
trophils and accumulation of monocytes and monocytic precursors in the bone rescent reporter cell line for the widely used stem cell and progenitor marker CD34. Em-
marrow (BM). We report now that unlike Gfi1 KO mice, mice expressing a low level ploying the CRISPR/Cas9 gene editing system, we introduced an internal ribosome
of Gfi1 (Gfi1 KD) develop spontaneously a fatal myeloproliferative disease (MPD) entry site (IRES) followed by the coding sequence for enhanced green fluorescent pro-
resembling leukemia when the mice are getting old, predisposing KD mice to tein (GFP) containing a nuclear localization signal, and an independent floxed puromy-
myeloid leukemia after additional mutations. KD mice present a similar block of cin resistance cassette into the 3’ untranslated region of CD34. After removal of the
myeloid cell differentiation as the KO mice but KD BM cells have a better reconsti- selection marker by transient Cre recombinase expression, we obtained isogenic hiPSC
tution capacity and survival due to a lower p53 activity. We found that a reduced clones harboring the IRES-GFP insertion in one of the two CD34 alleles. The reporter
expression of p53 in KO mice lead to the emergence of MPD with the same latency hiPSCs displayed a normal karyotype and retained the expression profile typical of plu-
than observed in KD mice confirming the role of p53 in the disease severity. Thus it ripotency. Upon differentiation of the reporter hiPSCs in Albumin Polyvinylalcohol
suggests that a knockout, but not a knockdown of Gfi1 initiates a p53 dependent cell Essential Lipids (APEL) medium supplemented with appropriate growth factors and cy-
death in myeloid cells and prevents the emergence of myeloid disease. In parallel, we tokines, a significant fraction of the emerging cells began to express GFP, most notably if
also found that the metabolism especially pathways related to radical oxygen specie a short term inhibition of Glycogen Synthase Kinase 3 (GSK3) using CHIR99021 was
formation, was deregulated in sick KD BM cells suggesting a direct or indirect role of performed. Specific antibody staining of the resulting differentiated cells demonstrated
Gfi1 in metabolism regulation. This metabolism deregulation could increase the concomitant expression of cell surface CD34 and nuclear GFP by confocal microscopy
severity of the disease in the KD by supporting the tumorigenesis potential of the and flow cytometry, confirming the functionality of the reporter. Thus, this reporter line
KD BM cells. now enables time lapse microscopy to track the differentiation process and facilitates
fluorescence-activated cell sorting of CD34+ hemato-endothelial progenitor cells. The
fast readout of this novel cellular tool also allows for a simplified monitoring and opti-
mization of endothelial and hematopoietic differentiation protocols. This project is
funded by the Austrian Research Promotion Agency (FFG #843456).
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136 S107
and autophagic flux. Together all, our data revealed the role of FLCN-TFE3 axis for distributions, as an approximation of cell populations dynamics in ALL. This tool
regulation of both lysosomal and mitochondrial biogenesis, autophagic process and may allow the study of the normal hematopoiesis replacement mediated by microen-
alternative activation in macrophages. Finally, we show the increase in CD11c vironment remodeling driven by leukemic cells.
expression and nuclear translocation of TFE3 in hemophagocytes from HLH (hemo-
phagocytic lymphohistiocytosis) patients, suggesting this axis could be a common
responsible mechanism for HLH.
3184 - A MULTI-MODULAR BOOLEAN NETWORK FOR 3185 - SINGLE-CELL ANALYSES SHOW TGF-B1 REDUCES
THE STUDY OF ACUTE LYMPHOBLASTIC LEUKEMIA SELF-RENEWAL AND MYELOID DIFFERENTIATION
Jennifer Enciso1,2,3, Elena Alvarez-Buylla4
, and Rosana Pelayo1 POTENTIALS IN HEMATOPOIETIC STEM CELLS
Xiaofang Wang1, Fang Dong1, Sen Zhang1, Wanzhu Yang1, Zhao Wang1,
1
Oncology Research Unit, Mexican Institute for Social Security, Mexico City,
Mexico; 2Biochemistry Sciences Program, Universidad Nacional Autonoma de Shanshan Zhang1, Jinhong Wang1, Yun Gao2, Ji Dong2, Fuchou Tang2,
Mexico, Mexico City, Mexico; 3Centro de Ciencias de la Complejidad (C3), Cheng Tao1, and Hideo Ema1
Universidad Nacional Aut, Mexico City, Mexico; 4Centro de Ciencias de la 1
State Key laboratory of Experimental Hematology, Institute of Hematology and
Complejidad (C3), Universidad Nacional Autonoma de Mexico, Mexico City, Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union
Mexico; Departamento de Ecologıa Funcional, Instituto de Ecologıa, Universidad Medical College, Tianjin, China (People’s Republic); 2Biodynamic Optical Imaging
Nacional Aut
onoma de Mexico, Mexico City, Mexico Center, College of Life Sciences, Peking University, Beijing, China (People’s
Cell fate decisions through the hematopoiesis progression depend on intrinsic factors Republic)
and extrinsic signals provided by the bone marrow (BM) microenvironment. Abnor- Aim: Quiescence is important for HSCs to maintain their pool. Transforming growth
malities in external regulatory factors appear to be key contributors of hematological factor-b1 (TGF-b1) is a candidate cytokine to keep HSCs quiescent. HSCs consist of
malignancies, including acute lymphoblastic leukemia (ALL). B-cell precursor ALL multiple subtypes with distinct functional potentials. It has been reported that TGF-
is the most common cause of death of pediatric cancer. Unfortunately, despite all the b1 at a low concentration stimulates proliferation of myeloid-biased HSCs but in-
intense research of the last years, the etiology and leukemia progression have not hibits that of lymphoid-biased HSCs. This study addressed whether TGF-b1 can
been completely solved. Leukemogenesis appear to be an emergent event of deregu- be used to control the quiescence and fate decision of HSCs by single-cell techniques.
lated biological modules involved in determination of cellular processes (prolifera- Methods: Single CD150+CD41-CD34-Lin-Sca-1+c-Kit+ HSCs were cultured in
tion, differentiation or apoptosis), genetic regulatory network of early B SCF and TPO with or without TGF-b1 (10pg/ml to 10ng/ml) for 5 days. The number
lymphopoiesis and intercommunication with the BM microenvironment. Here, we of cells was daily monitored. 10 HSCs were cultured in SCF, TPO, and TGF-b1 (0,
propose the reconstruction of these three modules merged in a complex network 10, 100 pg/ml and 1, 10 ng/ml) for 5 days and transplanted; cultured in SCF, TPO,
incorporating representative molecules in order to generate an in silico tool to under- and TGF-b1 (100 pg/ml) and transplanted on days 3, 5, and 7; cultured in SCF
stand basic processes leading to B-ALL development. The attractors obtained by the and TPO to which TGF-b1 (100 pg/ml) was added on days 0, 3, and 5 and trans-
computational simulations of the network as a boolean model are compatible to in planted on day 7. Single-cell culture of HSCs was performed with 10 and 100 pg/
vivo and in vitro cellular responses to microenvironmental cues in homeostasis and ml TGF-b1 and transplanted on day 5. Single-cell RNA-sequencing was performed
upon TLR–induced emergency settings. Attractors interpretation is targeted by ana- on cultured cells. Results: Single cell culture showed significantly slower division
lyses of cell fate decision nodes (e.g. cell growth arrest, proliferation and apoptosis). from second division and fewer cells by TGF-b1. 10-cell transplantation (8 months)
Moreover, our model is also being simulated with the tool MaBoSS, using Monte- showed that TGF-b1 between 10 pg/ml and 10 ng/ml similarly reduced the reconsti-
Carlo simulations to compute an estimate of the temporal evolution of probability tution levels and the myeloid-outputs, and the effect of TGF-b1 was detected as early
S110 Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
as day 3. Surprisingly, the similar effect of TGF-b1 was also detected when TGF-b1 3187 - IMMUNOSURVEILLANCE DURING THE
was present only on days 5-7, indicating that TGF-b1 similarly suppresses the func- FORMATION AND PROGRESSION OF AML
tion of cycling HSCs throughout culture. Single cell transplantation (6 months) Monika Dudenh€offer-Pfeifer, Amol Ugale, and David Bryder
showed that both total and myeloid reconstitution levels were dramatically reduced Lund University, Lund, Sweden
by 10 and 100 pg/ml TGF-b1. Single-cell RNA-sequencing showed that SCF+TPO
drove HSCs into cycling, while TGF-b1 inhibited their cycling in about half of the Despite progress in the treatment of acute leukemia, specific subtypes still have a
cells. Conclusion: TGF-b1 can let cycling HSCs go back into the quiescent state, very bad prognosis, highlighting the need for increased knowledge that can be har-
but in these quiescent HSCs, the self-renewal potential is reduced and the differenti- nessed to develop novel therapeutic strategies. While experimental data from the
ation potential is biased towards lymphoid lineage. last decade have conclusively established that the immune system can critically influ-
ence on the development of cancer, with a range of immunomodulatory strategies
reaching clinical use, most studies have been conducted using solid cancer models
of non-hematological origin. Such cancers evidently have requirements very distinct
from hematological cancers that pertain to initiation, maintenance and spreading
(metastasis). Also, constraints of experimental models precluding spatiotemporal
control have to a large extent hindered investigations of immunosurveillance and im-
munoediting mechanisms during the multistep progression of acute leukemia. Here,
we make use of a recently described mouse model of acute myeloid leukemia AML
(Ugale et al, 2014) in which physiologically relevant expression levels of the fusion
oncogene MLL-ENL can be conditionally activated in vivo as a leukemic ‘‘first-hit’’.
This model associates with a phase of ‘‘pre-leukemic’’ contraction after which overt
clonal transformation commences, highlighting the critical influence of secondary but
less defined events during the course of disease. By investigating aspects of the im-
mune system during the formation and progression of AML, we show that transfor-
mation follows similar kinetics regardless of developing in wild type (WT) or
immune-deficient (Rag2-/- or Rag2-/gc-/-) environments. By contrast, immunity criti-
cally influences on the propagation of established leukemia, but with little evidence
of primary immune-editing. Rather, intrinsic properties of individual leukemic
clones, regardless of their origin, underlie the aggressiveness of arising disease. Anti-
body-mediated in vivo depletion experiments revealed a critical role for CD8+ cells
in this process. Ongoing investigations focus on analyzing and characterizing CD8+
T lymphocyte subsets during the disease course of AML.
3189 - MARKING OF HUMAN MULTIPOTENT vated. In group B proportion of HLA-DR CD4+ and CD8+ cells was significantly
MESENCHYMAL STROMAL CELLS BY LENTIVIRAL lower, compared to group A and control samples. At the same time the number of
BARCODED LIBRARY REVEALED DYNAMIC CM and PD-1+ CD4+ cells was lower in group A, but number of TE was increased.
These data indicate that MSCs from group A became more immunogenic after inter-
POLYCLONALITY IN THEIR POPULATION THROUGH
action with lymphocytes and could not show immunomodulating properties in the
PASSAGES same way as MSCs from group B. Data obtained can explain why administration
Alexey Bigildeev1, Artem Pilunov2, Natalia Sats1, Nataliya Petinati1, of MSCs is not always successful. Preliminary study of MSCs prior to their admin-
Vadim Surin1, and Nina Drize1 istration may be used to predict their efficiency in the future.
1
National Research Center for Hematology, Moscow, Russia; 2Moscow State
University, Biology dep., Moscow, Russia
maturation into more than 95% GPA+ erythroid cells, iPSC- and ESC-derived RBCs 3193 - TWO HEMATOPOIETIC TRANSCRIPTION
failed in enucleation ( ! 15%) and expressed embryonic and fetal hemoglobin rather FACTORS, RUNX1 AND FUBP1, CONTROL THE
than HbA. Performed DNA methyation analysis allowed for the interrogation of EXPRESSION OF KIT ONCOGENE IN PRE-B
850,000 methylation sites across the genome. Principle component analysis demon-
LYMPHOBLASTS
strates tight clustering of samples derived from identical origin. PB-RBCs clustered
closely with CB-RBCs, although both cell types formed distinct populations. ESC-
Lydie Debaize1,2,3, Helene Jakobczyk4, Anne-Ga€elle Rio4,
RBCs clustered with iPSC-RBCs, both with great distance to CB- and PB-RBCs. Dif- Stephane Avner4, Aurelien A. Serandour5, Frederic Chalmel6,
ferential gene methylation analysis revealed a minority of 16,726 differentially methyl- Gilles Salbert7, Marie-Dominique Galibert8, Virginie Gandemer8, and
ated genes (DMG) bewteen PB-RBCs and CB-RBCs. This is in contrast to 190,000 Marie-Berengere Troadec4
1
DMGs between CB-RBcs and iPSC-RBCs and 228,963 DMGs between PB-RBCs CNRS UMR 6290 - IGDR, Rennes, France; 2Universite de Rennes 1, Rennes,
and iPSC-RBCs. Our preliminary data implicate great differences between cultured France; 3SFR Biosit UMS 3480, Rennes, Rennes, France; 4IGDR UMR 6290 CNRS-
RBCs from HSCs and iPSCs/ESCs. These differences might be the reason for impaired UR1, Rennes, France; 5Inserm U1232, Nantes, France; 6Inserm U1085-Irset, Rennes,
ex vivo erythropoiesis from the latter sources. Our ongoing work focusses on the iden- France; 7IGDR UMR 6290 CNRS-UR1, Rennes, France; 8IGDR UMR 6290 CNRS-
tification of affected pathways during erythroid maturation in order to overcome exist- UR1, Centre Hospitalier Universitaire, Rennes, France
ing hurdles in the ex vivo manufacturing of RBCs from human iPSCs.
RUNX1 encodes a key transcription factor for hematopoiesis and is implicated in cancer
notably in pediatric B-precursor acute lymphoblastic leukemia. To understand the mecha-
nisms behind the control of RUNX1 transcriptional activity, we performed RUNX1 chro-
matin immunoprecipitation (ChIP) experiments followed by mass spectrometry and
identified FUBP1 as a potential functional cooperator of RUNX1. The helicase and the tran-
scriptional regulator FUBP1 has recently been described to be essential for expansion and
self-renewal of hematopoietic stem cells and to function as a potential cancer driver gene
in leukemia. Co-immunoprecipitation and colocalization experiments demonstrate that
RUNX1 colocalizes with FUBP1 in pre-B lymphoblasts from leukemic patients and in
two pre-B cell lines. ChIP of FUBP1 combined with sequencing (ChIP-seq) reveals that a
fraction of the signal is enriched within the enhancer regions characterized by H3K4me1
marks. Our data show also that FUBP1 and RUNX1 are often retrieved on same chromatin
regions bound by FUBP1. Comparison of the ChIP-seq from FUBP1, RUNX1 or H3K27ac
allows us to identify potential target genes positively regulated by FUBP1 and RUNX1
within the same chromatin region. One enhancer of the oncogene KIT was identified to
be positively regulated by both RUNX1 and FUBP1 and was validated by reporter gene assay
and ChIP-qPCR. Then, we demonstrated that FUBP1 increases KIT protein expression and
exacerbates one of the KIT downstream pathway. Finally, FUBP1 overexpression in a pre-B
cell line increases cell proliferation by promoting cell cycle progression in vitro. In vivo, xen-
ografted immunodeficient mice injected with cells overexpressing FUBP1 presente a shorter
survival time than control mice. To conclude, we demonstrate that FUBP1 and RUNX1 can
colocalize on chromatin and regulate KIT transcription by binding a common KITenhancer.
These new findings suggest a new mechanism involved in proliferation of pre-B lympho-
blasts. Because FUBP1 and KITare overexpressed in some type of leukemia, the implication
of RUNX1 in cancer could involve FUBP1 and KIT oncogenes.
3192 - EBF1 FUNCTION IN THE HEMATOPOIETIC STEM 3194 - LENS EPITHELIUM-DERIVED GROWTH FACTOR/
CELL NICHE P75 IS DISPENSABLE FOR HEMATOPOIESIS BUT
Marta Derecka, Pierre Cauchy, Senthilkumar Ramamoorthy, ESSENTIAL FOR MLL-REARRANGED
Josip Herman, Dominic Gr€un, and Rudolf Grosschedl LEUKEMOGENESIS
Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany Jan De Rijck1, Sara El Ashkar1, J€urg Schwaller2, Tim Pieters3,
Hematopoietic stem and progenitor cells (HSPC) are among the best studied and clinically Steven Goossens3, Jonas Demeulemeester1, Pieter Van Vlierberghe3, and
relevant cells due to their ability of replenishing entire blood system. However, in vitro Zeger Debyser1
1
expansion of HSCs is still a major challenge because the cues from the bone marrow micro- KU Leuven, Leuven, Belgium; 2University of Basel, Basel, Switzerland; 3U Gent,
environment, including mesenchymal stem and progenitor cells (MSPC) that regulate he- Gent, Belgium
matopoietic homeostasis remain largely elusive. Here, we show that early B-cell factor 1
Mixed-lineage leukemia (MLL) represents a genetically distinct and aggressive sub-
(EBF1) functions as an important regulator of MSPCs activity. Mesenchymal progenitors
set of human acute leukemia carrying chromosomal translocations of the MLL gene.
isolated from Ebf1-/- mice show diminished capacity to form fibroblastic colonies (CFU-
These translocations result in the formation of oncogenic fusions that mediate aber-
F) and impaired in vitro differentiation towards osteoblasts, chondrocytes and adipocytes.
rant recruitment of transcription machinery to MLL target genes. The N-terminus of
Adoptive transfers of wild type HSPCs to Ebf1+/- recipients showed a decrease of the abso-
MLL and MLL-fusions forms a complex with the lens epithelium-derived growth
lute numbers of HSPCs in primary recipients. Moreover, we observed reduced donor chime-
factor (LEDGF/p75; encoded by the Psip1 gene) and MENIN. This complex contrib-
rism within the HSCP compartment in competitive secondary transplant experiments. Prx1-
utes to the association of MLL and MLL-fusion multiprotein complexes to chro-
Cre-mediated deletion of Ebf1 in MSPCs leads to reduced frequencies and numbers of
matin. Several studies have shown that both MENIN and LEDGF/p75 are required
HSPCs and myeloid cells in the bone marrow. We also observed a reduced ability of
for efficient MLL fusion-mediated transformation and for the expression of down-
HSCs from Ebf1 fl/fl/Prx1-Cre mice to form colonies in methylcellulose. HSCs exposed
stream MLL-regulated genes like HOXA9 and MEIS1. In the light of the develop-
to the Ebf1-deficient niche exhibit changes in chromatin accessibility with reduced occu-
ment of a therapeutic strategy targeting this complex, understanding of the
pancy of AP-1, ETS, Runx and IRF motifs, which is consistent with the decreased myeloid
function of LEDGF/p75 in normal hematopoiesis is crucial. We generated a condi-
output seen in Ebf1 fl/fl/Prx1-Cre mice. Finally, single-cell and bulk transcriptome analysis
tional Psip1 knockout mouse model in the hematopoietic compartment and examined
of EBF1-knockout MSPCs revealed differences in the niche composition and a decreased
the effects of LEDGF/p75 depletion in postnatal hematopoiesis and the initiation of
expression of lineage-instructive signals for myeloid cells. Thus, our study establishes
MLL leukemogenesis. Psip1 knockout mice were viable but showed several defects
EBF1 as a novel regulator of MSPCs playing a crucial role in the maintenance of HSPCs
in hematopoiesis, reduced colony forming activity in vitro, decreased expression of
and their differentiation towards myeloid lineage.
Hox genes in hematopoietic stem cells and decreased MLL occupancy at MLL target
gene promotors. Finally, in vitro and in vivo experiments showed that Ledgf/p75 is
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136 S113
dispensable for steady-state hematopoiesis but essential for the initiation of MLL- 3196 - OPTIMIZED INDUCTION OF HUMAN
mediated leukemia. These data corroborate the MLL-LEDGF/p75 interaction as HEMOGENESIS IN ADULT FIBROBLASTS
novel drug target for MLL-rearranged leukemia. Michael Daniel1, Jeffrey Bernitz1, Yesai Fstkchyan1, Namita Satija1,
Kenneth Law2, Andreia Gomes3, Carlos-Filipe Pereira3, Benjamin Chen1,
Ihor Lemischka1, and Kateri Moore1
1
Icahn School of Medicine at Mount Sinai, New York, United States; 2Rocket
Pharmaceuticals, New York, United States; 3University of Coimbra, Coimbra, Portugal
The inability to culture hematopoietic stem cells (HSCs) in vitro remains a significant prob-
lem in HSC biology. This dilemma hampers study of various hematologic pathologies, as
well as disease modeling and drug testing efforts to understand and treat these disorders.
Recent studies focus on reprogramming pluripotent stem cells or somatic cells to generate
HSCs, with most success found in those that attempt to follow the known details of devel-
opmental hematopoiesis. These studies, however, fall short of generating the bona fide
HSC. This may be due to an incomplete understanding of hematopoiesis and the microen-
vironmental signals required for HSC induction and maintenance. We recently demonstrated
our ability to induce a hemogenic program in mouse embryonic fibroblasts through overex-
pression of the transcription factors (TFs) Gata2, Gfi1b, and cFos (GGF). Cells reprog-
rammed with these factors traverse an endothelial intermediate that gives rise to CD45+
hematopoietic cells upon prolonged culture. Optimization of hemogenic induction in adult
human dermal fibroblasts (HDFs) would allow creation of patient- and disease-specific he-
matopoietic cells for HSC transplants, in vitro disease modeling, and drug testing platforms.
Although we show some success in reprogramming HDFs into HSC-like cells with the GGF
cocktail, the yield and functional potential of the derived cells must be improved. Addition of
GFI1 to our TF panel significantly improves the yield and function of the induced HSCs (the
new cocktail is now termed 3GF). The derived cells possess a surface phenotype highly
similar to HSCs (CD49f+CD34+CD90+BB9+), and the yield of these cells is significantly
greater than those reprogrammed with GGF. These cells generate colonies in CFU and
CAFC assays after co-culture with OP9-DL1 and AFT024 stroma, respectively, denoting
their in vitro hematopoietic function. 3GF cells reprogrammed to day 15 and sorted for
CD49f display multilineage engraftment potential upon intrahepatic transplant in newborn
NSG mice. These results together demonstrate our improvements to human hemogenic in-
duction, bringing us one step closer to applying these findings to translational medicine.
were more compatible with initiation of embryonic thymopoiesis by more lineage- only low marking for B cells with MPP contribution was detectable at day 21 and
restricted progenitors. Using different reporter mouse lines, whole-mount imaging, HSC appearing on day 57. Contribution from MPP and HSC to CD11b+ and
clonal assays and fate mapping analysis we have showed that the first hematopoietic CD11b+/Ly6G+ myeloid populations is clearly detectable at day 21, with a higher
progenitors migrating to the E11.25 thymic rudiment uniformly express Rag1 prior percentage for MPP progeny. Conclusions: This still ongoing study holds the possi-
to their thymus entry and possess combined lymphoid and myeloid (GM), but no bility to simultaneously track clonal contributions of 4 different subpopulations in
erythroid and megakaryocytic potentials at the single cell level. These studies parallel. Further analysis of the barcodes will reveal the clonal composition of the
establish the exact lineage commitment step at which the T-lymphocyte lineage re- blood, which can be traced back to the originally transduced stem cell/progenitor
striction process must migrate to the thymus. Moreover, global RNA sequencing of population.
embryonic thymopoiesis-initiating progenitors established their unique molecular
signature and insights into pathways critical for the thymus-seeding and T lineage
restriction processes. Moreover, Studies on Rbpj-deficient embryos unequivocally
disproved claims that canonical Notch-signaling is involved in pre-thymic T-line-
age-restriction and in the initial colonization of the embryonic thymus.
3204 - WHOLE EXOME SEQUENCING IDENTIFIED most frequently co-occurred ones. Their effect on myeloid lineage markers including pu.1,
CANDIDATE GENE MUTATIONS IN A PEDIGREE OF mpo, l-plastin, mpeg1 and c-myb will be studied.
FAMILIAL MYELOPROLIFERATIVE NEOPLASM
Chae Yin Cher1, Nelson Ka Lam Ng1, Stephen Sze Yuen Lam1,
Bai-Liang He1, Dingge Ying1, Brian Hon-Yin Chung1, Chun Hung Au2,
Tsun Leung Chan2, Edmond Shiu Kwan Ma2, Raymond Hin Suen Liang2,
Yok Lam Kwong1, and Anskar Yu Hung Leung1
1
The University of Hong Kong, Hong Kong, Hong Kong; 2Hong Kong Sanatorium &
Hospital, Hong Kong, Hong Kong
3207 - DEFECTIVE MEGAKARYOPOIESIS IN ACUTE p50.0184) but this defect could be rescued by an ALK5 inhibitor, SB431542.
MYELOID LEUKEMIA Administration of SB431542 to AML mice could increase the proportion and
Ai Gao1,2, Yuemin Gong3, Fang Dong3, Shihui Ma3, Hui Cheng3, numbers of normal hematopoietic progenitors, especially megakaryocyte/erythroid
progenitors (0.01160.005% vs 0.04860.001%, p50.017). Next, we examined if
Sha Hao3, and Tao Cheng3
1 TGFb1 could act via the Egr3/p21 pathway. Interestingly, TGFb1 treatment led to
State Key Laboratory of Experimental Hematology, Institute of Hematology &
a prompt increase of Egr3 and downstream p21 expression in HSCs. Moreover,
Blood Diseases, Tianjin, China (People’s Republic); 2Department of Stem Cell and
ChIP-PCR demonstrated a direct binding of SMAD3 to the promoter of Egr3 gene
Regenerative Medicine, Peking Union Medical College, Tianjin, China (People’s
in HSPCs. To define the source of excessive TGFb1 in leukemic microenvironment,
Republic); 3State Key Laboratory of Experimental Hematology, Institute of
TGFb1 expression was measured at both mRNA and intracellular protein levels in
Hematology & Blood Diseases Hospital, Tianjin, China (People’s Republic)
multiple types of niche cells including megakaryocytes, endothelial cells and mesen-
Acute myeloid leukemia (AML) is characterized by an increased number of myeloid chymal stem cells. Interestingly, increased production of TGFb1 was observed most
cells in the marrow and an arrested maturation, frequently resulting in hematopoietic prominently in megakaryocytes. Moreover, in vitro transwell co-culture of megakar-
insufficiency. Major causes of death in AML are infection in 70% of patients and yocytes with leukemic blasts resulted in increased expression of TGFb1, Egr1 and
hemorrhage in 52%. However, the mechanisms underlying defective thrombocyto- p21, suggesting the signals from leukemic blasts might cause proinflammatory re-
poiesis in leukemia have not been fully elucidated. In this study, we established a sponses and senescence-like changes in megakaryocytes. In summary, our current
non-irradiated AML mouse model, namely the MLL-AF9-induced AML, and study demonstrates that megakaryocyte-derived excessive TGFb1 contributes to sup-
observed a significant reduction in the number of platelets in peripheral blood of leu- pression of normal HSCs via Egr3/p21 in leukemic bone marrow.
kemia mice (1315680.65 vs 143.3668.01103 /ml, p50.0004, n53). Ploidy anal-
ysis and bone marrow frozen section indicated a decreased number of
megakaryocytes (28.2563.497 vs 15.0063.055/visual field, n53, p50.416) and
maturation disorder during AML development (DNA content >5 8N on CD41+
megakaryocytes: 85.7362.348 vs 62.6760.5548%, n53, p50.0007). On the other
hand, the majority of megakaryocytes in leukemic environment exhibited higher
HP1gamma foci (8.83560.1318 vs 46.6567.005%, n53, p50.0057), which sug-
gested the increased senescence of megakaryocytes under leukemia. Furthermore,
CFU-MK assays demonstrated that leukemic bone marrow plasma inhibited mega-
karyocytes differentiation from megakaryocyte erythroid progenitors (MEPs) and
megakaryocyte progenitors (MkPs) to megakaryocytes (MEPs: 36.2563.750 vs
15.7561.931, n54, p50.0028; MkPs: 101.564.735 vs 68.5063.524, n54,
p50.0014). Cell cycle analysis showed that compared with control group, MEPs un-
der leukemia were gradually accumulated in the G0 status (22.0361.046 vs
38.664.200%, n54, p50.0053), whereas MkPs became more proliferative in
leukemic mice (45.1361.137 vs 57.6560.7500% , n54, p50.0021). Single cell
PCR and transplantation assay proved that long-term hematopoietic stem cells (LT-
HSCs) in leukemia preserved their differentiation potential for megakaryocytes (fre-
quency of platelets at 16weeks after transplantation: 40.7363.517 vs 41.0062.923%,
n54, p50.9540). In view of the increased TGFb1 in the leukemia mice plasma
(2.3folds, n52, p50.016), the treatment of TGFbRI (ALK5) inhibitor SB431542
partially rescued the number of CFU-MK suppressed by leukemic bone marrow
plasma (MEPs: 15.7561.931 vs 31.0061.985, n54 ,p50.0015; MkPs:
68.5063.524 vs 92.5064.628, n54, p50.0062). In summary, we demonstrated
that defective megakaryopoiesis under leukemia was caused by the obstacle of
MEPs and MkPs differentiation. Moreover, TGFb may contribute to the blockage
of megakaryopoiesis in AML.
3208 - MEGAKARYOCYTE-DERIVED EXCESSIVE TGFB1 3209 - THE EARLY RESPONSE CELLS TO BONE MARROW
INHIBITS PROLIFERATION OF NORMAL INJURY
HEMATOPOIETIC STEM CELLS IN LEUKEMIC BONE Chia-ling Chen, Katerina Faltusova, Petr Paral, Martin Molik,
MARROW Tomas Heizer, Filipp Savvulidi, and Emanuel Necas
Yuemin Gong1,2,3, Ai Gao1, Wanzhu Yang1, Mei Zhao1, Xiuxiu Yin1, Institute of Pathological Physiology, 1st Faculty of Medicine, Charles University,
Linping Hu1, Xiaofang Wang1, Xiaobing Zhang1, Sha hao1, Hui Cheng1, Czech Republic, Prague, Czech Republic
and Tao Cheng1 We have previous studied the bone marrow response in submyeloablatively (6 Gy)
1
State Key Laboratory of Experimental Hematology, Institute of Hematology & irradiated mice. This allowed a detailed analysis of immature Lin-c-Kit+ (LK) cells
Blood Diseases Hospital, Tianjin, China (People’s Republic); 2Center for Stem Cell only after approximately two weeks. These studies revealed significant expansion of
Medicine, Chinese Academy of Medical Sciences, Tianjin, China (People’s Sca-1+ (S+) LK CD150+CD48+ cells with decreased c Kit expression level (Blood
Republic); 3Department of Stem Cell and Regenerative Medicine, Peking Union 2014 124:5112). Furthermore, they showed that not only LS+K cells responded to the
Medical College, Tianjin, China (People’s Republic) injury but also Sca 1- (S-) late myeloid progenitors were activated and some of them
Leukemia cells thrive in the bone marrow at the expense of normal hematopoiesis. were induced to re-expressed Sca 1 antigen. In present study, we focused on the
Our recent study showed a cell cycle arrest and differentiation blockade of normal response of LK cells occurring shortly after bone marrow damage by analyzing
hematopoietic stem cells (HSCs) in leukemic bone marrow, which is attributable to bone marrow 1-5 days after irradiation of mice with 2-4 Gy. The number of bone
elevated expression of the transcription factor, Egr3 (Cheng H et al, Blood 2015). marrow cells significantly decreased after 2-4 Gy irradiation during one day. In
However, the upstream signaling from the malignant microenvironment remains un- LK cells, the LS-K/LS+K ratio decreased (below 2) due to Sca-1 induction. However,
known. By analyzing the gene expression profile of Lin-c-Kit+Sca-1+ hematopoietic it recovered to normal level in a range of 5-7 after 3-5 days. We used CD150 and
stem and progenitor cells (HSPCs) from MLL-AF9 induced acute myeloid leukemia CD48 SLAM markers to distinguish between various subtypes of LSK cells, and
(AML) mice, we found an activation of TGFb signaling pathway. As measured by CD34 and CD16/32 markers to characterize various types of LS-K myeloid progen-
ELISA, total TGFb1 was significantly elevated in early-phase of leukemic bone itor cells. This revealed an early activation of megakaryocyte-erythroid and granulo-
marrow plasma (2.3 folds, p50.016). Notably, HSCs cultured in leukemic bone cyte macrophage myeloid progenitors. The activation was characterized by re-
marrow plasma exhibited a lower proliferation rate (11.461.4 vs 21.562.2, expression of Sca 1 in a part of these cells and by significantly decreased c-Kit
S118 Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
expression level. During 3-5 days, c Kit recovered fully in 2 Gy- and partly in 4 Gy- of E13.5 PDGFRA+ FL MSCs showed up-regulation of genes and pathways associated
irradiated mice. The transcription factor SCL/TAL1 with other co-factors is known to with haematopoiesis when compared with E18.5 PDGFRA+ FL MSCs. Taken together,
upregulate c-Kit gene and c-Kit expression. Therefore, we examined SCL/TAL1 we have identified a population of FL MSCs that contribute to the development of the
mRNA level in LK cells in bone marrow of irradiated mice. vascular architecture of the FL and can also support AGM HSCs.
Necas E, Szikszai Forgacova K, Faltusova K, et al: Lin-Sca-1+c-Ki-
tlowCD48+CD71+ Cells Are the Engine of Bone Marrow Regeneration. Blood
2014 124:5112 (abstract).
Research was supported by the Grant Agency of the Czech Republic (GACR 17-
01897S).
Murine haematopoiesis progresses in waves through several anatomical sites including During embryonic development, the first hematopoietic stem cells (HSCs) arise from
the yolk sac, aorta-gonad-mesonephros (AGM), foetal liver (FL), placenta and the bone a transient population of endothelial cells lining the ventral surface of the dorsal
marrow. The number of haematopoietic stem cells (HSCs) increases dramatically in the aorta, via a process of endothelial to hematopoietic transition (EHT) at embryonic
FL during mid-gestation. The cellular and signalling processes that govern HSC expan- day (E) 10.5. The aorta-gonad-mesonephros (AGM) region has resident mesen-
sion and maturation in the FL are not entirely understood. Mesenchymal stem cells chymal stem cell-like cells (MSC-LCs), but their identity and role in HSC generation
(MSC) are known to provide a niche for HSC maintenance in the adult bone marrow. are not well defined. Using a library of compound transgenic mice, we identified a
Therefore, we hypothesized that FL MSCs may also play a role in the maturation and population of PDGFRA+/Nestin-GFP (N-GFP)-/PDGFRB-/CD31- cells with MSC-
expansion of HSCs during embryonic development. Due to the lack of bona fide LC activity in the E10.5 and E11.5 mouse AGM. These freshly isolated MSC-LCs
markers for FL MSCs, we first estimated the number of FL MSCs between E11.5 were adept at forming blood vessels with CD31+ luminal endothelium enveloped
and E18.5 using colony forming unit- fibroblast (CFU-F) assays. FL CFU-Fs demon- by PDGFRB+ pericytes, when transplanted subcutaneously into mice. Conditional
strated serial clonogenicity, long-term self-renewal, and in vitro multipotency and their ablation of PDGFRA+ or Nestin+ cells led to either complete or partial loss of
number progressively increased with FL development. Using two different germ layer AGM MSC-LCs respectively, with severe loss of endothelial and pericyte-like cells,
specific Cre- transgenic mouse lines, we demonstrated that at early (E13.5) and late and concomitant loss of blood formation. Lineage tracing studies using tamoxifen in-
time points (E18.5) most FL MSCs were derived from MesP1 (mesoderm) but at duction on PDGFRACreERT2/R26eYFP embryos showed that stromal, sub-endothe-
E18.5, there is an additional contribution from Wnt1 (neural crest) to FL MSCs. FL lial, endothelial and long-term repopulating hematopoietic stem cells (LT-HSCs) in
MSCs at both early- and late-time points express PDGFRA, and PDGFRA+ cells the E11.5 AGM were progeny of PDGFRA cells. Using transgenic reporter mice,
reside near the peritoneal surface of the hepatic lobes and in the peri-sinusoidal and we showed that MesP1 derived PDGFRA+ cells dominated the sub-endothelial and
peri-vascular regions. Lineage tracing studies coupled with confocal microscopy re- deeper ventral stroma in the AGM at E10.5 and E11.5 but were replaced by Wnt1
vealed that PDGFRA+ cells directly contributed to hepatic mesothelial and endothelial derived cells by E13.5. Co-aggregation of E11.5 Mesp1 derived MSC-LC cells
cells during FL development. Transplantation assays performed with co-aggregates of (PDGFRA+/PDGFRB-/CD45-/CD31-/VE-CAD-) with E13.5 aortic or adult cardiac
E13.5 PDGFRA+ FL MSCs, and E11.5 AGM derived CD45+ cells, showed that the non-hemogenic endothelial cells (PDGFRA-/PDGFRB-/CD45-/CD31+/VE-CAD+)
former supported long-term transplantable HSCs. Genome-wide expression analysis resulted in the generation of endothelial cell derived LT-HSCs. RNA-seq analysis
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136 S119
of non-hemogenic E13.5 endothelial cells showed up-regulation of EHT genes when sue engineered vessel to mimic vascular niche of the HSCs. Our next aim is to inves-
co-aggregated with E11.5 Mesp1 derived MSC-LCs. LT-HSC generation from these tigate the interaction between HSCs and our newly constructed vessel. The authors
co-aggregates was suppressed by dose-dependent inhibition of PDGF-AA/PDGFRA wish to thank The Scientific and Technological Research Council of Turkey (Project
signalling. Taken together, we report that MSC-LC populations in the AGM are Number: 113S815) and Hacettepe University, Scientific Research Project Coordina-
temporally dynamic, and that MesP1-derived MSC-LCs regulate hemogenic potential tion Unit (Project Number: THD- 2016-10504) for their financial support.
of the endothelium and that this cooperativity is dependent on PDGF-AA signalling.
3214 - ASPIRIN-TREATED ESSENTIAL subsequently mature cells. To explore essential HSC features, we recently integrated
THROMBOCYTHEMIA PATIENTS: quantitative proteome, transcriptome, and methylome analyses of five FACS-sorted
WHICH TIMING? HSCs and MPP populations (MPP1-4) and combined these OMICs analyses to their
functional potential (Cabezas-Wallscheid et al., Cell Stem Cell 2014; Klimmeck et
Rossella Cacciola1, Elio Gentilini Cacciola2, and Emma Cacciola3
1 al., Stem Cell Reports 2014; Lipka et al., Cell Cycle 2014). We have now expanded
Dep. of Clinical and Experimental Medicine, Haemostasis Unit, University of
this analysis to dormant HSCs (dHSCs) identified by label-retaining assays (Wilson
Catania, Catania, Italy; 2University of Catania, Catania, Italy; 3Haemostasis Unit,
et al., 2008). Rare dHSCs reside at the top of the blood hierarchy harboring the high-
Univ. of Catania, Catania, Italy
est long-term reconstitution capacity. However, till the date the molecular identity of
The essential thrombocythemia (ET) is a myeloid neoplasm characterized by dHSCs, as well as the mechanism regulating maintenance and the transition out of
platelet hyperreactivity and thrombotic risk. The treatment with aspirin (ASA) is dormancy remain unknown. Rare dormant hematopoietic stem cells (dHSCs)
recommended in ET patients at risk of first-time or recurrent thrombotic events. harboring the highest long-term reconstitution capacity define the top of the hemato-
An unexplored topic is the optimal timing of once daily ASA intake. On the basis poietic system. The molecular identity of dHSCs and immediate progeny, as well as
of the presumptions that 1) platelet aggregation is higher in the morning and that 2) the mechanism regulating maintenance and the transition out of dormancy remain un-
the platelet inhibitory effect of ASA is not sustained during the usual 24-hour (h) known. We now show by single-cell RNA-seq analysis that the transition from
dosing interval and that 3) a higher gastric mucosal resistance in the evening, we dormancy towards cell cycle entry is achieved by a continuous and coordinated
evaluated platelet count, b-thromboglobulin (b-TG) and platelet factor 4 (PF4), up-regulation of all major biosynthetic processes rather than a switch on/off mecha-
as markers of platelet activation, the platelet function activity (PFA), as indicator nism. We generate a novel transgenic reporter mouse model that specifically labels
of ASA platelet sensitivity. We studied 60 patients (20 men, 40 women; mean dHSCs avoiding label retention assays. Finally, we show that retinoic acid signaling
age 51 years, range 32-70) with ET according to WHO criteria. The mean duration is a key pathway for in vivo retaining HSC dormancy.
of disease was 11 years. All patients were on ASA 100 mg once daily. Of these, 30
took ASA on awakening and 30 took ASA at bedtime. Of the 60 patients, 45 were
on anagrelide hydrochloride (daily dose 1.5 mg) (10 men, 35 women), 15 were on
hydroxyurea (daily dose 2 mg) (10 men 5 women). None had inherited or acquired
thrombotic risk factors. Sixty subjects served as controls. Platelets were measured
by automated analyzer. b-TG and PF4 were determined by ELISA. ASA platelet
sensitivity was measured by Platelet Function Analyzer (PFA-100). The mean
platelet count was 4556200x109/L. The awakening ASA patients had normal b-
TG and PF4 (1265 IU/ml and 461 IU/ml) and prolonged C/EPI closure time (T,
unit: s, n.v. 84-160 s) (249640 s), whereas the bedtime ASA patients had high
b-TG and PF4 (200615 IU/ml vs 20611 IU/ml and 170650 IU/ml vs 662 IU/
ml, respectively) (p!.0001 and p!.0001, respectively) and normal C/EPI closure
time (T, unit: s, n.v. 84-160 s) (90610 s). These findings suggest that in ET patients
the optimal timing of once daily ASA intake is in the morning.
3215 - VITAMIN A/ RETINOIC ACID SIGNALING 3216 - GENERATION OF A NEW, HUMAN GROWTH
REGULATES HEMATOPOIETIC STEM FACTOR-DEPENDENT, DE NOVO
CELL DORMANCY MODEL OF AML FROM C-MYC-
Nina Cabezas-Wallscheid1,2, Florian Buettner3, Pia Sommerkamp4, TRANSDUCED NORMAL HUMAN CD34+
Daniel Klimmeck4, Luisa Ladel4, Frederic Thalheimer5, HEMATOPOIETIC CELLS
Daniel Pastor-Flores6, Leticia Roma7, Simon Renders4, Petra Zeisberger4, Elizabeth Bulaeva1,2, Naoto Nakamichi1, Philip Beer3, Andrew Weng1,
Adriana Przybylla4, Katharina Sch€onberger4, Roberta Scognamiglio4, and Connie Eaves1
1
Sandro Altamura8, Carolina Florian9, Malak Fawaz10, Dominik Vonficht11, Terry Fox Laboratory, BC Cancer Agency, Vancouver, Canada; 2Faculty of
Melania Tesio4, Paul Collier12, Dinko Pavlinik12, Hartmut Geiger9, Medicine, University of British Columbia, Vancouver, Canada; 3Terry Fox
Laboratory, BC Cancer Agency, Cambridge, United Kingdom
Timm Schr€ oder13, Vladimir Benes12, Tobias Dick6, Michael Rieger14,
Oliver Stegle3, and Andreas Trumpp4 A role of altered expression of the transcription factor c-MYC has been implicated in
1
German Cancer Research Center (DKFZ), Freiburg, Germany; 2Max-Planck- maintaining the leukemic blast population of patients with chronic myeloid leukemia,
Institute of Immunobiology and Epigenetics, Freiburg, Germany; 3European acute myeloid leukemia (AML) and lymphoma. However, a specific role of MYC in
Molecular Biology Laboratory (EBI), European Bioinformatics Institute, Wellcome the initial pathogenesis of human leukemias has remained poorly defined. To investigate
Trust Genome Campus, Hinxton, Cambridge, United Kingdom; 4Division of Stem this question, we first examined the effects of lentivirally-mediated overexpression of
Cells and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany; MYC in normal human hematopoietic CD34+ cells isolated from cord blood (CB).
5
4LOEWE Center for Cell and Gene Therapy and Department of Medicine, We found that sublethally irradiated, immunodeficient NOD-Rag1-/-IL2R-/- mice trans-
Hematology/Oncology, Goethe University Frankfurt, Frankfurt, Germany; 6Division genically producing human IL-3, GM-CSF and Steel factor (NRG-3GS mice) when
of Redox Regulation, DKFZ-ZMBH Alliance, German Cancer Research Center transplanted with MYC-transduced CD34+38- or various subsets of CD34+38+ CB cells
(DKFZ), Heidelberg, Germany; 7Division of Redox Regulation, DKFZ-ZMBH consistently succumbed to a fatal human CD33+CD123+CD14-CD15- leukemia within
Alliance, German Cancer Research Center (DKFZ), Heidelberg, Germany; 10 weeks. Similar results were obtained when MYC-transduced normal adult human
8
Department of Pediatric Hematology, Oncology and Immunology, University of bone marrow CD34+ cells were transplanted into sublethally irradiated NRG-3GS-
Heidelberg; 9Institute for Molecular Medicine, Stem Cells and Aging, Ulm W41 hosts. Some of the MYC-induced leukemic CB cells generated in primary mice
University, Ulm, Germany; 10LOEWE Center for Cell and Gene Therapy and also produced a similarly fatal leukemia in secondary transplanted NRG-3GS mice,
Department of Medicine, Hematology/Oncology, Goethe University Frankfurt, although only after a long latency. Interestingly, further experiments using NRG mice
Frankfurt am Main, Germany; 11Heidelberg, Germany; 12Genomics Core Facility, (i.e. not producing human 3GS) as primary hosts showed engraftment of the transduced
European Molecular Biology Laboratory (EMBL), Heidelberg, Germany; cells, but no evidence of leukemogenesis. Mechanistically, we found that immediately
13
Department of Biosystems Science and Engineering (D-BSSE), ETH Zurich, Basel, post-transduction, MYC-transduced CD34+ CB cells had produced 7-fold larger clones
Switzerland; 14LOEWE Center for Cell and Gene Therapy and Department of than control cells after 12 days in vitro. When co-cultured with stromal cells as well as
Medicine, Hematology/Oncology, Goethe University Frankfurt, Frankfurt am Main, growth factors, the MYC-transduced cells also markedly outcompeted the control cells
Germany over a period of 12 weeks. In summary, we describe a novel, developmentally unre-
stricted, de novo model of human AML. This model will be useful to elucidate the mech-
Hematopoietic stem cells (HSCs) harbor the capacity to generate a series of multipo-
anisms by which elevated MYC expression cooperates with growth factor stimulation to
tent progenitors (MPPs) that differentiate into lineage-committed progenitors and
create a leukemic phenotype in naive human hematopoietic cells.
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136 S121
3217 - IDENTIFICATION AND CHARACTERIZATION OF tor RUNX1 abrogated primitive blood cell formation, halting cells at a vascular core
NOVEL FUNCTIONAL MARKERS OF EHT stage. Our data indicate that while SOX17 expression is dispensable, RUNX1 expres-
Marleen B€ uchler1, Paul Kaschutnig1, Marleen B€uchler2, sion is absolutely required to permit the transition from endothelium to blood during
human primitive hematopoiesis. In addition, we examined vascular development dur-
Roshana Thambyrajah3, Wiebke Nadler4, Sabrina Hanke4,
ing definitive hematopoiesis in hPSCs lacking SOX17 or all SOXF genes. While
Stella Pfaffenholz1, Julius Gr€asel1, Jakob Kremer1, Milena Block1,
SOX17-deleted cells showed some defects in vascular organisation, these were
Irem Bayindir-Buchhalter2, David Hills5, Andreas Trumpp4, Marc Thier4, most striking in SOXF-deleted cultures, demonstrating the critical role for these tran-
Lo€ıc Maillard6, Nico Lachmann7, Mania Ackermann7, Thomas Moritz7, scription factors in vascular modeling. Our studies revealed that day15 hemogenic
Christoph R€ osli4, Alexander Medvinski5, Michele Souyri6, endothelium possessed a decreased ability to generate blood cells in the absence of
Georges Lacaud8, and Michael Milsom1 SOX17. However, preliminary studies of SOXF knockout lines indicate an increase
1
German Cancer Research Centre, Heidelberg, Germany; 2German Cancer Research in blood cell generation, suggesting that the SOXF genes are involved in complex
Institute, Heidelberg, Germany; 3Manchester Institute Cancer Research, Manchester, regulation of the endothelial to hematopoietic transition in human cells. Studies using
United Kingdom; 4Hi-STEM, Heidelberg, Germany; 5Institute for Stem Cell these novel reagents will assist in further dissection of the regulation of human
Research, Edinburgh, United Kingdom; 6French Institute of Health and Medical hematopoiesis.
Research, Paris, France; 7Hannover Medical School, Hannover, Germany;
8
Manchester Institute Cancer Research UK, Manchester, United Kingdom
3218 - DISSECTING THE ROLES OF RUNX1 AND SOXF 3219 - MURINE FETAL LIVER MICROENVIRONMENT
TRANSCRIPTION FACTORS IN HUMAN HEMATOPOIESIS SUPPORTS HUMAN MEGAKARYOPOIESIS
Freya Faith Bruveris1, Elizabeth Ng1, Lisa Azzola2, Camille Jost1,2,3, Francois Lanza4, Christian Gachet4, and
Ana Rita Leitoguinho3, Nadia Davidson3, Alicia Oshlack3, Nathalie Brouard4
1
Edouard Stanley4, and Andrew Elefanty4 Etablissement Francais du Sang, Strasbourg, France; 2INSERM, Strasbourg, France;
3
1
Murdoch Childrens Research Institute, Monash University, Melbourne, Australia; Universite de Strasbourg, Strasbourg, France; 4EFS / INSERM, Strasbourg, France
2
Monash University, Melbourne, Australia; 3Murdoch Childrens Research Institute,
The fetal liver is a major site of expansion of hematopoietic cells, including mega-
Melbourne, Australia; 4Murdoch Childrens Research Institute, Monash University,
karyocytes (MK). We hypothesized that the cellular microenvironment of the fetal
University of Melbourne, Melbourne, Australia
liver could be the source of specific determinants capable of supporting a large
Human blood cells form via hemogenic endothelia at distinct embryonic sites and expansion of MK and their progenitors. The present study focuses on defining
stages during development. Using in vitro differentiation of blast colony-forming the respective role of stromal cells of the fetal liver during the production of MK
cells as a model system for human primitive hematopoiesis, we examined the gener- from hematopoietic stem cells (HSC). We identified in the mouse fetal liver, pop-
ation of the first hematopoietic and endothelial lineages. We used human embryonic ulations of cells that can produce adherent layers. One of these populations
stem cells tagged with mCHERRY and GFP at SOX17 and RUNX1C loci, to mark (CD45-TER119-CD31-CD51+VCAM-1+PDGFRa-), supports the production of a
endothelia and hematopoietic cells, respectively. In addition to chronicling the large number of MK from mouse HSC. We then evaluated in a co-culture assay
expression of these genes during blast colony differentiation, we also explored the the capacity of this stromal cell population to support the production of MK and
consequences of SOXF (SOX17,SOX7,SOX18) transcription factor and RUNX1 MK progenitors (MKp) from human HSC (HuHSC). HuHSC were isolated as
gene knockout. We observed that differentiating blast colonies formed SOX17+ CD34+CD38-CD45RA- cells from leukodepletion filters and seeded 500 cells per
and SOX17- endothelial precursors with differing hemogenic capacity, with the ma- well onto fetal stromal layers in a serum free medium supplemented with low con-
jority of blood cells being generated from SOX17- endothelium. Downregulation of centration level of cytokines. The co-cultures were maintained for 7 to 14 days and
SOX17 accompanied primitive blood cell formation from SOX17+ endothelium, the MK and MK progenitors produced were identified by flow cytometry. From day
although deletion of SOX17 did not influence the propensity of these two endothelia 7, we observed an increased production of fully mature MK characterized as
to make primitive blood. Conversely, deletion of the hematopoietic transcription fac- CD41+CD42+ polyploid cells on stromal cells compared to the no stroma condition.
S122 Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
At day 14, 3048266764 MK were produced in the presence of stromal cells while ferase domain, did not evoke significant changes in DNAm. Furthermore, we
882684 MK were produced in absence of stromal layer. More interestingly, the cul- observed significant changes in global gene expression upon knockdown of
ture on stromal layers also generated a greater number of MKp when compared to DNMT3A transcripts, which were inversed for specific genes upon overexpression
the no stroma condition (874061600 and 170649 respectively). MKp were identi- (e.g. CD34). Lastly, the expression signature of Tr.2 correlated with survival of acute
fied as CD34+CD41low cells, a population that we previously identified to be highly myeloid leukemia patients (p50.0095). Our results demonstrate that specific
efficient to produce proplatelets (Strassel et al, Blood 2016). We propose that this DNMT3A transcripts impact on growth, differentiation of HSPCs, and display unique
stromal cell population represents an essential component of the fetal microenvi- DNAm patterns. Thus, the alternative splicing of DNMT3A is of functional relevance
ronment supporting the engagement toward the megakaryocytic lineage. The for the site-specific epigenetic modifications in hematopoietic development.
unique properties of this stroma may guide in the development of new tailored
methods for in vitro production of platelets.
3220 - THE FUNCTIONAL RELEVANCE OF DNMT3A 3221 - THE SPLICEOSOMAL COMPONENT SF3B1 IS
SPLICE VARIANTS IN HEMATOPOIETIC ESSENTIAL FOR PROPER ERYTHROID
DIFFERENTIATION DIFFERENTIATION AND GENE EXPRESSION IN
Tanja Bozic1, Joana Frobel2, Annamarija Raic2, Fabio Ticconi3, ZEBRAFISH
Stefanie Heilmann-Heimbach4, Tamme W. Goecke5, Ivan G. Costa3, Teresa Bowman1, Adriana De La Garza2, Varun Gupta2,
Edgar Jost6, and Wolfgang Wagner2 Rosannah Cameron2, and Sara Nik2
1 1
University Hospital of the RWTH Aachen, Stemm Cell Biology and Cellular Departments of Developmental and Molecular Biology and Medicine (Oncology),
Engineering, Aachen, Germany; 2Helmholtz-Institute for Biomedical Engineering – Gottesman Institute of Stem Cell Biology and Regenerative Medicine, Albert
Stem Cell Biology and Cellular Engineering, RWTH Aachen University Medical Einstein College of Medicine, Bronx, United States; 2Albert Einstein College of
School, Aachen, Germany; 3Interdisciplinary Centre for Clinical Research (IZKF) Medicine, Bronx, United States
Aachen, RWTH Aachen University Hospital, Aachen, Germany; 4Institute of Human
Genetics, Department of Genomics, Life & Brain Center, University of Bonn, Bonn, Myelodysplastic syndromes (MDS) are a group of hematologic disorders characterized by
Germany; 5Department of Obstetrics and Gynecology, RWTH Aachen University ineffective hematopoiesis and cytopenias. Mutations in the spliceosomal component SF3B1
Hospital, Aachen, Germany; 6Clinic for Oncology, Hematology, and Stem Cell (Splicing Factor 3B, subunit 1) are prevalent in MDS and correlate strongly with refractory
Transplantation, RWTH Aachen University Medical School, Aachen, Germany anemia with ringed sideroblasts. How altered SF3B1 function affects erythropoiesis is un-
clear. To address this question, we determined the in vivo consequences of sf3b1 misregu-
DNA methyltransferase 3A (DNMT3A) plays a pivotal role for de novo DNA lation on red blood cell maturation using a zebrafish sf3b1 (sf3b1hi3394) loss-of-function
methylation (DNAm) during development. It can be spliced into various different mutant. We found that sf3b1 mutants develop macrocytic anemia with diminished hemoglo-
transcripts, and it is unclear how it governs the multitude of lineage-specific binization. Early hematopoietic progenitor markers scl and gata1 as well as beta-globin are
DNAm patterns. In this study, we followed the hypothesis that specific splice variants expressed normally showing initiation of erythropoiesis is unaffected in mutants. We did not
of DNMT3A are of particular relevance for hematopoietic differentiation. We have observe an increase in the pro-apoptotic marker caspase-3 in gata1:gfp+ erythroid cells or
modulated expression of DNMT3A transcripts in hematopoietic stem and progenitor suppression of anemia when Tp53-mediated apoptosis was blocked, indicating the anemia
cells (HSPCs) from cord blood: transcript 1+3 (Tr.1+3), transcript 2 (Tr.2), or tran- in sf3b1 mutants is not due to heightened cell death. Anemia can also be caused by defects in
script 4 (Tr.4) of DNMT3A were knocked down by short hairpin RNA or constitu- heme biosynthesis or iron transport. We found that treatment with exogenous iron did not
tively overexpressed by lentiviral infection. Downregulation of either Tr.2 or Tr.4 rescue hemoglobin production, ruling out defective iron transport as an underlying cause
reduced the proliferation rate of HSPCs significantly (n53, p ! 0.05). HSPCs main- for the observed anemia. To discover the underlying mechanism that could be driving ane-
tained CD34 expression for a higher number of cell divisions upon knockdown of mia in sf3b1 mutants, we performed RNA-sequencing of purified gata1:gfp-positive eryth-
Tr.2 (n53; p ! 0.05). In colony forming unit (CFU) assays downregulation of roblasts from sf3b1 mutants and wild-type siblings. Over 2000 genes were differentially
Tr.4 resulted in a clear bias towards erythroid colonies (n53, p ! 0.05). Overall, expressed between mutants and wild-type siblings, including diminished expression of
CFU frequency was reduced by knockdown of DNMT3A transcripts, whereas it two indispensable factors for heme biosynthesis, alad and urod. Additionally, the gene
was increased by overexpression. Subsequently, we analyzed global DNAm patterns signature in mutant erythroblasts was consistent with a block in cell cycle, a process known
by Illumina 450k BeadChip technology: several CpG sites revealed DNAm changes to be important for erythroid differentiation. DNA content analysis revealed an accumula-
upon knockdown of Tr.2 and Tr.1+3 (8,905 and 352 CpGs, respectively; n53, tion of sf3b1 mutant erythroblasts in G0/G1, confirming the gene expression data. Together,
adjusted p ! 0.05). Notably, these patterns were reversed upon overexpression of these data suggest that macrocytic anemia arising when Sf3b1 is defective is likely due to
the same transcripts. Knockdown of Tr.4, which does not have the DNA-methyltrans- pleiotropic effects on cell cycle progression and hemoglobin production.
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136 S123
3222 - INVESTIGATING THE MECHANISM OF emphasizes that different types of AML, in spite of being a disease of one specific
THROMBOCYTOPENIA RESULTING FROM MUTATIONS differentiation pathway are maintained by highly diverse transcriptional networks
IN ETV6 OR RUNX1 USING A HUMAN STEM CELL and it highlights the complexities we have to face in our quest for personalized
medicine.
MODEL SYSTEM
Sara Borst1,2, Paul Gadue3, and Jean Ann Maguire2
1
University of Pennsylvania, Philadelphia, United States; 2Children’s Hospital of
Philadelphia, Philadelphia, United States; 3University of Pennsylvani and Children’s
Hospital of Philadelphia, Philadelphia, United States
3225 - HSCS COMPLETELY FAIL TO REGENERATE 3227 - ELUCIDATING THE ROLE OF THE LONG
FOLLOWING INFLAMMATORY CHALLENGE, LEADING NON-CODING RNA DANCR IN LEUKEMIC STEM CELLS
TO AGED HEMATOPOIESIS IN ACUTE MYELOID LEUKEMIA (AML)
Ruzhica Bogeska1, Paul Kaschutnig1, Stella Paffenholz1, Julia Knoch2, Marius Bill1,2, Malith Karunasiri1, Matthew Burke1, Allison Walker1,
Dagmar Walter2, Jan-Philipp Mallm1, Felix Frauhammer1, Stefano Volinia3, Clara Bloomfield1, Ramiro Garzon1, and
Sandra Blaszkiewicz2, Tim Holland-Letz1, Noboru Asada3, Julius Gr€asel1, Adrienne Dorrance1
Prendergast1, Simon Haas1, Daniel Lipka1,
able1, Aine
Sina St€
1
The Ohio State University, Comprehensive Cancer Center, Columbus, United States;
2
Karsten Rippe1, Benedikt Brors1, Paul Frenette3, Marieke Essers1, and Division of Hematology, Department of Internal Medicine, The Ohio State
Michael Milsom1 University, Columbus, United States; 3Department of Morphology, Surgery and
1
German Cancer Research Center, Heidelberg, Germany; 2Heidelberg Institute for Experimental Medicine, Ferrara, Italy
Stem Cell Technology and Experimental Medicine gGmbH, Heidelberg, Germany; Prognosis for AML patients (pts) is still dismal. Therapeutic strategies are needed to
3
Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine target not only the rapidly dividing AML bulk blasts but also the distinct LSCs. Long
Research, Albert Einstein College of Medicine, New York, United States non-coding (lnc) RNAs regulate several cellular functions but a role in LSC has to be
HSCs are thought to possess extensive self-renewal capacity, underlying their ability elucidated. We derived a LSC-specific lncRNA profile using whole transcriptome
to sustain hematopoiesis throughout the lifetime of an organism. However, the func- sequencing (RNA-seq) of 377 pts with cytogenetically normal AML. Data were corre-
tional HSC pool contracts in elderly humans, suggesting that the canonical concept of lated with a ’core enriched’ (CE) gene expression signature (GES) representing 44 genes
HSC self-renewal may be incorrect. Inflammatory stress results in depletion of func- activated in LCSs (Eppert Nat Med 2011). Correlation revealed a set of 161 lncRNAs
tional HSCs in wild type(WT) mice, or exhaustion of the HSC pool and induction of deregulated in CE-GEShigh pts at a p ! .001 & correlation coefficient O.5 (Spearmen
aplastic anemia in a mouse model of Fanconi anemia (Walter et al, 2015, Nature). To correlation test). Among the top upregulated lncRNAs we identified DANCR. Published
assess regenerative self-renewal of HSCs in the native setting, WT mice were subject works have suggest a role for DANCR in promoting stemness through putative interac-
to polyinosinic:polycytidylic acid(pI:C) dose escalation, to mimic chronic viral infec- tions with b-catenin (Yuan Hepatology 2016), a key factor contributes to LSC mainte-
tion and inflammation. 1-3 rounds of pI:C had no effect on peripheral blood(PB) pa- nance through Wnt signaling. Using qRT-PCR, we found variable levels of DANCR
rameters, but the frequency of functional HSC progressively decreased approximately expression in the AML pts (n56) but significantly elevated values compared to healthy
2-fold per round of pI:C, as assessed by competitive repopulation. The in-vitro clonal controls (n53; p5.01). The variation was expected since these analyses were performed
proliferative capacity of HSCs also decreased and was skewed towards depletion of on whole pts blasts & not sorted LSC. Importantly, we showed that DANCR expression
multi-potent clones. To directly assess the regenerative capacity of HSCs in vivo, was significantly higher in functionally validated LSC enriched populations (n54) as we
mice were treated with 3 rounds of pI:C followed by 5-20 weeks of recovery. reported before (Dorrance Leukemia 2015). To assess the role in LSC self-renewal, we
Competitive repopulation and limiting dilution assays indicated absolutely no evi- knocked down DANCR in CD34+ selected AML pts (n53) using a Transferrin conju-
dence for HSC recovery with increasing time post-treatment. Likewise, pI:C medi- gated nanoparticle & found a significant decrease in colony numbers after replating
ated depletion of endogenous HSCs facilitated transplantation into these mice up (average decrease: 38.9%, p5.03). Currently we are performing long term culture initi-
to 20 weeks post-treatment, in the absence of any irradiation conditioning. Taken ating cell assays, Carboxyfluorescein succinimidyl ester (CSFE) & in vivo xenotransplant
together, inflammatory stress led to a progressive and irreversible depletion of func- experiments. Results will be updated at the meeting. Our data show that LSC have a
tional HSCs, suggesting that cumulative exposure to environmental agonists might be distinct lncRNA expression profile. DANCR is higher expressed in AML-LSC enriched
a mediator of age-associated pathologies. Indeed, aged WT mice treated with pI:C in populations & in AML pts compared to healthy donors. Preliminary functional data sug-
early to mid-life displayed PB cytopenias, BM hypoplasia and increased adipogene- gest that DANCR expression has an impact on self-renewal capacity, a property of LSC.
sis, that are clinical features of aged human hematopoiesis which never spontane- Further experiments are needed to define whether DANCR has a key role in LSC func-
ously develop in laboratory mice. We propose that environmental agonists are a tion & whether could be a novel target for treatment.
missing ingredient in the study of aged hematopoiesis in experimental mice, and hy-
pothesize that HSCs are not preserved during ageing by engaging in serial self-
renewal divisions, but rather by extended quiescence.
3226 - EFFICIENT ISOLATION OF HUMAN CD34+CD38- 3228 - TFEC, A MASTER REGULATOR OF THE HSC
HEMATOPOIETIC STEM CELLS USING NICHE DURING EMBRYOGENESIS
IMMUNOMAGNETIC SORTING Julien Bertrand
Dariusz Krenz, Faidra Aivazidou, Andreas Bosio, Sebastian Kn€obel, and University of Geneva - School of Medicine, Geneva 4, Switzerland
Ute Bissels During embryogenesis, the hemogenic endothelium produces a limited number of
Miltenyi Biotec GmbH, Bergisch Gladbach, Germany Hematopoietic Stem Cells (HSCs). This small subset needs to expand in order to
Hematopoietic stem and progenitor cells (HSPCs) are an important source for cell-based maintain regeneration of the blood tissue for the entire life. In mammals, this initial
therapies and are routinely used to reconstitute the immune system after treatment of he- expansion takes place in the fetal liver. Understanding the microenvironment driving
matological malignancies. In vitro, the CD34+ population constitutes the main cell HSC expansion would be beneficial for ex vivo HSC expansion, a key step in the pro-
source for functional assays, although the majority of the CD34+ cells are rather progen- cess of regenerative medicine and gene therapy. We use the zebrafish model to under-
itor than stem cells. In contrast, the CD34+ cell subpopulation with low CD38 expression stand this process, which occurs in the caudal hematopoietic tissue (CHT) between
(CD34+CD38- cells) is highly enriched for stem cells and, thus, much more suitable for 48 and 96 hours post fertilization (hpf). We previously showed that TFEC, a tran-
functional analysis of stem cells, e.g. in expansion protocols and engraftment assays. scription factor belonging to the MITF family, is specifically expressed in endothelial
Also, the more primitive CD34+CD38- cells are being discussed as an alternative cell cells from the CHT, and is required for HSC expansion in this site. Indeed, tfec gain-
source for stem cell gene therapy approaches with the potential to improve transduction of-function leads to higher HSC proliferation and our CRISPR tfec-/- mutant dies
protocols. Most separation approaches utilize flow-based sorting of CD34+CD38- cells from anemia, due to a complete loss of HSCs. To better understand the molecular
from pre-enriched CD34+ cells. We developed an easy two-step immunomagnetic isola- network controlled by TFEC, we analyzed the transcriptome of endothelial cells
tion protocol: The CD34+ cells are positively enriched in a first separation step, followed sorted from the CHT after tfec gain-of-function. This allowed us to identify new po-
by enzymatic release of the CD34 MultiSort MicroBeads and depletion of CD38+ cells in tential players involved in HSC expansion. One of them, si:ch73-47f2.1 was coding
a second separation step. Starting from 1x10^8 mononuclear cord blood cells with a for a protein containing a four-helical cytokine-like domain. We show that this gene
CD34+ cell frequency from 0.1% to 1%, we typically isolated 9.9x10^3 to 1.1x10^5 is the orthologue of the mammalian Onconstatin M (osm), a cytokine known to
CD34+ cells with low CD38 expression and a mean CD34+ cell purity of 80%. Func- enhance HSCs, but which actions are controversial. We show here that osm can
tional analysis of the isolated cells confirmed the more primitive phenotype of the induce HSC proliferation, while restricting lymphoid commitment. In parallel, we
CD38- subpopulation, e.g. a higher fold expansion of CD34+CD38- cells compared to are using human Endothelial feeders stably overexpressing hTFEC to expand human
CD34+CD38+ cells during in vitro cultivation. cord blood HSCs ex vivo. Altogether, our data show a crucial role for TFEC in regu-
lating the endothelial HSC niche during embryogenesis.
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136 S125
3229 - ENHANCED EX VIVO ERYTHROPOIESIS FROM treatment with the tranylcypromine derivatives, we again observed granulocytic differen-
HUMAN INDUCED PLURIPOTENT STEM CELLS UNDER tiation and a reduction in leukemic stem cell frequency. MN1-transformed cells again
SIMPLIFIED CONDITIONS exhibited no signs of granulocytic differentiation. The differentiating effect of LSD1 in-
hibition in our model was limited to the irreversible inhibitors. We therefore investigated
Claudia Bernecker1,2, Nico Lachmann3, Mania Ackermann3,
if the effect was due to interference with the enzymatic activity of LSD1. We used cells
Holm Zaehres4, Peter Schlenke1, and Isabel Dorn1
1 from mice carrying a conditional knock-in (KI) of an enzymatically inactive form of
Medical University Graz, Graz, Austria; 2Department of Blood Group Serology and
LSD1 (LSD1mut) and assessed its consequence both in vitro and in vivo in the H/M
Transfusion Medicine, Graz, Austria; 3Hannover Medical School, Hannover,
model. Similar to the effect seen with the conditional KO we observed granulocytic dif-
Germany; 4Ruhr University Bochum, Bochum, Germany
ferentiation upon induction of the conditional LSD1mut KI, but no survival benefit in
Until now, cell culture models for the ex vivo erythropoiesis from human induced plurip- vivo. In summary, LSD1 depletion or inhibition using tranylcypromine or its derivatives
otent stem cells (iPSC) are very complex and expensive due to high amounts of cyto- induces granulocytic differentiation, reduces LIC frequency and induces a survival
kines. Furthermore, ex vivo erythropoiesis from iPSC fails in terms of proliferation, benefit in H/M-driven AML. Our data further suggest that interfering with enzymatic
hemoglobin switching and enucleation. Underlying reasons might be a rather primitive and scaffolding functions of LSD1 mediates the effect of LSD1 inhibition.
than definitive phenotype of erythroid cells or a bypass of the highly proliferative progen-
itor stage. Based on a previous work focusing on the generation of myeloid cells, we
adjusted our embryoid body (EB) based ex vivo erythropoiesis model from human
iPSC. We allow the EBs to adhere in a completely defined culture media with much
reduced cytokine support. After three weeks, hematopoietic cells harvested from the su-
pernatant are brought into a three phase erythropoiesis model. We are able to generate a
homogenous population of 95% CD43 positive hematopoietic cells. In contrast to our
previous culture model, harvested cells show a high colony forming potential with a ma-
jority of BFU-E formation. This is reflected by a near 103-fold proliferation in the eryth-
ropoiesis culture (n54). The new protocol further leads to a homogenous 98% GPA
positive population with a high rate of averaged 50% enucleation until day 18 (n54).
In contrast, erythropoiesis from our previous protocol showed 200-fold proliferation
and an averaged enucleation of 20%, in line with reports by others. This new simplified
and cost saving method generates a homogenous population of hematopoietic- and
finally erythroid cells. The formation of stromal layers with associated organoid struc-
tures due to EB adherence might more closely simulate the physiological niche and
therefore enhance BFU-E potential, proliferation and enucleation. Further experiments
investigating the primitive vs. definitive nature of erythroid cells are ongoing.
3230 - LSD1 INHIBITION INDUCES DIFFERENTIATION 3231 - CLONAL DYNAMICS OF SINGLE-DONOR DERIVED
AND DECREASES LEUKEMIC STEM CELL FREQUENCY HUMAN CORD BLOOD HEMATOPOIETIC PROGENITOR
IN HOXA9/MEIS1-DRIVEN AML CELLS IN MURINE XENOGRAFTS REVEALED BY
Jessica Barth1,2,3, Anna-Maria Scheder4, Sebastian Mohr4, CELLULAR BARCODING
Johannes Schulz-Fincke5, Martin Schmitt5, Alexandra Walter5, Mirjam Belderbos1,2, Taco Koster1, Sabrina Jacobs1,
Milica Tosic6, Eric Metzger6, Michael L€ubbert7, Manfred Jung5, Bertien Dethmers-Ausema1, Erik Zwart1, Gerald De Haan1, and
Hubert Serve8, Roland Sch€ule9, and Tobias Berg8 Leonid Bystrykh1
1 1
Department of Medicine II, Hematology/Oncology, Goethe University, Theodor- European Research Institute for the Biology of Ageing, Groningen, The
Stern-Kai 7, Frankfurt, Germany; 2German Cancer Consortium (DKTK), Frankfurt, Netherlands; 2Princess Maxima Center for Pediatric Oncology, Utrecht, The
Germany; 3German Cancer Research Center (DKFZ), Heidelberg, Germany; Netherlands
4
Department of Medicine II, Hematology/Oncology, Goethe University, Frankfurt,
Germany; 5Institute of Pharmaceutical Sciences, University of Freiburg, Freiburg, Human cord blood (CB) provides an attractive source of hematopoietic stem cells for
Germany; German Cancer Consortium (DKTK) Freiburg and German Cancer allogeneic transplantation of patients with a variety of diseases. CB units are selected
Research Center (DKFZ), Heidelberg, Germany; 6Department of Urology and Center for transplantation based upon donor-recipient HLA-matching and on their CD34+ he-
for Clinical Research, University of Freiburg Medical Center, Freiburg, Germany; matopoietic progenitor cell content. Nevertheless, the frequency of hematopoietic stem
7
Department of Medicine I, Hematology, Oncology and Stem Cell Transplantation, cells (HSC) within the CD34+ population and the dynamics of their clonal offspring
University of Freiburg, Freiburg, Germany; German Cancer Consortium (DKTK), remain poorly understood and may differ between donors. Here we use cellular barcod-
Freiburg and German Cancer Research Center (DKFZ), Heidelberg, Germany; ing combined with multiplex high-throughput sequencing to characterize the clonal
8
Department of Medicine II, Hematology/Oncology, Goethe University, Frankfurt, behavior of CB CD34+ cells in murine xenografts. Hereto, CD34+ cells were isolated
Germany; German Cancer Consortium (DKTK), Frankfurt and German Cancer from CB from 10 individual donors, exposed to a lentiviral barcode library and trans-
Research Center (DKFZ), Heidelberg, Germany; 9Department of Urology and Center planted into immune deficient Nod/Scid/IL2Ry-/- mice at doses of 107-108 CD34+
for Clinical Research, University of Freiburg Medical Center, Freiburg, Germany cells/mouse. All transplanted CB units engrafted successfully and produced B-cells, T-
cells and myeloid cells, yet with different kinetics of engraftment. In xenografts that en-
Lysine specific demethylase 1 (LSD1) represents a promising epigenetic target in the grafted with GFP+ cells (n54), we identified an average of 9.3 (range 2.5-15) clones in
treatment of Acute Myeloid Leukemia (AML) and inhibition of LSD1 has been shown blood and 8.8 (range 2.5-15) clones in bone marrow. The majority of clones were
to facilitate a response of AML cells to all-trans retinoic acid (ATRA). In this project, we observed at multiple time points, indicating their steady contribution to hematopoiesis
have now modeled the effect of pharmacological or genetic inactivation of LSD1 in two over time. The contribution of individual clones to the production of B-, T-, and myeloid
different murine leukemia models based on the retroviral overexpression of either Hoxa9/ cells varied substantially. Importantly, the frequency of retrieved HPC clones differed up
Meis1 (H/M) or MN1. We transformed bone marrow cells from conditional LSD1 to 4-fold between different CB units and did not correlate with CD34+-cell content. Alto-
knock-out (KO) mice. Induction of the knock-out in vitro resulted in marked granulocytic gether, cellular barcoding provides a simple, sensitive method to characterize the clonal
differentiation documented by cytological and surface marker changes in H/M-trans- behavior of human hematopoietic stem cells, which may be used to understand and opti-
formed cells and increased the survival of mice transplanted with these cells. MN1-trans- mize CB CD34+ hematopoietic stem cell transplantation protocols.
formed cells did not show signs of granulocytic differentiation. We also treated H/M- and
MN1-transformed cells with different irreversible (tranylcypromine and more potent de-
rivatives) and reversible LSD1 inhibitors. When analyzing H/M-transformed cells after
S126 Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
3233 - IL7R-ALPHA FATE-MAPPING LABELS DISTINCT 3235 - FUNCTIONAL ANALYSIS OF GERMLINE ETV6
ADULT TISSUE RESIDENT MACROPHAGES VARIANTS ASSOCIATED WITH FAMILIAL
Anna E. Beaudin1, Taylor McCann2, Gabriel Leung3, and THROMBOCYTOPENIA AND ACUTE LYMPHOBLASTIC
E. Camilla Forsberg4 LEUKEMIA
1
Molecular and Cell Biology, University of California, Merced, Merced, United Rebekah Baskin1,2, Rina Nishii3, Javad Nadaf3, Katherine Verbist3,
States; 2San Jose State University, San Jose, United States; 3UC Merced, Merced, Paige Tedrick3, Keito Hoshitsuki4, Maoxiang Qian3, Takaya Moriyama3,
United States; 4UC Santa Cruz, Santa Cruz, United States Chunliang Li3, Lewis Silverman5, Melissa Burns5, Ching-Hon Pui3,
Recent studies have revealed that adult hematopoietic stem cells (HSCs) do not Charles Mullighan3, Kim Nichols3, and Jun Yang3
1
contribute substantially to all immune cells, including tissue-resident macrophages. St Jude Children’s Research Hospital, Cordova, United States; 2Tennessee, Cordova,
This delineation has prompted intensive investigation of the contribution of fetal he- United States; 3St Jude Children’s Research Hospital, Memphis, United States;
4
matopoiesis to the establishment of tissue-resident macrophage populations, with the University of Pittsburgh, Pittsburgh, United States; 5Dana-Farber Cancer Institute,
aim of defining the cellular and molecular mechanisms underlying their specification. Boston, United States
Fetal hematopoiesis comprises a series of developmentally-restricted progenitors that
Acute lymphoblastic leukemia (ALL) is the most common childhood cancer and a lead-
drive overlapping waves of hematopoietic cell generation. However, a knowledge gap
ing cause of pediatric morbidity and mortality despite high cure rates. Common and rare
exists between the identification of developmentally limited hematopoietic progeni-
germline genetic variants can influence the risk of developing childhood ALL. Recently,
tors and their specific contribution to the developing and adult immune system.
we and others identified heterozygous germline ETV6 mutations associated with hered-
Here, we compared the contribution of cells with an expression history of IL7ra,
itary thrombocytopenia and increased risk for hematologic malignancies, particularly
Rag1, or Flk2 – markers of recently identified hematopoietic progenitors – to adult
ALL (also known as Thrombocytopenia 5). Sequencing of 4,405 childhood ALL cases
tissue-resident macrophage subsets in vivo. Comparison of cre-mediated recombina-
identified 30 potentially damaging germline ETV6 variants in 35 patients. We conducted
tion within adult tissue macrophages across these three lineage-tracing models re-
a systematic in vitro evaluation of these variants by examining transcriptional repressor
vealed strikingly different labeling patterns. Surprisingly, whereas the majority of
activity, DNA binding, nuclear localization and homo-dimerization. We found that of the
circulating monocytes and BM myeloid progenitors were unlabeled, IL7Ra-Cre
30 variants, 18 including 13 of the 14 variants in the ETS/DNA binding domain impaired
led to almost complete labeling of macrophages in some tissues and intermediate
transcriptional repression activity via a putative dominant-negative mechanism. These 18
levels of labeling in other tissues. Examination of IL7ra-cre-driven labeling during
variants also showed impaired nuclear localization and DNA binding capacity yet main-
fetal development revealed significant labeling of multipotent and myeloid progeni-
tained the ability to dimerize with wild-type ETV6. By whole genome sequencing, we
tors in the fetal liver, despite an inability of IL7ra-marked progenitors to sustain mul-
identified several recurrent somatic variants, including activating KRAS or NRAS muta-
tilineage reconstitution upon transplantation. Remarkably, investigation of fetal
tions in 3 of 6 leukemia samples from patients harboring damaging germline ETV6 var-
macrophage development revealed highly regulated and dynamic expression of
iants. To further understand the biological role of ETV6 mutations in the development of
Il7ra message and protein upon acquisition of macrophage identity within specific
thrombocytopenia and ALL, we used a CRISPR-Cas9 mediated homology directed
tissues. Analysis of IL7ra-/- mice indicated a functional role for IL7ra signaling in
repair (HDR) strategy to create a genetically modified mouse model expressing the mu-
tissue resident macrophage development. These data provide novel evidence for a
rine equivalent of the damaging variant R359X, which introduces a stop codon within the
distinctly fetal function of IL7ra in specifying tissue-resident macrophages during
ETS domain of ETV6. We found that heterozygous mice harboring this truncating mu-
fetal liver hematopoiesis, and identify IL7 as a cytokine that directly and selectively
tation displayed thrombocytopenia consistent with patients carrying the deleterious
regulates tissue-resident macrophage identity.
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136 S127
ETV6 variants. In summary, we have systematically characterized 30 germline ETV6 mation. Moreover, identification of altered regulatory states may contribute to the iden-
variants associated with ALL risk, identified potential cooperating somatic events in tification of potential therapeutic targets that might be used to block AML.
ALL with germline ETV6 variants, and established a mouse model of ETV6-associated
thrombocytopenia. This mouse model is a critical tool to further assess the relationship
between germline ETV6 mutations and development of thrombocytopenia and/or ALL.
BM cells transduced with MPLW515L rapidly develop MF, myeloproliferation and 3239 - CHARACTERISTICS OF RECIPIENT’S BONE
splenomegaly. Flow cytometry showed an expansion of immature and mature megakar- MARROW STROMAL CELLS PRIOR TO ALLO-BMT
yocytes with overexpression of TGF-ß1 in MPLW515L cells. Treatment from day 12 to Ildar Barkhatov1, Nikolai Tsvetkov1, Daniil Ershov1, Alena Shakirova1,
day 26 after transplantation significantly reduced fibrosis as measured by reticulin stain-
Ludmila Zubarovskaya2, and Boris Afanasyev1
ing and hydroxyproline quantification. In vitro, Galunisertib counteracted the pro- 1
Academician I.P.Pavlov First St.Petersburg State Medical University, St. Petersburg,
fibrotic effects of TGF-ß1 on both murine and human MSC by antagonizing its stimula-
Russia; 2M. Gorbacheva Memorial Institute of Children Oncology, Hematology and
tory effects on collagen I and III production. Analysis of MSCs from Galunisertib-
Transplantation, St. Petersburg, Russia
treated MPLW515L mice also showed reduced collagen I and III production compared
to vehicle controls. In conclusion, we offer further evidence that aberrant megakaryopoi- Introduction: Deficient graft functioning observed in some cases necessitates devel-
esis drives TGF-ß1 overproduction in MPN by increasing megakaryocyte numbers and opment of functional tests for the stromal cells, in order to provide clinical indica-
overexpressing TGF-ß1 in mutant MKs. Galunisertib counteracts TGF-ß1 induced tions for co-transplanting of hematopoietic and bone marrow stromal cells
fibrosis in two MPN mouse-models and is a promising candidate for symptomatic treat- (BMSC). The aim of this study was to investigate the role of bone marrow stromal
ment of myelofibrosis. cells in the course of donor marrow cells engraftment and their significance in
post-transplant complications. Materials and methods: The study included clinical
observation of the post-transplant course in 38 patients with acute myeloid leukemia
(AML) and 20 healthy donors. Bone marrow nucleated cells were selectively har-
vested prior to BMT and followed by monolayer culture. Upon growth of fibro-
blast-like cell colonies (CFU-F), their hematopoiesis-supporting activity was
determined, as well as their differentiating ability. Results: When comparing func-
tional characteristics of BMSC from healthy donors and AML patients, an increased
hemostimulatory activity of the latter was noted, as reflected by an increase in large
and small CFU-GM numbers (p ! 0.02). In addition, an increase in differentiation
along adipogenic and osteogenic pathways was observed in AML patients (p 5
0.03). Moreover, the number of CFU-Fs, capable for adipogenic differentiation
was inversely correlated with platelet recovery time (p 5 0.05). In contrast, higher
numbers of osteogenic colonies in culture were associated with an increased time
to leukocyte lineage recovery. When analyzing gene expression in BMSC population,
a decreased expression of the CXCR4 gene responsible for the homing effect, with
age of the patient’s was noted. Conclusion: Stromal cells derived from the patients’
bone marrow exhibit higher proliferative activity and marked expression of mole-
cules mediating HSC homing, as compared with a group of healthy donors. BMSC
from AML patients taken before bone marrow transplantation are characterized by
a more pronounced capacity to osteogenic and adipogenic differentiation than those
from healthy donors. Increased adipogenic differentiation ability of the BMSCs is
associated with a more rapid recovery of hematopoiesis.
3238 - BONE MARROW STROMAL CELLS ARE CAPABLE 3240 - RECIPIENT ORIGIN FETAL MICROCHIMERISM IN
TO GAIN GENETIC ABERRATIONS IN A CULTURE DEVELOPMENT OF ACUTE GVHD AFTER
Ildar Barkhatov, Daniil Ershov, Nikolai Tsvetkov, Alena Shakirova, HAPLOIDENTICAL BMT
Tatiana Gindina, Ludmila Zubarovskaya, and Boris Afanasyev Ildar Barkhatov, Alena Shakirova, Olesya Smykova, Sergey Bondarenko,
Academician I.P. Pavlov First St. Petersburg State Medical University, St. Petersburg, Ludmila Zubarovskaya, and Boris Afanasyev
Russia Academician I.P.Pavlov First St. Petersburg State Medical University, St.Petersburg,
Russia
Introduction: Recent data supports the idea that bone marrow stromal cells (BMSC)
also have genetic aberrations and may tightly involved in the pathogenesis of HSCT Introduction: Haploidentical transplantation of hematopoietic stem cells (haplo-
complications. The aim of this study was to evaluate genetic aberrations in BMSC HSCT) is a feasible therapeutic approach in patients lacking an HLA-identical donor.
and check the ability to gain them in coculture system. Methods: The interaction of Currently, there exist some data concerning associations between maternal microchi-
BMSC with hematopoietic tumor cell lines bearing specific genetic aberrations (K-562 merism levels (detection of maternal cells in recipient’s blood) and increased risk for
and Uke-1) was investigated in stromal cells harvested from 17 patients and 8 healthy do- GVHD development. Meanwhile, the effects of microchimerism upon GVHD prob-
nors. We cultivated BMSCs and tumor cells using semipermeable membrane inserts with ability are still controversial. The aim of our study was to adapt a technique for quan-
different pore size (0.4 mm and 3.0 mm). We looked also forexisting specific genetic ab- titative evaluation of fetal (recipient-cell) and mathernal (donor-cell) microchimerism
errations in leukemic clone (fusion transcripts) in BMSC of patients. Results: We exam- and investigation of its effects upon actual risk of common complications after
ined BMSC from leukemia patients bearing recurrent genetic abnormalities and in one HSCT. Methods: Determination of microchimerism was carried out by real-time
case the leukemia-specific ETV6-RUNX1 fusion gene was detected by RQ-PCR alle-specific polymerase chain reaction (AS RQ-PCR). The analytic panel included
(z0.02%) in BMSC by ALL patient. When BMSCs and Uke-1 cell line were cocultured 20 InDel markers located at different chromosomes. Evaluation of PCR sensitivity
by using of 3.0 mm pore culture dishes membrane inserts, the BMSC population gained was carried out using DNA mixtures from several established cell lines at serial di-
the Jak2V617F mutation (allele burden z 30.39%). We reproduced similar experiments lutions. The samples from 20 donor-recipient pairs were analyzed. The patients’ age
with the K-562 cells and got similar results – the BCR-ABL gene expression in BMSC ranged from 2 to 27 years. Results: The sensitivity thresholds for the InDel-based test
was detected (BCR-ABL/ABL*100519). We repeated same test with 0.4 mm pore inserts panel ranged from 10^- 4 to 10^-5. We have found out that detection of fetal microchi-
and without them in order to check implication of cell-to-cell interaction. We didn’t obtain merism in the donor organism is associated with lower risk, or lower degree of acute
any similar results with smaller pores, but the fusion transcript was detected in CD45- GVHD (p50.01). Moreover, we observed a trend to higher timeframe for transplant
BMSC population when these two cell populations weren’t devided. Both findings point engraftment, and lower probability of full donor chimerism achievement, if the donor
out at possible horizontal gene transfer mediated by membrane vesicles larger than 0.4 mm exhibited fetal microchimerism (p50.12). The patients in our setting transplanted
and direct whole cell fusion. Conclusions: Our data stands for the existence of horizontal from donors with detectable fetal microchimerism had a tendency to a higher overall
gene transfer between leukemic clone and BMSC. This process seems to be mediated by survival (p50.14). Hovewer, we did not reveal any significant association between
membrane vesicles larger than 0.4 mm in size. We also confirmed the fact BMSCs can microchimerism levels (fetal and maternal), and higher probability of chronic
bear clonal genetic rearrangements which are not specific to tumor cell populations. These GVHD development, as well as any differences in other posttransplant complica-
findings show tight interaction between tumor and microenvironment cells and may partly tions for recipients with maternal microchimerism. Conclusions: Evaluation of
explain nature of PCR-based MRD persistence in complete remission.
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136 S129
fetal microchimerism may be considered a useful and informative approach to significantly impaired proliferation and clonogenic growth in vitro and delayed onset
selection of potential donors, or a method of GVHD prediction in haploidentical of leukemia in NSG mice. PIWIL4 KD in primary AML patient BM cells lead to 5-
HSCT. fold decrease in clonogenicity, but had no impact on clonogenicity of healthy stem
progenitors in vitro. Western blot and ChIP-seq in THP-1 cell line revealed a marked
global reduction in repressive H3K9me3 marks upon PIWIL4 KD. Over 500 pro-
moter and 600 gene body associated loci exhibited loss of H3K9me3 marks. RNA-
seq analyses revealed over 4000 differentially expressed genes upon PIWIL4 deple-
tion. 30% of the loci that lost H3K9me3 marks at promoters and gene body were
differentially expressed in RNA-seq. These genes belonged to pathways associated
with RNA metabolism, transcription and cell death. Moreover, these genes were en-
riched for binding sites of SETDB1. Notably, PIWIL4 was found to associate with
SETDB1 in 293T cells. 560 unique piRNAs were found to physically bind with PI-
WIL4 and 981 unique piRNAs were differentially expressed upon PIWIL4 depletion
in THP-1 cells. Thus, collectively, we could show for the first time that PIWIL4
expression is deregulated in human AML and acts as a piRNA binding, epigenetically
active growth regulatory protein in human AML.
3241 - ABERRANTLY EXPRESSED STEM CELL 3242 - ABERRANTLY EXPRESSED TET1 IN T-ALL
ASSOCIATED PROTEIN PIWIL4 ACTS AS A PIRNA REGULATES DNA REPAIR AND LEUKEMIC GROWTH VIA
BINDING, EPIGENETICALLY ACTIVE AND GROWTH MAINTENANCE OF 5-HYDROXYMETHYLOME AND CAN
REGULATORY FACTOR IN HUMAN ACUTE MYELOID BE ANTAGONIZED BY THE PARP INHIBITOR OLAPARIB
LEUKEMIA Shiva Bamezai1, Deniz Demir1, Alex Jose1, Fabio Ciccarone2,
Shiva Bamezai1, Medhanie Mulaw1, Naidu Vegi1, Fengbiao Zhou2, Elena Fischbein1, Fabian Mohr1, Medhanie Mulaw1, Paola Caiafa3,
Christian Rohde2, Konstanze D€ohner3, Hartmut D€ohner3, L€uder Meyer4, Klaus-Michael Debatin4, Chiara Borga5,
Michaela Feuring-Buske3, Jessica H€oll4, Andreas Kloetgen4, Geertruy te Kronnie6, Konstanze D€ohner7, Hartmut D€ohner7,
Arndt Borkhardt5, Alexei Aravin6, Christoph Plass7, Michaela Feuring-Buske7, Christian Buske8, and Vijay Rawa8
1
Carsten M€uller-Tidow8, Vijay Rawat1, and Christian Buske1 Institute of Experimental Cancer Research, University Hospital of Ulm, Ulm,
1
Institute of Experimental Cancer Research, University Hospital of Ulm, Ulm, Germany; 2Department of Cellular Biotechnology and Haematology, Medicine 2,
Germany; 2Department of Medicine IV, Hematology and Oncology, University University of Rome ‘La Sapienza’, Rome, Italy; 3Department of Cellular
Hospital of Halle (Saale), Halle (Salle), Germany; 3Department of Internal Medicine Biotechnology and Haematology, Medicine 2, University of Rome ‘La Sapienza’,
III, University Hospital of Ulm, Ulm, Germany; 4Department of Pediatric Oncology, Rome, Italy, Rome, Germany; 4Department of Pediatrics, University Hospital Ulm,
Hematology and Clinical Immunology, Heinrich-Heine-University, Medical Faculty, Ulm, Germany; 5Department of Pediatrics, University of Padova, Padova, Italy;
6
D€usseldorf, Germany; 5Clinic for Child Oncology, Hematology and Clinical Department of Pediatrics, University Hospital Ulm, Ulm, Germany, Padova, Italy;
7
Immunology, D€ usseldorf, Germany; 6Division of Biology and Biological Department of Internal Medicine III, University Hospital Ulm, 89081, Ulm,
Engineering, California Institute of Technology, Pasadena, CA, United States; Germany; 8Institute of Experimental Cancer Research, CCC and University Hospital
7 of Ulm, Ulm, Germany
Division of Epigenomics and Cancer Risk Factors, German Cancer Research Center
(DKFZ), Heidelberg, Germany; 8Medizinischen Klinik V Schwerpunkt H€amatologie,
The Ten-eleven translocation 1 (TET1) enzyme catalyzes the oxidation of 5-methyl-
Onkologie und Rheumatologie, Heidelberg, Germany
cytosine (5mC) into 5-hydroxymethylcytosine (5hmC) and plays a key role in active
Piwi proteins are essential for maintaining the germ stem cell population in lower or- DNA demethylation and regulation of gene expression. TET1 has been shown play a
ganisms through epigenetic silencing of transposable elements via DNA methylation tumor supper function in B-cell malignancies however, its roles in T-cell acute
and H3K9me3 marks, in close interaction with non-coding RNA called piwi interact- lymphoblastic leukemia (T-ALL) is yet unknown. qRTPCR revealed that TET1
ing RNA (piRNA). There are neither precise data on the function of Piwi proteins in was significantly higher expressed in all human T-ALL cell lines and 95% of primary
human acute myeloid leukemia (AML), nor reports on expression of piRNAs in this T-ALL patients compared to B-ALL cell lines, other primary leukemia types and
disease. Among the family of human PIWIL genes, PIWIL4 showed the highest healthy T-cells. This was further confirmed by cDNA microarray analysis on larger
expression level and was ubiquitously expressed in healthy hematopoietic stem/pro- cohort of primary leukemia patient samples. Knockdown (KD) of TET1 using
genitors, mature lymphoid and myeloid cells. PIWIL4 was aberrantly higher ex- shRNAs in T-ALL cell lines adversely affected their cell growth and clonogenicity
pressed in more than 89% of the AML patients compared to healthy CD34+ BM in vitro, and significantly reduced their leukemic engraftment potential in xenografts.
and total BM cells. Overexpression of AML specific oncogenes in murine stem pro- Global analysis of 5hmC and 5mC marks using hMeDIP-seq and MeDIP-seq per-
genitors, within 96h post-transduction, induced O6 fold increase in Piwil4 expres- formed on TET1 depleted JURKAT cells revealed lower global 5hmC levels and
sion compared to GFP control. Knockdown (KD) of PIWIL4 in AML cell lines increased 5mC levels (n52). Promoters (-5kbTSS) demonstrating loss of 5hmC or
S130 Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
gain of 5mC marks belonged to gene families involved in DNA repair, Cell cycle, T- ropoiesis. These data suggest that RUNX3 plays an integral role in megakaryocy-
cell development, Cancer and Wnt signaling. Changes in promoter 5hmC/5mC marks te-erythroid specification, but that CBFb is required only for the megakaryocyte
were paralleled by changes in expression of DNA repair and T-cell associated genes lineage.
as measured by RNA-Seq, and protein levels as measured by western blot. Further-
more, KD of TET1 in T-ALL cell lines induced DNA damage and cell cycle arrest in
the G2M phase. Aberrant expression of TET1 could be reverted by Olaparib, an in-
hibitor of DNA repair enzyme PARP, which induced a reduction of euchromatic
marks on the TET1 promoter accompanied by decrease in TET1 mRNA and protein
levels and global reduction of 5-hmC levels in T-ALL cell lines. Olaparib treatment
adversely affected cell growth, clonogenicity and leukemic engraftment potential of
T-ALL cell lines. In conclusion, these data demonstrate for the first time that TET1
plays an important oncogenic role in T-ALL. We also identify PARPs as upstream
regulators of TET1 in T-ALL, opening the way to antagonize TET1 pharmacologi-
cally in this poor prognosis leukemia.
3243 - RUNX3 IS REQUIRED FOR MEGAKARYOCYTE- 3244 - A CLUSTER OF ENHANCER MODULES DIRECTS
ERYTHROID SPECIFICATION IN PRIMARY HUMAN DIFFERENTIAL MYC EXPRESSION ALONG THE
PROGENITORS NORMAL AND LEUKEMIC HAEMATOPOIETIC STEM
Peter Balogh and Adam Goldfarb CELL HIERARCHIES
University of Virginia, Charlottesville, United States Carsten Bahr1,2, Lisa von Palekse3, Veli Uslu4, Silvia Remeseiro4,
Naoya Takayama5, Stanley Ng6, Alex Murison7, Katja Langenfeld4,
The RUNX family of transcription factors is comprised of three RUNX proteins and a
common binding partner, CBFb, that enables DNA binding. These factors regulate Massimo Petretich4, Roberta Scognamiglio8, Petra Zeisberger3,
stem cell maintenance, lineage specification, and cellular differentiation. In the Amelie Benk3, Ido Amit9, Peter Zandstra6, Mathieu Lupien10, John Dick11,
context of malignancies, RUNX1 and CBFb are the most common targets for fusion Francois Spitz12, and Andreas Trumpp13
1
and point mutations that give rise to such disease states as familial platelet disorder, German Cancer Research Center (DKFZ), Heidelberg, Germany; 2Heidelberg
acute myelogenous leukemia, and myelodysplastic syndromes. By contrast, RUNX3 Institute for Stem Cell Technology and Experimental Medicine (HI-STEM),
dysregulation has been observed in solid tumors. In healthy tissue, RUNX1 is Heidelberg, Germany; 3Division of Stem Cells and Cancer, Deutsches
required for the ontogenic switch from primitive to definitive hematopoiesis. In Krebsforschungszentrum (DKFZ) & Heidelberg Institute for Stem Cell Technology
mature organisms, RUNX1 mediates transitions between non-lineage stem cell states, and Experimental Medicine (HI-STEM gGmbH), Heidelberg, Germany;
4
protects against inappropriate stem cell expansion, and plays discrete roles in Developmental Biology Unit, European Molecular Biology Laboratory (EMBL),
myeloid differentiation. RUNX2 and RUNX3 have known roles in bone and Heidelberg, Germany; 5Princess Margaret Cancer Centre, University Health Network,
lymphoid cell specification, respectively. The role of CBFb in lineage specification Toronto & Department of Molecular Genetics, University of Toronto, Toronto, Canada;
6
is not well understood. It is known that erythroid cells down-regulate CBFb during Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto,
differentiation, but megakaryocytes, a lineage derived from a common progenitor, Canada; 7Princess Margaret Cancer Centre, University Health Network, Toronto &
do not. Therefore, we investigated whether CBFb is required for this lineage bifurca- Department of Medical Biophysics, University of Toronto, Toronto, Canada; 8Division of
tion in primary human progenitors. We found that lentiviral-mediated knock-down of Stem Cells and Cancer, Deutsches Krebsforschungszentrum (DKFZ), Heidelberg
CBFb blocked formation of CD41+ megakaryocytes, but spared formation of glyco- Institute for Stem Cell Technology and Experimental Medicine (HI-STEM gGmbH),
phorin A+ erythroid cells. When screening knock-downs of each of the RUNX Heidelberg, Germany; 9Department of Immunology, Weizmann Institute of Science,
factors, we discovered that only loss of RUNX3 resulted in a similar blockade of Rehovot, Israel; 10Princess Margaret Cancer Centre, University Health Network, Toronto
megakaryopoiesis. Surprisingly, we observed that loss of RUNX3 also blocked eryth- & Department of Medical Biophysics, University of Toronto, Toronto, Canada;
Poster Presentations/ Experimental Hematology 53 (2017) S54-S136 S131
11
Princess Margaret Cancer Centre, University Health Network, Toronto & Department together, our results indicate that Fli1 is required for maintenance of HSC homeosta-
of Molecular Genetics, University of Toronto, Toronto, Toronto, Canada; 12(Epi) sis and function, making it one of the unique transcription factors to play a critical
genomics of Vertebrate Development, Department of Developmental Biology and Stem role in HSC function in adults as well as during development.
Cells, Institut Pasteur & Developmental Biology Unit, European Molecular Biology
Laboratory (EMBL), Paris, France; 13Division of Stem Cells and Cancer, Deutsches
Krebsforschungszentrum (DKFZ) & Heidelberg Institute for Stem Cell Technology and
Experimental Medicine (HI-STEM gGmbH), Heidelberg, Germany
The ETS family of transcription factors is known to play an essential role in hemato-
poietic and vascular development. One such factor that is widely expressed in all
vascular beds and almost all hematopoietic lineages, extending from long term he-
matopoietic stem cells to terminally differentiated peripheral blood cells, is Fli1.
Global deletion of Fli1 leads to embryonic lethality at E12.5 due to dramatic hemor-
rhaging caused by poor vascular integrity and platelet dysfunction. The role of Fli1 in
adults has been explored in several different cells types ranging from megakaryocytes
to B and T cells. It has also been implicated in transcriptional regulation of hemato-
poietic stem and progenitor cells through combinatorial analysis, however its exact
contribution to stem cell maintenance and function remains unclear. We hypothesized
that Fli1 plays a dual role in regulating both the seed and the soil – specifically he-
matopoietic stem cell (HSC) function in a cell autonomous manner as well as the
niche required for nurturing these cells. In this study, we focused our attention on
the cell autonomous function of Fli1. We found that global deletion of Fli1 leads
to lethality as a result of complete peripheral blood failure in addition to aberrant
vasculature. Using the cre-lox system and various transplantation strategies, we iden-
tify Fli1 as one of the critical regulators of adult hematopoietic stem cell function.
Specific deletion of Fli1 in the hematopoietic compartment alone is sufficient to
induce significant reduction in peripheral blood counts, accompanied by hemorrhage,
resulting in lethality. On further inspection, Fli1-/- HSCs are unable to expand ex
vivo or engraft in a competitive setting. Additionally, Fli1 is essential for hematopoi-
etic reconstitution post radiation and its deletion abolishes the ability of stem cells to
reconstitute the bone marrow and contribute to peripheral blood lineages. Taken
S132 Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
3247 - EMBRYONIC HEMATOPOIETIC PROGENITORS 3249 - HUMAN HSPC HARBOR ROS-DEPENDENT SELF-
DEPEND ON KIT LIGAND FOR IN VIVO EXPANSION AND RENEWAL DEFECTS AFTER EXPOSURE TO A SINGLE
DIFFERENTIATION ACUTE LOW DOSE OF IONIZING RADIATIONS
Emanuele Azzoni1,2, Kathleen McGrath3, Vincent Frontera1, Marie-Laure Arcangeli1,2, Ines Souissi-Sahraoui2, Elia Henry3,
Joe Harman1, Joana Carrelha4, Claus Nerlov1, James Palis3, Margaux Deynoux3, Andreas Lefevre2, Vilma Barroca3, and
Sten Eirik Jacobsen5, and Marella Dr Bruijn1 Francoise Pflumio3
1 1
MRC Molecular Hematology Unit, Weatherall Institute of Molecular Medicine, INSERMU967, FONTENAY-AUX-ROSES, France; 2CEA, FONTENAY-AUX-
University of Oxford, Oxford, United Kingdom; 2Oxfordshire, Oxford, United ROSES, France; 3INSERM U967, FONTENAY-AUX-ROSES, France
Kingdom; 3Center for Pediatric Biomedical Research, University of Rochester
Medical Center, Rochester, United States; 4Hematopoietic Stem Cell Laboratory, Hematopoietic stem cells (HSC) are responsible for life-long blood cell regener-
Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United ation. In the adult, HSC quiescence, self-renewal and differentiation capacities
Kingdom; 5Wallenberg Institute for Regenerative Medicine, Department of Cell and are tightly regulated by intrinsic and extrinsic signals provided by the bone
Molecular Biology and Department of Medicine Huddinge, Karolinska Institutet and marrow (BM) microenvironment. Nowadays, HSC endure more and more
Center for Hematology and Regenerative Medicine, Karolinska University Hospital genotoxic stresses such as radiations or chemotherapy that can alter their self-
Huddinge, Stockholm, Sweden renewal properties. Indeed, people are increasingly exposed to low doses of
ionizing radiations (LDIR), mostly provided by medical imaging procedures. It
The micro-environmental factors orchestrating the establishment of a functional he- is therefore of major importance to evaluate the biological consequences of
matopoietic system are poorly understood. Kit ligand (Kitl; also known as Stem Cell such procedures. Here, we studied the consequences of a single acute 20mGy
Factor/SCF, or steel factor) is a pivotal cytokine in adult hematopoiesis. Its role in LDIR on differentiation and self-renewal capacities of human cord blood-
embryonic hematopoiesis, however, is still unclear. We investigated in detail the he- derived HSC using ex-vivo and in vivo (xenograft models) LDIR protocols.
matopoietic phenotype of early Steel (Sl/Sl) mutant embryos, completely lacking We observed that LDIR have no or little impact on HSC differentiation and
Kitl. We show that in the absence of Kitl, erythro-myeloid progenitor (EMP) expan- reconstitution potentials but impair their self-renewal capacities. Single
sion in the yolk sac and fetal liver is severely compromised due to a decrease in pro- 20mGy LDIR do not induce DNA double strand breaks, suggesting no major
liferation and an increase in apoptosis. Mutant embryos show a dramatic decrease of defect in DNA repair, but results in an increase in ROS production in HSC imme-
fetal liver erythroid cells downstream of EMPs, already evident prior to the onset of diately following LDIR and the activation of p38MAPK pathway. Importantly
hematopoietic stem cell (HSC)-derived erythropoiesis. We also observed a depletion ex-vivo treatment of HSC with ROS or p38MAPK inhibitors prior to radiation
of tissue macrophages, known to be derived, at least in part, from EMPs. In addition allows rescuing the 20 mGy-induced-HSC defects. Altogether our results indi-
to EMPs, yolk sac-derived immune-restricted progenitors were decreased in the Sl/Sl cate that a single 20 mGy LDRI induces HSC self-renewal defect through a
embryo. In the aorta-gonad-mesonephros (AGM) region, Sl/Sl embryos display a ROS/p38MAPK pathway.
reduction of HSCs and progenitors, with an apparent defect in the maturation of
pre-HSC type II cells. This differentiation block is also underlined by RNA
sequencing of Sl/Sl AGM hematopoietic clusters. Furthermore, by using a Kitl trans-
genic reporter line, we show that Kitl-expressing cells are found in all sites of he-
matopoietic emergence and expansion. Thus, our in vivo data demonstrate a
previously unrecognized early role for Kitl in the expansion of yolk sac-derived
EMPs and maturation of the AGM HSC lineage and identify this cytokine as a com-
mon regulator in embryonic hematopoietic niches. Interestingly, it is the EMP defect
that underlies the early erythroid defect in the Sl/Sl fetal liver.
Methyladenosine (2-CM) a nucleoside analogue which inhibits mitochondrial replating capacity was not affected suggesting no significant effects on normal
POLRMT. We could see a significant decline in proliferation in both human cell lines HSPC. In both murine congenic as well as xenotransplantation models of AML, treat-
and in murine leukemic cells in a concentration-dependent manner, measured by the ment with our novel PU.1 inhibitors decreased tumor burden and resulted in
MTT assay. Next we have started to investigate the effect of 2-CM in vivo in our BM increased survival. Our study establishes proof-of-concept for PU.1 inhibition as a
transplantation mouse model. The drug has been administrated for eleven days two novel therapeutic strategy for the treatment of AML, and shows that it is feasible
weeks post transplantation and compared with mice treated with only the vehicle. to pharmacologically target a transcription factor through minor groove-directed
The drug has been well tolerated by the mice and is now being followed to determine approaches.
whether the drug can delay or prevent onset of acute leukemia. In conclusion, our in
vitro experiment show promising results suggest that inhibiting the mitochondrial
transcription can be used as a potential therapeutic strategy in the treatment of
AML but our in vivo results are still pending but will be presented.
3257 - ASSESSMENT OF ER STRESS IN MESENCHYMAL formed typical CFU-GM and BFU-E colonies in Methocult (Stem Cell Technologies). In
STEM CELLS FROM HEALTHY AND OBESE BONE conclusion, iPS cells from a Griscelli type 2 patient could be obtained from mononuclear cells
MARROW DONORS using LV-OSKM. Cells showed typical iPS morphology and expression of pluripotency
markers and could be redifferentiated towards hematopoietic stem cells.
Baris Ulum1, Hikmet Taner Teker2, G€unay Balta1,
Duygu Uçkan-Çetinkaya1, and Fatima Aerts-Kaya1
1
Hacettepe University, Institute for Health Sciences, Dept of Stem Cell Sciences,
Center for Stem Cell Research and Development, Ankara, Turkey; 2Middle East
Technical University, Department of Biological Sciences, Ankara, Turkey
3258 - DEVELOPMENT AND CHARACTERIZATION OF IPS 3259 - HUMAN MESENCHYMAL STEM CELLS SUPPORT
CELL LINES FROM A PATIENT WITH GRISCELLI TYPE 2 B-CELL AND T-CELL DIFFERENTIATION OF CORD
DISEASE BLOOD CD34+ CELLS UNDER OPTIMIZED CONDITIONS
G€
ulen G€ €
uney2, Burcu Pervin2, Ozge Burcu Sahan2, Gerard Wagemaker, Duygu Uçkan-Çetinkaya, and Fatima Aerts-Kaya
€
Duygu Uckan-Cetinkaya2, Ayşen G€unel-Ozcan2
, Axel Schambach3, and Hacettepe University Center for Stem Cell Research and Development, Ankara, Turkey
Fatima Aerts-Kaya1 In vitro assays for T/B/NK lymphopoietic differentiation could be helpful to assess the differ-
1
Hacettepe University, Institute for Health Sciences, Dept of Stem Cell Sciences, Center
entiation potential of hematopoietic stem cells (HSCs) from patients with immune defi-
for Stem Cell Research and Development, Ankara, Turkey; 2Hacettepe University Center
ciencies before and after genetic correction. Current protocols involve the use of murine
for Stem Cell Research and Development, Ankara, Turkey; 3Hannover Medical School
Op9/Op9-DL1 cell lines. However, these protocols are suboptimal for differentiation of hu-
Institute for Experimental Hematology, Hannover, Germany
man HSCs. NOTCH1 signaling occurs through its ligands Delta-Like 1 (DL1) and is impor-
Griscelli Syndrome Type 2 (GS-2) is a rare, genetic immune deficiency, caused by mutations tant for T cell differentiation. Here, we developed immortalized cell lines using human bone
in the RAB27A gene, which causes functional impairment of in particular cytotoxic T cells marrow-derived mesenchymal stem cells (MSCs) and 3rd generation self-inactivating (SIN)
(CTL) and Natural Killer (NK) cells. GS-2 is a rare disease and diagnosed in ! 1/1000.000 lentiviral vectors carrying a codon-optimized gene for human telomerase reverse transcrip-
newborns. Here, we aimed to develop and characterize induced Pluripotent Stem (iPS) cells tase (hTERTco) and eGFP. Subclones were transduced with codon-optimized human
from a patient with GS-2. Bone marrow mononuclear cells were isolated and pre-cultured in hDL1co-Tomato. The hTERTco cell line was used for B-cell differentiation; the
StemMACS HSC expansion medium (Miltenyi) supplemented with SCF, TPO and Flt3- hTERTco/hDL1co cell line was used for T-cell differentiation. CD34+ UCB cells were
ligand for 5 days. Cells were subjected to two days of transductions with lentiviral vectors cultured for 6 weeks in presence of differentiation medium containing Flt3 ligand, IL-7
carrying Oct3/4, Klf4, Sox2 and c-Myc (LV-OSKM). Cells were cultured for an additional and c-fms inhibitor III, to inhibit M-CSF signaling. Once weekly, 10% of the hematopoietic
5 days in HSC expansion medium and then plated on CTS-coated 24-well dishes. Media cells was collected and analyzed, remaining cells were replated on fresh hTERTco or
were changed to StemMACS iPSBrew (Miltenyi), supplemented with bFGF. After approx- hTERTco/hDL1co feeder layers. After 6 weeks of co-culturing on hTERTco, O20% of cells
imately 2 weeks typical colonies appeared and 3 clones were selected and characterized for expressed the B-cell marker CD20. Co-culturing on hTERTco/hDL1co resulted in robust
pluripotency markers using immunofluorescence staining and flow cytometer (FACS). T-cell differentiation (CD3+/CD4+ and CD3+/CD8+ cells) and when stimulated for
Pluripotent cells were then co-cultured on Op9 stromal cell layers for 9 days in StemMACS 4 days with PHA, these cells displayed typical clonal expansion. In conclusion, functional
HSC expansion medium, supplemented with SCF, TPO, Flt3-ligand and BMP-4 and B and T-cell differentiation on human MSC cell lines is feasible under controlled culture
analyzed by FACS and Methocult (Stem Cell Technologies) assay. All colonies displayed conditions.
typical iPS cell morphology and stained positive for Oct3/4, Sox2, TRA-1-60 and SSEA4
(immunofluorescence). Cells were positive for Oct3/4 and SSEA4 by FACS analysis, and
negative for hematopoietic and mesenchymal markers, including CD45, CD73 and CD90.
Up to 15% of iPS cells co-cultured on Op9 stromal cells expressed the CD34 antigen, and
S136 Poster Presentations/ Experimental Hematology 53 (2017) S54-S136
3260 - HYPOTHERMIC STORAGE OF HEMATOPOETIC the question whether PKNOX2 has a role on HSC self-renewal and differentiation, PKNOX2
STEM CELLS CAN BE USED AS AN ALTERNATIVE TO overexpression plasmid was transfected into BM-MSCs and CD34+ HSCs were co-cultured
SHORT-TERM CRYOPRESERVATION with pPKNOX2:MSCs. Flow cytometer analysis showed that CD34+ HSCs co-cultured with
PKNOX2 overexpressing BM-MSCs had higher CD38+ cells but lower CD34+ cells
Trui Visser2, Burcu Pervin2, Duygu Uçkan-Çetinkaya2,
compared to controls. Colony forming assays revealed an increase in CFU capacity, especially
Gerard Wagemaker2, and Fatima Aerts-Kaya1
1 in BFU-E. Taken together, our results suggest that hyperactive secretion of TGFbeta1 from FA
Hacettepe University, Institute for Health Sciences, Dept of Stem Cell Sciences, Center
HSCs may change PKNOX2 expression levels in FA BM-MSCs, resulting in the increase of
for Stem Cell Research and Development, Ankara, Turkey; 2Hacettepe University
HSC self-renewal and differentiation to myeloid lineage that will eventually exhaust HSC pool.
Center for Stem Cell Research and Development, Ankara, Turkey
This study was supported by TUBITAK Research Fund (Project no. 110S021 and 214Z033).
Hematopoietic Stem Cells (HSC) give rise to all mature blood and immune cells. Currently,
more than 40.000 HSC transplantations are carried out annually in Europe. Clinical protocols
for storage of HSCs include use of preservatives, such as DMSO, which prevent formation of
ice crystals in cells during freezing, but also result in cell toxicity after thawing and a variety of
side effects after infusion in patients. There is a need for the development of novel protocols,
which can be used to store HSCs for a limited period of time, in the absence of toxic cryopre-
servatives. Storage of HSCs under hypothermic conditions, could be an alternative to short
term cryopreservation and would be advantageous, since cells would not require DMSO or
freezing, and transport would be less challenging, without the requirements of liquid nitrogen
or cooling systems. Hypothermic storage might significantly affect current clinical storage
and transport protocols of HSCs. SUL-109 (RokepieÒ, Sulfateq) is a recently developed
6-chromanol derivate, that promotes storage of several cell lines under hypothermic condi-
tions. Here, we cultured CD34+ cord blood cells up to 7 days in serum-free medium (Stem-
MACSTM HSC expansion medium XF, Miltenyi), with SCF, TPO, IGF-2, aFGF and
Angptl3. SUL-109 was added directly to the media and culture vessels were sealed air-tight
and stored at 4oC for up to 14 days. Cells were tested for viability, apoptosis, cell cycle and
colony assays. Hypothermic storage of HSCs in presence of SUL-109 resulted in a significant
effect on viability, with cells viable and capable of colony formation after maintenance for 14
days at 4oC. After 3 days of hypothermic storage, more than 30% of cells was still dividing (S
or G2/M phase), whereas after 7 days of storage at 4oC, increasing numbers of cells were held
up in G0/G1 phase. In contrast, in the absence of SUL-109, hardly any viable cells were de-
tected after 3 days of hypothermic storage. These data show that SUL-109 is a potent chem-
ical, protecting HSCs effectively from cell death during short-term storage under hypothermic
conditions and may be used as an alternative to short-term HSC cryopreservation.
Author Index
Bernstein, Irwin 3171 Brown, John 2012, 2036 Caudrier, Pierre 2027
Bertrand, Julien 3013, 3228 Brown Lee, Chris 3211 Cautive, Kelly M. 3098
Bevington, Sarah 3200 Brϋggemann, Monika 3012 Çelebi Saltik, Betül 3212, 3213
Bhatt, Sima 3050 Brunetti, Lorenzo 2014 Cha, Soungchul 3058
Bhayadia, Raj 2037 Bruveris, Freya Faith 1013, 3218 Chabot, Ashley 2005
Bieker, Jim J. 3254 Bryder, David 1017, 2028, Chahabi, Sara Zai 3023
Bigildeev, Alexey 3189 3025, 3154, 3187 Chalmel, Frederic 3193
Bill, Marius 3227 Büchler, Marleen 3217 Champlin, Richard 3058
Binagui-Casas, Anahi 3234 Budhathoki, Anjali 3237
Chan, Tsun Leung 3204
Bindels, Eric 3167 Buettner, Florian 2002, 3215
Chandrakanthan, Vashe 3210, 3211
Birkhofer, Carina 3172 Buisman, Sonja 2029
Chang, Wing 3020
Bisig, Bettina 3031 Bulaeva, Elizabeth 3216
Chang, Yang 3183
Bissels, Ute 3226 Burke, Matthew 3227
Chau, Crystal 3020
Blackburn, Clare 3197 Burns, Melissa 3235
Burri, Olivier 3031 Chavakis, Triantafyllos 3161
Blagborough, Andrew 3158 Chen, Benjamin 3196
Busch, Hauke 3101
Blake, Amanda 3095 Chen, Chia-ling 3209
Busch, Katrin 1006
Blank, Thomas 3089 Chen, Fangli 3117
Buske, Christian 3092, 3132,
Blaszkiewicz, Sandra 3225 Chen, Jenny 3020
3133, 3241, 3242
Block, Milena 3217 Chen, Jiahao 3251
Buske, Michaela 3132
Bloomfield, Clara D. 3227
Busque, Lambert 2027 Chen, Jie 3106, 3107
Bock, Christoph 1027
Butler, Jason 3100 Chen, Jonathan 2010
Boehm, Thomas 3041 Butylin, Pavel 3040 Chen, Min 3054
Bogeska, Ruzhica 3225 Bystrykh, Leonid 2023, 2029, 3231 Chen, Riyan 3173
Boggio Merlo, Maria Elena 3224
Chen, Shaohua 3105, 3106,
Böiers, Charlotta 2012, 3197 C 3107, 3109
Bokemeyer, Carsten 3017
Cabezas-Wallscheid, Nina 2030, 3038, Chen, Ting 3206
Bolan, Timothy 2003
3114, 3215 Chen, Yufei 1018
Boller, Soren 3120
Cacciola, Emma 3214 Chen, Zhyjang 1023
Bondarenko, Sergey 3240
Cacciola, Rossella 3214 Chen, Zizhen 3006
Bonifer, Constanze 3223
Cagnan, Ilgin 3261 Cheng, Hui 3207, 3208
Bönig, Halvard 3010, 3015, 3130
Caiafa, Paola 3132, 3242 Cheng, Tao 3109, 3207, 3208
Bonnet, Dominique 2002, 3021, 3086
Calero-Nieto, Fernando 3060, 3135, 3236 Cheng, Yik Ling Bowie 3205
Borga, Chiara 3242
Cales, Carmela 3043 Chennupati, Vijaykumar 3252
Borkhardt, Arndt 3241
Calleri, Angelica 3224 Cher, Chae Yin 3204
Börries, Melanie 3101
Calvanese, Vincenzo 1013, 2003 Chi-Wang Yu, Eric 3252
Borst, Sara 3222
Calvo, Julien 2032 Chin, Desmond W.L. 2025
Bosio, Andreas 3226
Camargo, Fernando D. 2033 Chin, Paulynn 3223
Bothur, Sabrina 2037
Cameron, Rosannah 3221 Chiu, Sung Kai 3030, 3203
Boukarabila, Hanane 3197
Cammenga, Jörg 3025 Chong, Yang 3183
Bouriez-Jones, Tiphaine 3197 Campbell, Peter 2001
Boussema, Chiheb 3031 Chung, Brian Hon-Yin 3204
Campos, Vasco 3031 Chung, Stephen 3202
Bouwers-Vos, Annet 3048 Cantor, Alan 2035
Bowman, Teresa V. 3221 Churchill, Gary 1021
Cao, Benjamin 3067
Boykin, David W. 3251 Ciccarone, Fabio 3132, 3242
Cao, Huimin 3156
Božić, Tanja 3172, 3220 Clapes, Thomas 3059, 3112, 3123
Carapito, Raphael 3118
Brandts, Christian 3004, 3077 Claus, Rainer 3101
Carlsson, Göran 3127
Braun, Armin 3062 Clements, Wilson K. 3201
Carrelha, Joana 3197, 3247
Brdicka, Tomas 3001 Carter, Gregory W. 1021 Clever, Hans C. 3081, 3161
Bresnick, Emery H. 1010 Carvajal, Luis A. 2015, 3251 Cloonan, Nicole 3141
Brocks, David 3101 Cascante, Marta 3182 Cockerill, Peter 3200
Brors, Benedikt 3225 Catala, Albert 3101 Coetzee, Simon G. 2009
Brouard, Nathalie 3219 Cauchy, Pierre 3112, 3192, Cohen, Adam 3058
Brown, Andreas 3022, 3049 3200, 3223 Cohen, Sandra 2027
Author Index / Experimental Hematology 2017;53: S137-S148 S139
Coleman, Robert 2015 Derecka, Marta 3111, 3192 Eich, Christina 2004
Collier, Paul 3215 Desai, Niruba 3073 Eitler, Jiri 3063
Collombet, Samuel 1031 Dethmers-Ausema, Bertien 2023, El Ashkar, Sara 3194
Colombo, Emanuela 3224 2029, 3231 Elefanty, Andrew 1013, 3218
Comoglio, Federico 3096 Dettinger, Philip 2007, 3116 Elemento, Olivier 3100
Conde, Lucia 2036 Devadas, Prithvia 1010 Ellis, Sarah 3156
Contavalli, Paola 3058 Dewey, Colin 1010 Ema, Hideo 3185
Conway, Ashlee 3199 Deynoux, Margaux 3249 Emmrich, Stephan 2037
Coon, Joshua 1010 Di Ruggiero, Elodie 3013 Enard, Wolfgang 3067, 3143
Cooper, Leanne 3141 Di Stefano, Bruno 1031 Enciso, Jennifer 3184
Cornillet-Lefebvre, Pascale 3070 Dias, Sheila 3178 Endo, Itsuro 3034
Cornils, Kerstin 3017, 3198 Dick, John E. 2011, 3128, 3244 Endo, Mitsuhiro 3183
Cortes, Mauricio 3033 Dick, Tobias P. 3215
Endoh, Tamie 3183
Cortes, Roldan 3182 DiPaola, Jorge 3061
Enver, Tariq 2012, 2036
Cosgun, Erdal 3261 DiPersio, John F. 3130
Epling-Burnette, Pearlie 3237
Costa, Ivan, G. 3220 Ditadi, Andrea 2011
Erdem, Ayşegül 3182
Coutu, Daniel 3121 Dobrzycki, Tomasz 3082
Ernst, Patricia 1018
Curtis, David 3030, 3203 Doench, John 3087
Ershov, Daniil 3238, 3239
Dohner, Hartmut 2037, 3132, 3133,
D 3241, 3242 Errami, Youssef 3096
Dohner, Konstanze 2037, 3132, Esain, Virginie 3174
Dagati, Gianluca 3112 Esser, Andre 3172
3133, 3241, 3242
Daley, George Q. 2031
Domingues, Melanie 3076 Essers, Marieke 1009, 3143,
Damm, Erick W. 3201 3160, 3225
Daniel, Michael 3196 Dong, Fang 3185, 3206, 3207
Dong, Ji 3185 Etten, Richard 3115
Danek, Peter 3001
Doondeea, Jessica 2032 Etzrodt, Martin 2007, 3116, 3256
Danlin, Yao 3108
Dorantes-Costa, Elisa 2035 Eugster, Anne 3161
Dannenmann, Benjamin 3195
Danner, Eva 3015 Dorn, Isabel 3191, 3229
Dorrance, Adrienne 3227 F
Daunt, Carmel 3178
Davidson, Nadia 3218 Dosquet, Christine 3086
Doyle, Alexander 1017 Fadeel, Bengt 3127
Davydova, Yulia 3190
Dozal, David 3024 Falchi, Mario 3085
Dawson, Kevin 2001
Draber, Petr 3131 Falkenburg, J.H. Frederick 3012
de Boer, Bauke 3048
Draberova, Lubica 3131 Faltusova, Katerina 3065, 3181,
de Bruijn, Marella 1016, 3197, 3247
Drize, Nina 3189, 3190 3209
De Conti, Giulia 3224
Du, Wei 3188 Fan, Rong 2010
De Gorter, David 3015
Duchosal, Michel 3252 Fang, Yixuan 3180
de Haan, Gerald 2023, 2029, 3231
Dudenhöffer-Pfeifer, Monika 3187 Farahat, Abdelbasset 3251
De La Garza, Adrianna 3221
Duhrsen, Ulrich 3125 Fasel, Nicolas 3252
De Leval, Laurence 3031
de Looper, Hans 3167 Dupuis, Eleonore 3013 Fassoni, Artur 3165
De Moerloose, Barbara 3101 Durham, Benjamin 3089 Fawaz, Malak 3010, 3215
de Pater, Emma 3167 Dworzak, Michael 3101 Federici, Giulia 3085
De Rijck, Jan 3194 Dzierzak, Elaine 2004, 3091, Feinberg, Andrew 3232
Debaize, Lydie 3193 3104, 3167 Fenaux, Pierre 3086
Debatin, Klaus-Michael 3242 Feng, Wenli 3002
Debyser, Zeger 3194 E Fernandes, Sophia S. 3179
Dedhar, Shoukat 3054 Earp, Eleanor 3096 Ferrando, Adolfo A. 3251
Delisle, Jean Sebastein 2027 Eaves, Allen 3020 Ferrari, Francesco 3090
Delwel, Rudd 3001 Eaves, Connie 1002, 3054, Ferrero, Giuliano 3013
Demeulemeester, Jonas 3194 3094, 3162, 3216 Feuring-Buske, Michaela 3133, 3241,
Deng, Deborah 3162 Ebinger, Sarah 3067 3242
Dengel, Karen 3058 Eckert, Elias S.P. 2020, 3186 Feyerabend, Thorsten 1006
Deniz, Demir 3242 Eckert, Paul G. 3178 Fidanza, Antonella 3176
S140 Author Index / Experimental Hematology 2017;53: S137-S148
Kapranov, Nikolay 3190 Knezevic, Kathy 3210, 3211 Lachmann, Nico 3217, 3229
Karamitros, Dimitris 2032 Knobel, Sebastian 3226 Ladel, Luisa 2030, 3038,
Kardel, Melanie 3020, 3094 Knoch, Julia 3225 3114, 3215
Kardosova, Miroslava 3001, 3131 Knoess, Sabine 3051 Ladopoulo, Vasileios 3135
Karigane, Daiki 3035 Kobayashi, Michihiro 3007 Lai, Jing 3107, 3109
Kariya, Ryusho 3068 Kohlhofer, Ursula 3133 Lai, Courtney K. 2037
Karlsson, Stephan 2012 Kokkaliaris, Konstantinos D. 3121 Lam, Stephen Sze Yuen 3204
Karpova, Darja 3130 Koliqi, Tereza 3031 Lan, Yu 3113
Karrasch, Charles 3100 Komatsu, Norio 3157 Landais, Severine 2027
Karsan, Aly 3162 Kometani, Kohei 3120 Landers, Cameron 3124
Karunasiri, Malith 3227 Konantz, Martina 3118, 3119 Lane, Steven W. 3141
Kaschutnig, Paul K. 3217, 3225 Konig, Heiko 3117 Lang, Fabian 3012
Kato, Kota 3129 Koniski, Anne 1016 Lang, Stefan 1017
Kato, Takashi 3071, 3074, 3129 Konno, Shungo 3071 Langa Olivia, Javier 3059
Katsumura, Koichi 1010 Konu, Özlen 3261 Langenfeld, Katja 3244
Kaufmann, Kerstin B. 3128 Koren-Michowitz, Maya 3084 Lanza, Francois 3219
Keeshan, Karen 3052 Koschmieder, Steffen 3172 Larochelle, Fannie 2027
Keles, Sunduz 1010 Koseki, Haruhiko 3145 Laurenti, Elisa 2001
Keller, Gordon 2011 Koster, Taco 3231 Lausen, Jörn 3010, 3164
Keller, John 3050 Kovtonyuk, Larisa V. 2026, 3146 Law, Kenneth 3196
Kent, David 2001, 2021, Kraus, Thomas 3172 Laycock, Emma 2012
3072, 3073 Krause, Daniela 3023, 3115, Lazare, Seka 2023, 2029
Kenworthy, Charles 2015 3150, 3164 Lazarov, Tomi 3089
Kerr, Naseem 3058 Kräutler, Nike 3146 Lazzaretto, Beatrice 3127
Khandagale, Avinash 3127 Kramer, Ashley 3095 Le Bechec, Antony 3070
Khandanpour, Cyrus 3125, 3126, Krecsmarik, Monika 3082 Leboulch, Philippe 1024
3173 Lechman, Eric 2011
Kremer, Jakob 3217
Kikushige, Yoshikane 3053 Lee, Brendan 3210, 3211
Krenz, Dariusz 3226
Kim, Hye Mi 3251 Lee, Chen 3171
Kristiansen, Trine A. 1017, 3137
Kim, Jin-Soo 1010 Lee, Jerry 2031
Kronnie, Geertruy 3242
Kim, Kunhwa 3058 Lee, Lydia K. 3168
Krowiorz, Kathrin 2037
Kindinger, Tim 3155 Lee, Xue 3188
Kuchenbauer, Florian 2037
Kindler, Thomas 3037 Lee-Six, Henry 2001
Kuldanek, Susan 3061
King, Katherine 3124 Lefevre, Andreas 3249
Kull, Tobias 3116
Kinnebrew, Garrett 3117 Lefkopoulos, Stylianos 3112
Kuller-Müller, Uta 3150
Kinston, Sarah J. 2021 Lehner, Ben 1031
Kumar, Arvind 3251
Kirkpatrick, Gregory 3061 Lehrke, Michael 3095
Kumar, Atul 3140
Kirschner, Kristina 2017 Leite, Joana 3251
Kumar, Rahul 3023, 3115
Kirsten, Nicole 3092 Leitoguinho, Ana Rita 3218
Kumar Jha, Deepak 2031
Kiss, Thomas 2027 Lemieux, Sebastein 2027
Kunar, Balvir 3100, 3245
Kitano, Hiroaki 3046 Lemischka, Ihor 1012, 3196
Kunisaki, Yuya 3149
Klauke, Karin 2029 Lenaerts, Aurelie 3111
Kunz, Leo 3121
Klaus, Anna 3123 Lengerke, Claudia 3066, 3118, 3119
Kurasawe, Romy 1021
Klein, Allon M. 2033 Lenz, Michael 3172
Kurrle, Nina 3036
Klein Geltink, Ramon I. 3059 Leuitoguinho, Ana Rita 1013
Klimek, Virginia 3202 Kuskonmaz, Baris 3261
Leung, Anskar Yu Hung 3204, 3205
Klimiankou, Maksim 3083, 3195 Kuzmina, Larisa 3190 Leung, Gabriel 3233
Klimmeck, Daniel 2030, 3215 Kwak, Larry 3058 Leung, Wing Yan 3076
Kloetgen, Andreas 3241 Kwong, Yok Lam 3204 Levine, Ross 1029, 3237
Klose, Markus 3122 Lewandowski, Daniel 3110
Klusmann, Jan-Henning 2037, 3051
L Li, Bo 3106
Knapp, D.J.H.F. 1002 Lacaud, Georges 3217 Li, Chunliang 3235
Knapp, David 3162 Lachance, Silvy 2027 Li, Haiyan 3124
Author Index / Experimental Hematology 2017;53: S137-S148 S143
Plate, Karl Heinz 3010 Redmond, David 3100 Rudolph, Lenhard 1023
Polyzos, Alexander 3059 Rehage, Maike 3055 Ruf, Franziska 3144
Polyzou, Aikaterini 3059 Reimann, Andreas 3116 Ruivo, Nicola 3158
Poon, Gregory M.K. 3251 Reinhardt, Dirk 2037, 3044 Rybtsov, Stanislav 1004, 3234
Poot, Raymond 2029 Reinisch, Andreas 2032
Popescu, Michael 3058 Reis e Sousa, Caetano 2013 S
Porcher, Catherine 2032 Remeseiro, Silvia 3244
Porse, Bo 3029 Ren, Qian 3002 Sahan, Ozge Burcu 3258
Potuckova, Lucie 3131 Ren, Xiubao 3237 Sahin, Deniz 3132
Poulos, Michael G. 3100 Renders, Simom 2030, 3038, Sahm, Franziska 3093
Powell, David 3203 3114, 3215 Sakoda, Teppei 3053
Power, Carl 3210, 3211 Renesova, Nicol 3065 Salbert, Gilles 3193
Pradhan, Kith 3237 Rettig, Michael 3130 Salomonis, Nathan 2006, 2009, 2034
Prendergast, Aine M. 3225 Repapi, Emmanouela 2032 Samitsch Marina 2032
Prick, Janine C.M. 3073 Rice, Siobhan 3091 Sandberg, Rickard 3197
Prikhodko, Stanislava 3040 Richardson, Simon 2012 Sandoval, Antonio 3024
Prinz, Marco 3089 Rieger, Michael A. 2037, 3010, 3012, Sankaran, Vijay G. 3252
Pryzybylla, Adriana 2030, 3038, 3055, 3169, 3215 Sardina, Jose 1031
3215 Rink, Lothar 3172 Sarro, Rossella 3031
Ptasinska, Anette 3223 Rio, Anne-Gaelle 3193 Sarrou, Evgenia 3052
Pui, Ching-Hon 3235 Rippe, Karsten 3225 Satija, Namita 3196
Rispoli, Rossella 3082 Sato, Kei 3129
Q Ritchey, Julie 3130 Sats, Natalia 3189
Rittinghausen, Susanne 3062 Sauvageau, Guy 1033, 2027
Qazilbash, Muzaffar 3058
Qian, Maoxiang 3235 Roberts, Irene 3252 Savchenko, Valery 3190
Qiao, Qiao 3210, 3211 Robin, Catherine 3123 Savvulidi, Filipp 3181, 3209
Quadroni, Manfredo 3252 Robinson, Phillip 3030 Saw, Jesslyn 3030, 3203
Quek, Lynn 2032 Rodewald, Hans-Reimer 1006 Säwen, Petter 2028
Qui, Dan 3106 Rodriguez, Antony 3124 Scadden, David 1032, 3087
Quintanilla Martinez Fend, Leticia 3066, Rodriguez-Fraticelli, Alejo E. 2033 Scandura, Joseph M. 3100
3133 Roeder, Ingo 3005, 3122, Schachterle, William 3100
3165, 3170 Schambach, Axel 3062, 3258
R Roers, Axel 3081, 3170 Scheder, Anna-Marie 3230
Rogozea, Adriana L. 3117 Schenke-Layland, Katja 1013
Rabu, G.M. 1002 Rohde, Christian 3241 Schepers, Hein 2029
Raffel, Simon 3160 Roma, Leticia 3215 Schlenke, Peter 3191
Rafii, Shahin 3100, 3245 Romano, Nicola 3176 Schlesner, Matthias 3101
Rafii Tabrizi, Arash 3100 Romeo, Paul-Henri 3110 Schmidt, Manfred 2020, 3186
Raghupathy, Narayanan 1021
Romero, Sandra 3143, 3144 Schmitt, Martin 3230
Rahmig, Susann 3057
Romine, Molly 3050 Schmugge, Markus 3101
Raic, Annamarija 3220
Romling, Ute 3127 Schneider, Dorit 3051
Ramamoorthy, Senthilkumar 3120, 3191
Rosa, Fabio 2013 Schneider, Pascal 3252
Ramasz, Beáta 3056
Rosenwaks, Zev 3100 Schnoor, Michael 3024
Rambold, Angelika 3059
Rosli, Christoph 3217 Schnüttgen, Frank 3036, 3115
Ramiliason, Mirana 3156
Ross, Julie 2038 Schoedel, Kristina B. 3081, 3170
Ramirez, Dalia 3024
Ranheim, Erik 1010 Rossi, Derrick J. 2028, 3232 Schönberger, Katharina 2030, 3038,
Rao, Sheetal 3058 Rössler, Jens 1006 3114, 3215
Rapaport, Franck 3237 Rothe, Katharina 3028, 3054 Schorpp, Michael 3041
Rapin, Nicolas 3029 Rothe, Michael 3062 Schreck, Christina 3143, 3144
Rasche, Mareike 3044 Rouault-Pierre, Kevin 3021, 3086 Schröeder, Timm 2007, 3012,
Rashkovan, Marissa 3173 Rouhi, Arefeh 2037 3018, 3097, 3116,
Rawat, Vijay 3132, 3133, Roy, Denis Claude 2027 3121, 3215, 3256
3241, 3242 Roy, Jean 2027 Schuettpelz, Laura 3050, 3130
S146 Author Index / Experimental Hematology 2017;53: S137-S148
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