Articles

The genomic landscape of schwannoma

Sameer Agnihotri1, Shahrzad Jalali1, Mark R Wilson1, Arnavaz Danesh2, Mira Li1, George Klironomos1,
Jonathan R Krieger3, Alireza Mansouri1, Osaama Khan1, Yasin Mamatjan1, Natalie Landon-Brace1, Takyee Tung1,
Mark Dowar2, Tiantian Li2, Jeffrey P Bruce2, Kelly E Burrell1, Peter D Tonge1, Amir Alamsahebpour1,
Boris Krischek4, Pankaj Kumar Agarwalla5–8, Wenya Linda Bi5,6,8,9, Ian F Dunn5,9, Rameen Beroukhim5,6,8–10,
© 2016 Nature America, Inc., part of Springer Nature. All rights reserved.

Michael G Fehlings11, Vera Bril12, Stefano M Pagnotta13,14, Antonio Iavarone14, Trevor J Pugh2,15,
Kenneth D Aldape1,2,15–18 & Gelareh Zadeh1,2,11,18

Schwannomas are common peripheral nerve sheath tumors that can cause debilitating morbidities. We performed an integrative
analysis to determine genomic aberrations common to sporadic schwannomas. Exome sequence analysis with validation by
targeted DNA sequencing of 125 samples uncovered, in addition to expected NF2 disruption, recurrent mutations in ARID1A,
ARID1B and DDR1. RNA sequencing identified a recurrent in-frame SH3PXD2A-HTRA1 fusion in 12/125 (10%) cases, and
genomic analysis demonstrated the mechanism as resulting from a balanced 19-Mb chromosomal inversion on chromosome 10q.
The fusion was associated with male gender predominance, occurring in one out of every six men with schwannoma.  
Methylation profiling identified distinct molecular subgroups of schwannomas that were associated with anatomical location.
Expression of the SH3PXD2A-HTRA1 fusion resulted in elevated phosphorylated ERK, increased proliferation, increased  
invasion and in vivo tumorigenesis. Targeting of the MEK-ERK pathway was effective in fusion-positive Schwann cells,  
suggesting a possible therapeutic approach for this subset of tumors.

Schwannomas are tumors that arise from the transformation of
Schwann cells, the myelin-producing cells of the peripheral nervous
system. Substantial morbidity results from schwannomas occurring
in cranial and spinal nerves. Cranial nerve schwannomas represent
8% of intracranial tumors and usually affect the vestibular nerve,
often resulting in hearing loss and neurological defects. Vestibular
schwannomas are predominantly unilateral, although in ~10% of
cases they are bilateral, arising from the neurofibromatosis type 2
(NF2) cancer predisposition syndrome. Spinal schwannomas are the
most common primary spinal tumors, accounting for one-third of
all spinal tumors1. Early diagnosis and treatment of schwannoma is
essential to prevent serious morbidities including, but not limited
to, hearing loss, tinnitus, nerve paralysis, imbalance, hydrocephalus
and in some instances death. Surgical resection can also result in the
sacrifice of nerves adjacent to the tumor, resulting in life-changing
morbidities. Treatment options are limited, although there have been
a number of responses to various targeted agents2,3. At the molecular
level, loss of NF2 occurs in 60% of sporadic schwannomas, but the
genomic landscape of schwannoma has not been fully elucidated.
To gain further insight into the molecular mechanisms of schwan-
nomas, we performed an integrative analysis using whole-exome
sequencing, RNA sequencing and methylation profiling of both spinal
and vestibular schwannomas.
RESULTS
Exome sequencing identified new recurrent mutations in
schwannomas
We investigated the genetic landscape of 13 cranial and 13 spinal
sporadic schwannomas (Supplementary Table 1). We performed
whole-exome sequencing to a median depth of 80× (range, 62× to
88×), and, on average, 93.0 ± 1.71% of target bases had >20 reads
(range, 88.1–95.1%) (Supplementary Table 2). We identified 441
somatic single-nucleotide variants (SNVs) in the exons of our 26
samples, corresponding to 0.16 mutations per coding megabase,
which is comparable to low-mutation-rate tumors such as Ewing
and rhabdoid sarcomas4. The algorithms scale-invariant feature

1MacFeeters Hamilton Centre for Neuro-Oncology Research, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada. 2Princess Margaret

Cancer Centre, University Health Network, Toronto, Ontario, Canada. 3SPARC Biocentre, The Hospital for Sick Children, Toronto, Ontario, Canada. 4Department of
Neurosurgery, University Hospital of Cologne, Cologne Germany. 5Harvard Medical School, Boston, Massachusetts, USA. 6Broad Institute of MIT and Harvard,
Cambridge, Massachusetts, USA. 7Department of Neurosurgery, Massachusetts General Hospital, Boston, Massachusetts, USA. 8Dana-Farber Cancer Institute,
Boston, Massachusetts, USA. 9Department of Neurosurgery, Brigham and Women’s Hospital, Boston, Massachusetts, USA. 10Department of Medical Oncology,
Dana-Farber Cancer Institute, Boston, Massachusetts, USA. 11Department of Neurosurgery, University Health Network, Toronto, Ontario, Canada. 12Department of
Medicine (Neurology), and the Elizabeth Raab Neurofibromatosis Program, University of Toronto, Toronto, Ontario, Canada. 13Department of Science and Technology,
Università degli Studi del Sannio, Benevento, Italy. 14Department of Pathology and Cell Biology and Neurology, Columbia University, New York, New York, USA.
15Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada. 16Department of Pathology, Maryland Anderson Cancer Center, Houston,

Texas, USA. 17Department of Laboratory Medicine and Pathology, University of Toronto, Toronto, Ontario, Canada. 18These authors jointly directed this work.
Correspondence should be addressed to G.Z. (gelareh.zadeh@uhn.ca) or K.D.A. (kaldape@uhnresearch.ca).

Received 26 February; accepted 2 September; published online 10 October 2016; doi:10.1038/ng.3688

Nature Genetics  VOLUME 48 | NUMBER 11 | NOVEMBER 2016 1339

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a 18
Spinal Age <30
16 Location
14 Vestibular <50
Number of mutations

12 <70
10 Female
Gender
8 Male Mutation Missense
6 Nonsense
4 Neutral Indel
2 Chr 22q
Loss

% Altered # Cases Significant by MutSig

22q loss 70 18
NF2 77 20 *
DDR1 19 5 *
ALPK2 12 3 *
TAB3 12 3 *
CAST 8 2 *
LZTR1 8 2 n/a
TSC1 8 2 n/a
TSC2 8 2 n/a
© 2016 Nature America, Inc., part of Springer Nature. All rights reserved.

ARID1A 8 2 n/a
ARID1B 4 1 n/a
Location
Gender
Age

b Melanoma
WGS
0
WES
121
Lung adenocarcinoma 23 159
Squamous cell lung cancer 19 0
Colon and rectal 0 224
Small-cell lung cancer 2 29
Multiple myeloma 38 0
GBM 42 291
Ovarian cancer 0 316
Breast Cancer 0 547
Craniopharyngioma 0 15
Prostate 7 0
Neuroblastoma 18 240
Medulloblastoma 39 21
Paediatric rhabdoid cancer 0 35
T-cell precursor ALL 12 94
Schwannoma 0 26
Retinoblastoma 4 0
Ewing sarcoma 112 0
0 5 10 15
Nonsynonymous mutations/Mb

Figure 1  Mutational landscape of schwannoma. (a) Results from whole-exome sequencing (n = 26) samples with frequency and mutations identified (left).
Overview of the genes mutated in schwannomas and the significance of recurrent mutations as identified by MutSig (*P < 0.05; n/a, not applicable).
(b) Mutations per megabase from several cancer sequencing projects 42–58. Schwannoma represents data in this study. WGS, whole-genome sequencing;
WES, whole-exome sequencing.

transform (SIFT) and protein variation effect analyzer (PROVEAN)
predicted that one-third of protein-coding alterations have a del-
eterious effect (Supplementary Tables 3 and 4). We used mutation
significance (MutSig) analysis to identify genes that were mutated
more often than expected by chance (Supplementary Table 5). We
summarized the mutational landscape of schwannomas in Figure 1.
In keeping with previous studies, we identified NF2 as a frequently
altered gene, as 20/26 samples (77%) harbored NF2 mutation or
22q loss. We also found LZTR1 mutations, previously implicated in
schwannomatosis and sporadic schwannomas5, in our cohort (8%,
Fig. 1a). Additional recurrent but low-frequency mutations not pre-
viously reported in schwannomas included ARID1 family members
(ARID1B and ARID1A), DDR1, TAB3, ALPK2, CAST, TSC1 and
TSC2. To validate these findings, we sequenced all exons of these
genes in an additional 99 schwannomas (Fig. 2a). We observed NF2
inactivation through mutation or 22q loss in 77% of all cases (57%
mutation). NF2 mutations in general were significantly higher in
vestibular schwannomas compared to spinal schwannomas (67% vs.
33%, P < 0.0001, Fisher’s exact test). Furthermore, 29% (37/125) of
all schwannomas harbored inactivating missense, nonsense and/or
indel mutations in either ARID1A or ARID1B, members of the SWI-
SNF chromatin-remodeling complex (Fig. 2a and Supplementary
Fig. 1). We found mutations in DDR1, encoding a receptor tyrosine
kinase activated in lung cancer and other tumor types6, in 14/125
cases and included several point mutations in the kinase domain
sequence and a recurrent hotspot mutation in the discoidin domain
sequence (n = 4, p.Arg183His). Mutation locations relative to pro-
tein sequence for these and additional candidates are presented in
Supplementary Figure 1. Pathway analysis of nonsilent mutations
identified enrichment in the following pathways, which have been
implicated as core schwannoma pathways in previous studies: inflam-
matory response, MEK signaling, mTOR signaling, TNFα-NFκB and
ERBB2 signaling. In addition, we identified some lesser-implicated
pathways: epithelial-to-mesenchymal transition, estrogen response
and ATF2 activation (Supplementary Fig. 2a). 19/20 (95%) of TSC1
or TSC2 mutations occurred with either NF2 mutation and/or 22q
loss (P = 0.042). We observed only one TSC1 mutation with no evi-
dence of NF2 mutation or loss of 22q. DDR1 mutations (n = 14)
and the SH3PXD2A-HTRA1 fusion (n = 12, described below) were
exclusive events.

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Methylation profiling identified distinct schwannoma subgroups cohort of 125 schwannoma samples. Loss of 22q was the only recur-
We next used the Infinium HumanMethylation450 BeadChip to derive rent chromosomal aberration in schwannomas, seen in 76/125 (61%)
DNA copy number changes and global methylation patterns on our tumors, with slightly higher percentage seen in cranial (64%, 41/64)

a Age
Gender
Tumor size
Location
Methylation
group
NF2 77%

SH3PXD2A- 10%
HTRA1
ARID1B 18%

ARID1A 14%

DDR1 11%

TSC1 9%
© 2016 Nature America, Inc., part of Springer Nature. All rights reserved.

CAST 8%

ALPK2 8%

LZTR1 8%

TSC2 7%

TAB3 3%

Gender Male Female Tumor size Vestibular Reaching brain stem Intra meatal
Age 19–31 31–50 51–65 65–79 Tumor size Spinal Intraspinal Extraspinal
Subgroup Methylation group 1 Methylation group 2 n/a Genetic alteration Heterozygous deletion Missense mutation
Location Vestibular Spinal Fusion Truncating mutation
Indel and nonsense

b k=2 k=3 c 1.0 d 21

2 clusters, Gini = 0.455
3 clusters, Gini = 0.531 17
4 clusters, Gini = 0.612
0.8 5 clusters, Gini = 0.63
6 clusters, Gini = 0.65 13
7 clusters, Gini = 0.669
k=4 k=5 8 clusters, Gini = 0.716
0.6 9
L(p)

0.4 5
PC3

k=6 k=7 0.5

0.2
–4
0.0
–8
0.0 0.2 0.4 0.6 0.8 1.0
k=8
p
–12

f Group 1 Group 2 –16

e –20
–27 –22.3–17.6–12.9 –8.2 –3.5 1.2 5.9 10.6 15.3 20
2
PC

100 PC1 Group 1

Group 2
% Methylation

Group 1 vestibular

50 g 800 h 250
Group 2: spinal

200
# CpG Probes

600
# Genes

150
0 400
100
200 50
0 0
Group 1 Group 2 Group 1 Group 2

Figure 2  Molecular subgroups of schwannoma based on DNA methylation. (a) Integrated oncoprint demonstrating somatic landscape of schwannomas
including mutations, 22q loss, methylation subgroup, clinical features and SH3PXD2A-HTRA1 fusion (RNA sequencing identified recurrent fusion).
(b) Unsupervised consensus k-means clustering of 125 schwannoma samples for k = 2 to k = 8 using the 1,000 most variable methylated probes.
SigClust, a statistical method for testing the significance of clustering results confirmed the two clusters were significant at k = 2 (*P < 0.05).
(c) Lorenz curves supporting two molecular groups of schwannomas as supported by change in Gini coefficient. (d) Principal component analysis (PCA)
of schwannomas based on 1,000 variable methylated probes. Ellipsoids represent two s.d. of the data distribution for each, and lack of significant
overlap supports evidence for two molecular subgroups. (e) Anatomical distribution of schwannoma groups. Arrow points to cranial nerve VIII.
(f) Hierarchical clustering using Pearson correlation of significant differentially expressed CpG-methylated probes between group 1 (n = 64) and
group 2 (n = 61) schwannomas (P < 0.05, Wilcoxon rank-sum test). (g,h) CpG and gene count summary of group 1 and group 2 schwannomas.

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versus spinal (56%, 35/61) tumors (Supplementary Fig. 3a−d).
Samples with 22q loss had significantly lower NF2 RNA expression
(Supplementary Fig. 3b). Consensus k-means clustering and non-
negative matrix factorization (NMF) clustering identified two stable
DNA-methylation clusters using variable inputs of highly variant meth-
ylation probes (Fig. 2b,c and Supplementary Fig. 4a−d). Group 1
schwannomas were comprised mainly of vestibular schwannomas
(96%), whereas group 2 schwannomas were predominantly spinal (98%)
(Fig. 2d,e). To characterize biological differences between methylation
subgroups, we used the top differentially methylated probes between
groups 1 and 2, (Fig. 2f; Wilcoxon rank sum test, P < 0.05) correspond-
ing to 1,202 CpG probes and 395 genes (Fig. 2g,h). Gene-set enrichment
analysis (GSEA) identified significantly enriched pathways unique to
each group. Group 1 schwannomas were enriched for molecular path-
ways, including MAZ transcription targets, FOXA1 transcriptional
targets, and JNK and RAS signaling (Supplementary Fig. 5a). Group 2
schwannomas were enriched for molecular pathways including epithe-
© 2016 Nature America, Inc., part of Springer Nature. All rights reserved.
probe set tiling the introns of both fusion partners. We performed
targeted intron probe capture followed by Illumina sequencing in
addition to a sensitive large-insert and soft-clipped read detection
algorithm analysis to identify breakpoint-supporting reads in intron
6 of SH3PXD2A and intron 1 of HTRA1 in 10/12 samples; this con-
firmed that the resultant fusion rearrangement was formed through a
balanced translocation (Fig. 3e,f and Supplementary Table 7).
The SH3PXD2A-HTRA1 fusion had pro-growth function
The C-terminal portion of the fusion protein contains a trypsin-like
peptidase domain and PDZ domain from HTRA1, whereas the N
terminus contains a PXD2A domain from SH3PXD2A. To examine
and distinguish the fusion protein from its normal counterparts, we
performed protein blot analysis using both HTRA1 C-terminal and
N-terminal antibodies on available frozen lysates from nine schwan-
noma samples, including three with the fusion. Expression of a fusion
protein was suggested in these three samples via the use of a C ter-

lial-to-mesenchymal transition, NOTCH signaling and EGFR signal-
ing (Supplementary Fig. 5b). We further explored whether clinical
patterns and genetic mutations were associated with methylation
subgroups. The two groups were balanced for 22q loss, mutations in
ARID1A, ARID1B, TSC1, TSC2, DDR1, CAST and ALKPK2, global
methylation levels, gender and age (Supplementary Table 6).
RNA sequencing identified a recurrent fusion in schwannomas
We then examined RNA sequencing (RNA-seq) data for recurrent
gene fusions using deFuse7 (Supplementary Table 7). We identified
a previously uncharacterized in-frame fusion involving SH3PXD2A
(SH3 and PX domains 2A) and HTRA1 (high temperature require-
ment A serine peptidase 1, both on chromosome 10q), in 5/41 samples
that we subjected to RNA-seq, which we then confirmed with RT-PCR
(Fig. 3a,b). The resultant transcript sequence was identical in all five
cases at the fusion junction (exon 6 of SH3PXD2A fused to exon 2 of
HTRA1) (Fig. 3c). The predicted structure of the protein encoded by
the SH3PXD2A-HTRA1 fusion involved the PX domain encoded by
SH3PXD2A fused to the serine peptidase and PDZ domains encoded
by HTRA1 (Fig. 3d). We identified and validated the fusion in an
additional 7/84 samples (Supplementary Fig. 6a). We observed
a gender association among the 12 fusion-positive cases, with a sig-
nificant enrichment in men (11/12 fusion-positive cases were males,
P = 0.0044, Fisher’s exact test) and 11/64 (17%) of the total male
cohort. We also confirmed the reciprocal noncoding fusion transcript
with HTRA1 at the 5′ end and SH3PXD2A at the 3′ end in 5/41 sam-
ples, suggesting the fusion arises through a potential chromosomal
inversion (Supplementary Fig. 6b−d). We also identified the fusion
in 2/20 vestibular schwannomas but not matched blood from known
NF2 syndromic patients (Supplementary Fig. 6e,f). Examination of
exome sequencing data did not identify changes in fusion-positive
versus fusion-negative cases across the distance (19 Mb) between the
gene-fusion partners or at the exon boundaries at the fusion junction
(Supplementary Fig. 7). To identify the potential breakpoint that results
in the SH3PXD2A-HTRA1 fusion, we generated a targeted capture
minus HTRA1 antibody and absence of signal with an N-terminal
HTRA1 antibody (Fig. 4a). Additionally, liquid chromatography–
mass spectrometry (LC-MS) analysis identified peptides mapping
to the fusion in lysates from three fusion-positive samples but not
three fusion-negative cases (Fig. 4b,c and Supplementary Table 8).
Consistent with protein blot analysis, the N-terminal peptides of
HTRA1 and C-terminal peptides of SH3PXD2A were not identified
in fusion-positive cases, further suggesting that the peptides detected
were from only the fusion protein (Supplementary Fig. 8a,b). Fusion-
negative samples expressed both HTRA1 and SH3PXD2A, with no dif-
ferences in transcript levels when compared to fusion-positive samples
(Supplementary Fig. 8c). RNA-seq reads in HTRA1 and SH3PXD2A
exons, which were absent in the fusion RNA also support that fusion-
positive schwannoma patients still retain wild-type expression of these
genes at the RNA level (Supplementary Fig. 9a−d).
To characterize effects of the fusion on cellular function, we trans-
fected an HA-tagged SH3PXD2A-HTRA1 fusion construct into
Schwann cells and a schwannoma cell line. Stable expression of the
fusion promoted cell growth in the NF2 mutant schwannoma cell line
HEI-193 immortalized with viral proteins E6-E7 (Fig. 4d,e)8. Fusion-
positive cells had increased phospho-ERK and increased phospho-p70
S6 kinase, both a part of key signaling molecular pathways involved in
schwannomas (Fig. 4d). We also observed these findings in immortal-
ized human Schwann cells9 (Fig. 4f,g). As a relevant control, overex-
pression of full-length wild-type HTRA1 and SH3PXD2A in human
Schwann cells did not result in increased cell growth (Supplementary
Fig. 10a,b). SH3PXD2A and HTRA1 have been variably implicated in
regulating cell invasion and anoikis10,11. Expression of the SH3PXD2A-
HTRA1 fusion promoted an increase in cell invasion and resistance to
anoikis (Fig. 4h,i and Supplementary Fig. 10c). Furthermore, single
loss or combined loss of wild-type HTRA1 and SH3PXD2A did not
alter cell growth over 7 d as compared to overexpression of the fusion
(Supplementary Fig. 10d,e). As HTRA1 is a serine protease, we tested
the fusion protein using a serine protease assay; the purified fusion har-
bored comparable protease activity to recombinant HTRA1 purified

Figure 3  Structure of SH3PXD2A-HTRA1 fusion. (a) Alignment to an in silico reconstruction of the SH3PXD2A-HTRA1 fusion with reads spanning
the junction site of the two genes (red vertical line). (b) Validation RT-PCR using primers specific to the SH3PXD2A-HTRA1 fusion to confirm presence
of the fusion. (c) Chromosomal location of SH3PXD2A and HTRA1 on 10q24.1 and 10q26.3. Genes are ~19 Mb apart. Sanger sequencing confirms
the fusion sequence in five patient samples. (d) Domain structure of HTRA1, SH3PXD2A and fusion protein. (e) Schematic of balanced translocation
rearrangement between gene fusion partners SH3PXD2A and HTRA1. (f) Integrated Genome Viewer screen shot capture of SH3PXD2A-HTRA1
translocation sites (SH3PXD2A intron 6 and HTRA1 intron 1) from targeted intron capture confirms a chromosomal inversion. Genomic coordinates
for the fusion breakpoint were identified in 10/12 samples that had sufficient coverage around the breakpoint regions (see Supplementary Table 7
for coordinates of the fusion breakpoint).

1342 VOLUME 48 | NUMBER 11 | NOVEMBER 2016  Nature Genetics

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protein (Fig. 4j and Supplementary Fig. 10f,g). Site-directed muta- of the fusion (Fig. 4j). Furthermore, the p.Ser322Ala protease-dead
genesis to substitute the serine 322 with alanine (p.Ser322Ala) in the fusion construct allele did not increase cell proliferation, supporting
protease domain of the fusion construct abolished the protease activity the hypothesis that the protease domain of the SH3PXD2A-HTRA1

a SH3PXD2A HTRA1
b

n
co
51

22
06

38

24 2
94

89
10

21
00

31

A
3

N
25

18
30

23

24

37
26

29
18

18

D
200 bp
SH3PXD2A
100 bp -HTRA1

200 bp
β-actin
100 bp

RNA-seq + + + – – – – – – – –
RT-PCR + + + + + – – – – – –

Chromosome 10
c d
© 2016 Nature America, Inc., part of Springer Nature. All rights reserved.

PX SH3 SH3 SH3 SH3 SH3

PDZ domain
5–124
SH3PXD2A HTRA1 IGFBP binding domain
13 11 9 7 5 3 1 Exon 1 3 5 7 9

IBP KD TPD PDZ Trypsin-like peptidase domain

14 12 10 8 6 4 2 2 46 8 SH3 domain
105350 105650 124220 124280 Kb from pter 37–89 110–155 204–342 375–463
Kazal domain
PX TPD PDZ
Exon 1 6 2 9 PX domain

SH3PXD2A HTRA1
AA Ser Lys Arg Lys Ser Gly Gln Glu Asp Pro
(amino acids 1–141) (amino acids 142–465)
Patient 1

e Chromosome 10 10q24.3 10q26.3

Patient 2

HTRA1 (+)
Intronic breakpoints
Patient 3

SH3PXD2A (–)

Intron 6 Intron 1

Balanced inversion
Patient 4

2 fusion products
5′-SH3PXD2A-HTRA1-3′ fusion 5′-HTRA1-SH3PXD2A-3′ fusion
Patient 5

Chr10 Chr10
f
Chr10:105,434,397–105,435,617 Chr:124,221,396–124,222,616

[0–236] [0–236]

SH3PXD2A HTRA1

Nature Genetics  VOLUME 48 | NUMBER 11 | NOVEMBER 2016 1343

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a b c

HEI-193 + fusion
Fusion- Fusion- 30

Normalized spectrum count

*

HEI-193
negative positive

1 2 3 4 5 6 7 8
Fusion
* 20
HTRA1
(50 kDa) (52 kDa)
HTRA1 C-term 10

HTRA1 N-term
0
SH3PXD2A

e
si s ive

es tiv
Fu ple gat

pl si
β-actin

m po
m e
sa n n
SH3PXD2A: AA 1–141

sa on
o
si
HTRA1: AA 142–465

Fu
*: Breakpoint

d e f Immortalized Schwann cells g h

Clone A

Clone B
Clone 1

Clone 2
Control

Control
Control HEI
Control 3,000 Fusion clone A schwannoma cells
3,000 Fusion clone 1 Fusion clone B *

Cell count (×1,000)

2.0 *

relative fold increase

Fusion clone 2 * * *
Cell count (×1,000)

HA HA
* 2,000 1.5
© 2016 Nature America, Inc., part of Springer Nature. All rights reserved.

2,000

Invasion:
pERK pERK
1.0
ERK 1,000
ERK 1,000 0.5
pS6 Thr 389 pS6 Thr 389
0 0.0
0 0 1 2 3 5 7
pS6

or

2
pS6

e
ct
0 1 2 3 5 7

on

on
ve
Time (d)

cl

cl
y
GAPDH

pt

on

on
GAPDH

Em

si

si
Fu

Fu
i j k DAPI HA EEA1 Merge
HEI-fusion positive

Immortalized
Schwann cells 40
% Activity relative to
no protease control

3
relative fold increase

* * 30

2 20
Invasion:

10
1
0
HEI control cells

0 HTRA1 Fusion Fusion No

S322A protease
Fu n cl or

cl A
B
ct

on e

e
si on

on
ve
y
pt

70 kDa BSA
o
Em

si
Fu

Figure 4  Characterization of the SH3PXD2A-HTRA1 fusion. (a) Protein blot of patient samples with the fusion using N-terminal and C-terminal HTRA1
antibodies. (b) LC-MS of fusion peptides from fusion-positive cases. Gray highlighted boxes indicate amino acid coverage from LC-MS-identified
peptides mapping to the fusion. Amino acids in blue correspond to SH3PXD2A and those in black map to HTRA1. The asterisk represents the amino
acid sequence of fusion breakpoint. (c) Normalized peptide counts supports the fusion is present in fusion-positive patient samples and absent in
fusion-negative samples. Error bars, s.e.m.; n = 3. (d) Protein blot confirming expression of the HA-tagged SH3PXD2A-HTRA1 fusion and ERK, pS6
activity in HEI-193 cells. (e) Trypan blue cell counts of parental HEI-193 cells and two stably expressing fusion clones. Error bars, s.e.m.; n = 5.
(f) Protein blot of the HA-tagged SH3PXD2A-HTRA1 fusion and ERK, pS6 activity in immortalized Schwann cells. (g) Trypan blue cell counts of
parental Schwann cells and two stably expressing fusion clones. Error bars, s.e.m.; n = 5. (h,i) Invasion assay of HEI-193 and Schwann cells
comparing empty vector cells to fusion-positive cells. Error bars, s.e.m.; n = 3. (j) Serine protease assay with incubation of immune-precipitated
HTRA1, SH3PXD2A-HTRA1 fusion and peptidase inactivated fusion SH3PXD2A-HTRA1 (p.Ser322Ala) incubated with bovine serum albumin (BSA),
a protease substrate for the HTRA1 portion of the fusion. Error bars, s.e.m.; n = 5. (k) Immunofluorescence assay of HEI-193 cells to identify cellular
localization of fusion and co-localization with EEA1. Scale bar, 10 µm.

fusion is linked to its activity promoting cell growth (Supplementary nonobese diabetic severe combined immunodeficient (NOD-SCID)
Fig. 10a,b). SH3PXD2A is known to be located in endosomes, and mice (compared to 0/10 control cells). Histology analysis revealed
therefore we examined localization of the fusion in both cell lines. that these tumors were low in proliferative index (Ki67), robust in
Immunofluorescence analysis identified both a diffuse and punctate S100 staining, pERK-positive and in general had morphology more
cytoplasmic localization of the fusion in both cell lines (Fig. 4k and similar to benign schwannoma (Fig. 5a). To confirm this finding,
Supplementary Fig. 11a−d). The PXD2A domain targets proteins to HEI-193 fusion-positive cells injected into the flank of NOD-SCID
the endosome for protein trafficking. Consistent with this, the fusion mice formed tumors at a higher penetrance compared to immortal-
also localized with EEA1 (early endosome antigen 1), a marker of early ized Schwann cells without the fusion, 5/10 cases compared with 0/10
endosome formation (Fig. 4k and Supplementary Fig. 11a−d). empty vector control HEI-193 cells (Fig. 5b,c). Histology analysis con-
firmed transformation of HEI-193 cell line into a high-grade tumor
The SH3PXD2A-HTRA1 fusion promoted tumorigenesis (Supplementary Fig. 12a). All tumors in vivo expressed high phospho-
We also tested Schwann cells transfected with the fusion construct ERK and phospho-p70 with focal S100 protein positivity (a marker
in vivo. In 2/10 cases those cells formed tumors in the flank of for neural-crest-derived cells, positive in human schwannomas),

1344 VOLUME 48 | NUMBER 11 | NOVEMBER 2016  Nature Genetics

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a H&E HA-SH fusion Ki67 b c 500 d 100 * e Control Fusion

Tumour volume (mm3)

80 cells pos cells

% Patients
400
60
300 40
200 20
pERK S-100 0
100
Thr202 Tyr204

si ion

ga ion
e
e

tiv
po us

ne Fus
tiv
0

F
Z-score expression

on
tro
Extraspinal with bone erosion reaching

si
on

Fu
50 um brainstem with splaying of IAC

C
–3 0 3
n = 0/10 n = 5/10
Small: intraspinal/
not reaching brainstem

f g Fusion h i j Control HEI cells:

IC 50, 85 nM
EMT enriched 40 2 Fusion-positive
TNFα signaling 30 3 HEI cells: IC50 11 nM
4 IL2/STAT5 signaling 100
Myogenesis 20 5
6
Inflammatory response 10 Mitotic spindle
IL2/STAT5 signaling 0 1 Cyclin D1 activation 50

0 10 20 30 1 2 3 4 5 6 0 2 4 6
–Log FDR Number of clusters –Log FDR 0
Z-score expression –10 –9 –8 –7 –6 –5
10 10 10 10 10 10
Log (trametinib) (M)
© 2016 Nature America, Inc., part of Springer Nature. All rights reserved.

–3 0 3

k l Vehicle Trametinib m Vehicle Trematinib n HEI HEI HSC HSC

Control Schwann cells:
Clone A
Clone B

Clone A
Clone B

control fusion-positive control fusion-positive

Clone 1
Clone 2

Clone 1
Clone 2
IC 50, 120 nM
Fusion-positive
EV

EV

EV

Schwann cells: IC50 20 nM EV

Vehicle-
treated
pERK1/2 pERK1/2
100
Viability (%)

ERK1/2 ERK1/2
50 pMEK pMEK

Trametinib-
treated
MEK MEK
0
–10 –9 –8 –7 –6 –5
10 10 10 10 10 10 Cleaved PARP Cleaved PARP
Log trametinib (M)
β-actin β-actin

Figure 5  SH3PXD2A-HTRA1 fusion promoted tumorigenesis and sensitivity to a MEK-ERK inhibitor. (a) Hematoxylin and eosin (H&E) stain and
immunohistochemistry (IHC) analysis of p70SK6, pERK, Ki67 and S100 from Schwann cell fusion-positive tumors. (b) Volumetric measurement
represented as box blots with mean and whiskers of tumor volumes from HEI-193 fusion-positive cells injected into NOD-SCID mice at the 3-month
endpoint. (c) H&E stain and IHC analysis of p70SK6, pERK, Ki67, HA-tag and S100 from fusion-positive tumors in a. HA-IHC confirmed fusion
expression in xenografts. (d) Distribution of tumor size in SH3PXD2A-HTRA1 fusion-positive cases and fusion-negative cases, *P = 0.028 Fisher’s exact test.
(e) Hierarchical clustering of genes differentially expressed between control empty-vector-immortalized Schwann cells and Schwann cells expressing
SH3PXD2A-HTRA1 fusion. We identified 496 genes as significant and differentially expressed (fold change > 1.5, P < 0.05, FDR < 10%). (f) GSEA
identified several significantly enriched biological processes in fusion-positive Schwann cells. (g) Unsupervised hierarchical clustering based on ward
method and Euclidean distance of RNA-seq data supports identified two clusters, with the fusion enriched in the black cluster (4/5). (h) Calinsky and
Harabasz curve supporting two RNA-seq clusters. (i) GSEA results associated with the fusion enriched (black) cluster (minimum 1.5-fold change,
uncorrected P < 0.05. (j,k) IC50 curves of HEI and immortalized cells with and without expression of the fusion assessed at 48 h. (l,m) Protein blot
analysis of cells treated with vehicle and trametinib. EV, empty vector. (n) Colony forming assay of cells with and without fusion treated with trametinib
for 10 d at 50 nM. Error bars, s.e.m.; n = 3.

and all five xenografts were HA-positive (the N-terminal tag on the exact test P = 0.0291). Gene-expression profiling of fusion-positive
SH3PXD2A-HTRA1 fusion) (Supplementary Fig. 12a). Histologic human Schwann cells compared to control cells identified differen-
progression of nerve sheath tumors is correlated with loss of differ- tially expressed genes enriched for epithelial to mesenchymal tran-
entiation and a decrease in S100 expression12. Consistent with this sition (EMT) and IL2-STAT5 signaling as top molecular pathways,
finding, we confirmed relative S100 downregulation in HEI-193 corroborating the findings of increased invasion and phospho-ERK
cells expressing the fusion compared to controls (Supplementary signaling (Fig. 5e,f). RNA clustering of the top 50 most variable genes
Fig. 12b). In our cells, we observed are reduction in trimethylated identified two clusters of schwannomas from our 41 RNA-sequenced
histone lysine 27 (H3K27me3), a marker of reduced PRC2 activ- samples with five known fusion-positive cases. Among the five fusion-
ity, similar to the case in human malignant peripheral nerve sheath positive samples, four cases clustered into one group (Fig. 5g,h and
tumors13 (Supplementary Fig. 12b). Unlike HEI-193 cells, immortal- Supplementary Fig. 13a). GSEA of the top differentially expressed
ized Schwann cells stably expressing the SH3PXD2A-HTRA1 fusion genes in the fusion enriched cluster identified several significantly
had no reduction in S100 or H3K27me3 (Supplementary Fig. 12c). enriched pathways, including IL2-STAT5 signaling (which was com-
Lysates from fusion-positive patient samples had no differences in mon to both patient data and cell line data) as well as mitotic spindle
H3K27me3 or S100 levels compared to fusion-negative patients formation (Fig. 5i and Supplementary Fig. 13b). Given our in vitro
(Supplementary Fig. 12d). and in vivo data supporting upregulation of pMEK-pERK in fusion-
Extending these in vitro and in vivo findings, we examined char- positive cells, we hypothesized whether MEK and/or ERK inhibitors
acteristics of human tumors that correlated with the presence of the could provide a therapeutic benefit. Treatment of SH3PXD2A-HTRA1
SH3PXD2A-HTRA1 fusion. Fusion-positive clinical samples were fusion-positive cells with the MEK1/2 inhibitor trametinib resulted
significantly larger compared to fusion-negative cases, with evidence in an eightold and sixfold lower half-maximal inhibitory concentra-
of bone erosion and remodeling in spinal tumors (Fig. 5d, Fischer’s tion (IC50) for fusion expression in both HEI-193 and immortalized

Nature Genetics  VOLUME 48 | NUMBER 11 | NOVEMBER 2016 1345

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Schwann cells, respectively (Fig. 5j,k). Furthermore, treatment of both
HEI-193 and immortalized Schwann cells expressing the fusion with
50 nM trametinib abolished pMAPK and pERK expression, increased
apoptosis (as measured by cleaved PARP) and resulted in significantly
reduced cell growth as measured by colony forming assay compared
to both non-treated fusion cells and non-treated parental controls
(Fig. 5l–n). Wild-type HTRA1 or SH3PXD2A did not alter pMEK or
pERK, or alter response to trametinib as evaluated by IC50 measure-
ments compared to empty vector controls (Supplementary Fig. 14a,b).
Re-expression of wild-type NF2 in HEI-193 cells did not fully rescue
pro-growth advantage conferred by the fusion, and loss of NF2 in
combination with the fusion led to an additive growth advantage as
evaluated by cell doubling times (Supplementary Fig. 14c−f).
DISCUSSION
Schwannomas are traditionally managed by surgical resection or use
of radiation therapy14,15. However, complete resections of schwanno-
© 2016 Nature America, Inc., part of Springer Nature. All rights reserved.
mas can be difficult, and progression of residual or radiation-treated
schwannomas is a common clinical challenge. Bevacizumab treatment
has been shown to improve hearing and reduce tumor growth in some
but not all patients with vestibular schwannomas3, and this example
demonstrates the need to identify clinically relevant and actionable
therapeutic targets based on molecular analysis. Our current under-
standing of the molecular alterations in schwannomas is still limited,
but some studies have shown that schwannomas harbor aberrant
receptor tyrosine kinase activity and activation of the phosphoinositide
3-kinase-AKT-mammalian target of rapamycin (PI3K-AKT-mTOR)
signaling pathways16–18. In our study, we observed, as expected, fre-
quent NF2 alterations, present in 76% of all schwannomas, caused by
either chromosome 22q deletion or NF2 mutation. Additional findings
included loss of the chromatin-modifying genes ARID1A or ARID1B
in 29% of cases, as well as mutations in DDR1 in 11% of cases.
ARID1 proteins in the SWI-SNF complex allow for recruitment
of chromatin remodeling proteins through recruitment of specific
transcription factors and transcriptional co-activators or repres-
sors19,20. Three of our schwannoma cases had both ARID1A and
ARID1B mutations. One study had observed that loss of ARID1B in
cancers already deficient in ARID1A led to synthetic lethality, and
would support that mutations in these genes would be mutually exclu-
sive21. However, the authors and subsequent studies have shown that
ARID1A and ARID1B mutations can co-occur in both cancer cell lines
and primary tumors, but that all lines retained at least one allele of
either ARID1A or ARID1B, supporting that one functional ARID1A
or ARID1B allele is essential for survival21–23. In the samples we
tested, we observed protein staining for both ARID1A and ARID1B,
suggesting that schwannomas with single or dual ARID1 mutations
maintain ARID1A and ARID1B expression (perhaps the remaining
wild-type allele; Supplementary Fig. 15). Additional studies have
provided evidence for a haploinsufficient role for both ARID1A and
ARID1B in cancer and development24–27, and future functional work
in schwannoma will be required to determine the specific role of
ARID1 in schwannoma biology. Another recurrent mutation of inter-
est was in the gene encoding receptor tyrosine kinase DDR1, which
has previously been shown to represent a potential therapeutic target
in lung cancer, as pharmacological inhibition of DDR1 blocked tumor
progression in a mouse model6,28. DDR1 mutations and SH3PXD2A-
HTRA1 fusion events were exclusive in our data set, supporting that
these alterations may perform similar functions, which remain to be
fully elucidated. Additionally, 15% of schwannomas harbored muta-
tions in TSC1 or TSC2, representing a potential subset of patients that,
in principle, could be tested for potential benefit from mTOR inhibitors
1346 VOLUME 48 | NUMBER 11 | NOVEMBER 2016  Nature Genetics
such as rapamycin. TSC1 or TSC2 mutations co-occurred with NF2
mutation or 22q loss, suggesting that NF2 loss and TSC1 or TSC2 loss
could be cooperating events. It must be noted from our mutational
analysis that we did not have matched normal tissue for our valida-
tion cohort, and it is possible that a subset of our mutations represent
undiscovered germline SNPs.
Most notable in this study was the discovery of an in-frame fusion
involving SH3PXD2A and HTRA1, arising through a balanced translo-
cation on chromosome 10q in 12/25 cases. Fine mapping of the break-
points of this inversion using targeted DNA sequencing identified
recurrent hot-spot genomic breakpoints of the SH3PXD2A-HTRA1
fusion in intron 1 of HTRA1 and intron 6 of SH3PXD2A. Several
tumors with the fusion did not harbor NF2 mutations or chromosome
22q deletion, suggesting that its potential tumorigenic function may
not require loss of NF2 function. Our three fusion-positive samples
for which we possessed protein lysate were negative for wild-type
HTRA1 and SH3PXD2A protein, despite RNA-seq transcript data
supporting that these samples still expressed wild-type HTRA1 and
SH3PXD2A transcripts. This supports that the balanced fusion was
not a homozygous event, and we hypothesize that silencing of wild-
type HTRA1 and SH3PXD2A in these cases may occur at the post-
transcriptional level as both genes have been reported to be targets
of miRNA29,30. An elegant study of angiocentric glioma identified a
tripartite mechanism whereby MYB-QKI rearrangements led to gen-
eration of an oncogenic fusion protein, overexpression of the onco-
gene partner (MYB) and loss of a tumor suppressor (QKI)31. From our
results, combined loss of both SH3PXD2A and HTRA1 in Schwann
cells did not recapitulate the same effect as expression of the fusion
with respect to cell growth. However, additional work will be required
to fully elucicate the functional consequence of losing both fusion
partners in vitro and in vivo independent of the fusion. Screening of
additional fusion-positive cases at the protein level will be essential to
determine whether all fusion-positive samples lose both fusion part-
ners at the RNA level. Expression of the SH3PXD2A-HTRA1 fusion in
two different relevant cell models resulted in increased cell prolifera-
tion, invasion and resistance to anoikis. Overexpression of the fusion
conferred tumorigenicity, in contrast to cells with overexpression of
the wild-type gene partners, HTRA1 or SH3PXD2A. The fusion lacks
the N terminus of wild-type HTRA1 that contains the Kazal domain,
an inhibitory domain for the peptidase domain of HTRA1, but gains
a PXD2A domain from SH3PXD2A32. Is it is plausible that lack of the
Kazal domain in the fusion allows for aberrant peptidase and protease
activity, whereas the PXD2A domain in the fusion protein allows for
both novel interactions and sites of localization for the fusion. We
observed a common signaling pathway involving STAT5 in the fusion-
positive cells in vitro, as well as the fusion-enriched cluster based on
gene expression analysis in tumor specimens. Active STAT5 signaling
has been shown to promote migration and invasion of prostate cancer
cells, and induce EMT33,34, similar to the phenotypes we observed in
the schwannomas. STAT5 inhibitors have been shown to have effi-
cacy in several cancers35, and the mechanism by which the fusion
potentially activates STAT5 warrants future investigation. The fusion
promoted tumor formation in vivo in HEI-193 cells with reduction of
S100 expression and histone 3 trimethylation (H3K27me3) compared
to control cells, supporting that these cells have transformed to a more
malignant peripheral nerve tumor, as loss of S100 and H3K27me3
are observed in malignant peripheral nerve sheath tumors13,36.
Whether the fusion is present in malignant or other peripheral nerve
tumors remains to be determined. Our second xenograft model gen-
erated a more benign schwannoma-like tumor, which was positive
for H3K27me3, possessed a low proliferative index, and exhibited

robust S100 staining, consistent with another study that observed no
significant loss of H3K27me3 in schwannomas36. Our in vitro data
also supported that fusion-positive Schwann cells had no reduction
of S100 or H3K27me3 compared to controls, and our fusion-positive
primary tumor samples also had no reduction in S100 or H3K27me3
compared to fusion-negative samples. This discrepancy between our
two models could arise from the fact that HEI-193 cells were immor-
talized with E6-E7, which inhibits p53 and Rb signaling. This cell
line also harbors losses in chromosomes 8, 13, 15, 17, 18 and 22, and
possesses an NF2 mutation with no other mutations observed from
our targeted sequencing analysis8,37. We postulate that these genetic
events in HEI-193 cells in combination with the fusion result in malig-
nant transformation. Of note is that our control in both models did
not grow, supporting that the fusion itself has transformative ability.
Although the overall rate of fusion positivity was 10%, the presence of
the fusion was highly associated with sex, as 11/64 male schwannoma
patients were fusion-positive. Beyond noting the striking male pre-
© 2016 Nature America, Inc., part of Springer Nature. All rights reserved.
dominance, we currently do not have a specific hypothesis regarding
this bias. Sex differences in cancer incidence and susceptibility are well
established, and we require more fusion-positive cases to conclude
whether it truly is a sex-biased event38. Our methylation clustering
identified two molecular subgroups of schwannoma, with each sub-
group enriched for anatomical location, which suggests that they may
originate from different precursor or progenitor cells. A recent study
on methylation profiling of benign and malignant peripheral nerve
tumors reported similar grouping based on anatomical location, and
other CNS tumors such as ependymal tumors and atypical rhabdoid
tumors have been reported to be comprised of molecular subgroups
with enrichment for different tumor locations39–41. Given the evidence
that the SH3PXD2A-HTRA1 fusion is a potential driver present in a
subset of schwannomas, we provide here a rationale that fusion-posi-
tive cells are potentially sensitive to MEK inhibitors and may repre-
sent a therapeutic approach for treatment-refractory fusion-positive
schwannomas. Further work is required to identify whether addi-
tional pathways are activated or inhibited by the SH3PXD2A-HTRA1
fusion, but from our preclinical results, MEK inhibitors fully suppress
fusion-positive cell growth. Since the SH3PXD2A-HTRA1 fusion is
recurrent and contains an identical mRNA and protein sequence, this
also raises the possibility for simple detection in clinical samples, and
possible targeting with a fusion-specific inhibitor or antibody. The
finding of peptidase or protease activity in the fusion suggests that
this activity may be important for its growth-promoting activities,
and the loss of both protease and growth-promoting activity with
the protease-dead fusion p.Ser322Ala construct supports the concept
that the protease domain of the fusion accompanies the pro-growth
phenotype in schwannoma. It will be important to further identify
potential substrates of the fusion, and how it specifically regulates
EMT and MEK-ERK signaling pathways.
URLs. Picard tools, http://picard.sourceforge.net/; SAMtools, http://
samtools.sourceforge.net/; Genome Analysis Toolkit, http://www.
broadinstitute.org/gatk; Firehose, https://confluence.broadinstitute.
org/display/GDAC/Home; Oncotator, http://www.broadinstitute.org/
oncotator; Exome Aggregation Consortium, http://exac.broadinstitute.
org; Conumee, http://www.bioconductor.org/packages/release/bioc/
html/conumee.html; Hospital for Sick Children SPARC facility, http://
www.sickkids.ca/Research/SPARC/.
Methods
Methods and any associated references are available in the online
version of the paper.
Nature Genetics  VOLUME 48 | NUMBER 11 | NOVEMBER 2016 1347
Articles
Accession codes. Methylation data are accessible through the Gene
Expression Omnibus (accession GSE79009). Cell line gene expression
data are accessible through Gene Expression Omnibus (GSE81145).
Exome and RNA sequencing data are accessible through the European
Genome-phenome Archive (EGAS00001001886).
Note: Any Supplementary Information and Source Data files are available in the
online version of the paper.
Acknowledgments
This work was supported by the Canadian Institute of Health Research (CIHR)
post-doctoral fellowship (S.A.). The contributions of T.J.P., K.D.A. and G.Z. to this
project were supported by the Princess Margaret Cancer Foundation. We thank
the staff of the Princess Margaret Genomics Centre (N. Winegarden, J. Tsao and
N. Khuu) and Bioinformatics Services (C. Virtanen and Z. Lu) for their expertise
in generating the sequencing data used in this study. G.Z. is supported by the
Wilkins Family Chair in Brain Tumor Research, CIHR grants, and The Terry Fox
Research Institute. K.D.A. is supported by funding from the MacFeeters-Hamilton
Neuro-Oncology Research Program. The human immortalized Schwann
cells were a gift from A. Hoke (The Johns Hopkins School of Medicine,
Baltimore, Maryland, USA).
AUTHOR CONTRIBUTIONS
S.A., K.D.A. and G.Z. conceived the entire project, designed, performed and
analyzed the majority of the experiments in this study, prepared figures and
wrote the manuscript. M.R.W. and M.L. performed in vitro experiments
and in vivo studies. S.J., M.D. and T.L. performed QC and targeted sequencing
library preparations. T.T., G.K., A.M., O.K. and B.K. clinically annotated
schwannomas samples. J.R.K., Y.M., N.L.-B., A.A., M.D., T.L., T.J.P., S.M.P.,
K.E.B. and P.D.T. provided technical assistance and data interpretation.
A.D. and T.J.P. analyzed and interpreted fusion breakpoint, point mutations
and indels from targeted re-sequencing. K.D.A. and G.Z. funded the study.
J.P.B., P.K.A., W.L.B., I.F.D. and R.B. provided technical assistance and data
interpretation. A.I. and S.M.P. provided computation expertise and data
interpretation. M.G.F. and V.B. provided clinical expertise.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Reprints and permissions information is available online at http://www.nature.com/
reprints/index.html.
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 ONLINE METHODS
Patient samples. Schwannoma samples and peripheral blood of patients were
collected from the University Health Network Brain Tumor Bank (Toronto)
with informed consent reviewed by a Research Ethics Board. Additional
samples were obtained from Department of Pathology, MD Anderson
Cancer Centre (Houston). In summary, we used 126 schwannoma samples
in this study (64 vestibular schwannomas and 62 spinal schwannomas). We
used 26 patient samples and matched blood for exome sequencing, 41 for
RNA-sequencing, 125 for DNA methylation profiling and 125 for targeted
resequencing (see Supplementary Table 1 for additional information).
Patients analyzed by exome sequencing (n = 26) were confirmed to not have
germline mutations, deletions or loss of heterozygosity (LOH) for LZTR1,
SMARCB1 or SMARCE1.
Exome and targeted sequencing analysis. Whole-exome sequencing of 26
sporadic schwannoma and matched blood samples was performed by EA
Quintiles Genomic services (Durham, North Carolina, USA). Exonic regions
were isolated using the Agilent SureSelect 51 Mb Exome Capture and DNA Seq
Kit followed by sequencing on an Illumina Truseq (Illumina Inc.) to a median
© 2016 Nature America, Inc., part of Springer Nature. All rights reserved.
of 80× coverage. Read pairs were aligned to the hg19 reference sequence using
Burrows-Wheeler Aligner59. All sample pairs passed a quality control pipeline
to test for tumor-normal mix-ups. Somatic variants calls were performed on
tumor/normal variant pairs using Strelka 60. Variants were annotated using
Annovar (version 2014Nov12)61,62, including common databases (ClinVar63,
1000 Genomes64 (phase 3 variant set), dbSNP65 build 138 and COSMIC66
v73). To assess variant impact, we also scored variants using PROVEAN and
SIFT67,68. To identify significantly mutated genes, we applied the mutation
significance analysis (MutSigCV v1.3) algorithm to identify genes that were
mutated more often than expected by chance given background mutation rate4.
Copy number from exome data was inferred using Control-FREEC (Control-
FREE Copy number and allelic content caller)69.
Targeted sequencing and analysis. Targeted exon sequence was performed as
follows. Briefly, 100 ng of input DNA was fragmented by sonication (Covaris
Inc.) to 150 bp and purified using Agencourt AMPure XP beads. Size-selected
DNA was then subject to end repair and A-tailing using the KAPA Hyper Prep
Kit for Illumina Platforms (Kapa Biosystems, Inc.). Each tumor DNA sample
was then ligated to a unique barcoded NEXTflex adaptor (NEXTflex-96 DNA
Barcodes for Illumina, #514106, Bioo Scientific) using the KAPA Hyper Prep
Kit for Illumina Platforms (Kapa Biosystems, Inc.). Sample-specific barcoded
samples were amplified for four cycles with NEXTflex Primer Mix (for Illumina
sequencers, Bioo Scientific) to generate 100 ng of library-prepared samples
using KAPA HiFi HotStart ReadyMix PCR (Kapa Biosystems, Inc.) and puri-
fied using Agencourt AMPure XP beads followed by quantification by Qubit
Fluorometic Quantification (Thermo Fisher Scientific Inc). Targeted exon
capture and sequencing of the validation cohort was performed as follows.
A custom schwannoma xGen Lockdown Panel of DNA biotinylated capture
probes was generated from Integrated DNA Technologies (IDT) to enrich for
all exons of the following genes: ALPK2, ARID1A, ARID1B, CAST, DDR1,
FUBP1, LZTR1, NF2, HTRA1, SH3PXD2A, TAB3, TSC1 and TSC2. To detect
the breakpoint for the SH3PXD2A-HTRA1 fusion, we designed capture probes
spanning all exons of SH3PXD2A and HTRA1 and generated capture probes
tiling every 300−500 bp of all intronic of both genes. In total, 1,005 targeted
DNA capture probes were generated (the list and sequences are provided in
Supplementary Table 9). Targeted capture was performed by pooling on aver-
age eight individual library prepared DNA samples with unique barcodes to
a total of 500 ng DNA per pool. Target enrichment capture was performed at
65 °C for 12 h with 2 pmol of biotinylated capture probes, 5 µg Cot-1 DNA
(Thermo Fisher Scientific Inc.) and 1 µl each of xGEN universal blocking
Oligos (HT-i5, HTi7) using the NimbleGen SeqCap Hybridization and Wash
Kit (Roche NimbleGen). Captured DNA was recovered with Dynabeads M-
270 Streptavidin beads (Thermo Fisher Scientific Inc.) and washed with the
NimbleGen SeqCap Hybridization and Wash Kit. A final post capture PCR
on purified DNA bound beads as performed using the KAPA HiFi HotStart
ReadyMix PCR for 10 cycles (Kapa Biosystems, Inc.), followed by Agencourt
AMPure XP bead purification with a final Qubit Fluorometic Quantification.
Pooled library quantification, average library base-pair size and quality control
doi:10.1038/ng.3688
were determined using the Agilent 2200 TapeStation instrument and Agilent
High Sensitivity D1000 ScreenTape assay. Final capture pooled libraries
were further pooled, providing each pool maintained unique barcodes and
sequenced in three lanes using an Illumina HiSeq 2000 (Illumina Inc.) using
the sequencing system at the Princess Margaret Genomics Centre (PMGC,
http://www.pmgenomics.ca), Toronto.
After sequencing, read pairs were aligned to the hg19 reference sequence
using the Burrows-Wheeler Aligner59, and sample reads were demultiplexed
using Picard tools. Data were then sorted and duplicate-marked using Picard
and SAMtools. Biases in base quality score owing to flowcell, lane, dinucleotide
context and machine cycle were analyzed and recalibrated by members of the
Princess Margaret Genomics Centre. Local realignment around insertions or
deletions (indels) was achieved using the Genome Analysis Toolkit70. Somatic
variant calling was performed using the Firehose environment46,71, and
somatic variant/mutation and indel calls were made and post-filtered using
MuTect71 and IndelLocator70. Variants were annotated using Oncotator 72,
including common databases (ClinVar63, 1000 Genomes (phase 3 variant
set)64, dbSNP (build 138)65, COSMIC (v73)66), and problematic calls caused
by mismapping and other previously identified systematic errors were removed
using an established list of known problematic sites73–75. We excluded germ-
line variants found in the 1000 Genomes Project, dbSNP build 132. We also
screened out mutations against the Exome Aggregation Consortium (ExAC
version 0.3.1) and eliminated any mutation observed in that database with a
frequency >0.0001. All reads were output as bam files, and mutations/indels
were subjected to manual review using Integrated Genomics Viewer (IGV,
Broad Institute, Cambridge, Massachusetts, USA). FUBP1 was a false positive
from our original 26 exome sequencing analysis, and those two samples and
the remaining 99 samples for the validation cohort did not reveal any further
FUBP1 mutations in targeted resequencing.
Fusion breakpoints were mapped from targeted sequencing data using a
two-step algorithm. First, we isolated read pairs aligned to different chromo-
somes or separated by large genomic distances, as indicated by large insert sizes
that may indicate a genome rearrangement. Read pairs supporting a similar
rearrangement were grouped into clusters, and the minimum and maximum
genome coordinates were used to define two candidate break point regions,
one for each end of a mate pair. Next, we searched in each of these candidate
regions for single reads with >20 base quality, >30 mapping quality and >33%
soft-clipped (i.e., unaligned) bases that may indicate possible genomic break-
points. A combination of large-insert read pairs and breakpoint-spanning
reads was taken as indicative of a rearrangement. All reads were written to a
new bam file for manual review of supporting reads.
DNA methylation. DNA (0.5−1 µg) was used for bisulfite treatment (Qiagen,
EpiTect plus). Bisulfite-treated DNA was then quantified using spectropho-
tometry (Nanodrop) and sent to The Centre for Applied Genomics (TCAG;
Toronto) for hybridization on to the HumanMethylation450 BeadChip for 125
schwannoma samples. Arrays were also normalized using the open-source
statistical programming language R. Raw IDAT data files were processed
using the minfi Bioconductor package40,76 and normalized using Beta Mixture
Quantile dilation (BMIQ)77. Methylation values were then exported as β values
(CpG methylation levels). Array probes that overlapped with single-nucleotide
polymorphisms, mapped to chromosomes X and Y, or were Illumina control
probes, were removed from further analysis. In addition to the 125 samples, we
randomly ran and additional six in duplicate as controls for reproducibility. All
samples that were run in duplicate (n = 6) demonstrated near perfect correla-
tion with its duplicate. All sample had an r > 0.99; P < 1 × 10−15). Consensus
clustering, non-negative matrix factorization (NMF) and principal component
analysis (PCA) for subgroup identification was performed in GenePattern
and Partek genomics suite78. SigClust (version 1.1.0) was used to compute the
significance of the clusters identified79. Copy number profiles were generated
using the ‘conumee’ R package in Bioconductor as previously described40,41.
Whole-transcriptome paired-end sequencing (RNA-seq) and fusion
detection. RNA-seq was performed as previously described. We prepared
41 non-strand-specific sequencing libraries using the TruSeq Stranded
mRNA Library Prep Kit (Illumina). Sequencing was carried out on the HiSeq
2000 platform using the manufacturer’s instructions (EA Quintiles Genomic
Nature Genetics
 services). Short read sequences obtained from sequencing were mapped to
the reference human genome (hg19), and spliced (cDNA) and unspliced gene
sequences (Ensembl version 54) were aligned using the Bowtie algorithm80.
Fusion transcript discovery was performed using the deFuse algorithm 7.
Briefly, deFuse identifies fusion transcripts by clustering discordantly align-
ing paired-end reads as potential reads that span a potential fusion breakpoint.
Discordantly aligning cluster reads were then used to inform a targeted search
for reads split by fusion breakpoints. Results from deFuse were next filtered to
reduce false positives using several criteria: (i) predictions fusions had to have
a minimum of 10 reads spanning the fusion breakpoint; (ii) predictions involv-
ing ribosomal proteins or small nuclear ribosomal proteins were removed.
We performed further analysis and validation on a candidate list of fusions
(Supplementary Table 7). Fusion gene expression levels were determined
according to the total coverage of a gene, which was defined as the sum of
the coverage of each nonredundant exonic nucleotide normalized by total
mapped nucleotides.
To identify significant RNA expression changes between subgroups of schwan-
noma, we performed differential expression analysis from transcript sequence
count data using the DESeq and DESeq2 statistical packages in R81,82.
© 2016 Nature America, Inc., part of Springer Nature. All rights reserved.
Fusion validation by RT-PCR and qRT-PCR. We confirmed the SH3PXD2A-
HTRA1 fusions in tumor samples by RT-PCR. RNA was extracted from
snap-frozen tumor samples or FFPE samples, and reverse-transcribed using
SuperScript VILO (Life Technologies). PCR was performed on the cDNA using
primers described in Supplementary Table 10.
PCR products were purified using the Qiagen MinElute PCR purifica-
tion kit, run on a 1% DNA-agarose gel and sent for traditional direct Sanger
sequencing at TCAG (SickKids Hospital, Toronto). To identify fusions in
FFPE RNA extracted samples, a custom TaqMan (Life Technologies) probe-
based gene expression assay was performed using TaqMan universal master
mix II (Life-Technologies) using the manufacturers protocol with a StepOne
Real-Time PCR machine (Life Technologies). Fusion-negative cases and non-
reverse transcribed samples were used as negative controls. Sequences are as
follows with the TaqMan probe being labeled with a 5′ fluorescent dye (6-car-
boxyfluorescein; FAM) and a 3′-labeled quencher dye (tetramethylrhodamine,
TAMRA). Primer sequences are described in Supplementary Table 10.
Cell culture, cell growth, viability and IC50 assays. HEI-193 schwannoma
cells were grown in Dulbecco’s modified Eagle medium (DMEM, Wisent
Technologies) and supplemented with 10% fetal bovine serum (Wisent
Technologies)8. Immortalized primary human Schwann cells using SV40 large
T-antigen and human telomerase reverse transcriptase were grown in DMEM
(Wisent Technologies), supplemented with 10% fetal bovine serum (Wisent
Technologies) and supplemented with 2 µM Forskolin (Sigma F6886). HEI-
193 cells were validated and authenticated as previously described17, and the
immortalized primary human Schwann cells were generated and characterized
as previously described9. Cells were tested for mycoplasma using the myco-
plasma PCR facility at the Hospital for Sick Children (Toronto). For direct cell
counting, cells (1 × 105−2 × 105 cells) were plated into 6-well dishes in 2 ml of
cell-line-specific medium. After incubation times (days 1−7), empty-vector-
expressing cells or fusion-expressing cells were collected and analyzed for cell
count and cell viability using Trypan blue. Cells were directly counted using
the Beckman Coulter Vi-CELL (12-Sample Carousel) Cell Viability Analyzer
(Beckman Coulter). IC50 assays were performed in 96 wells using Alamar Blue
cell viability reagent (Invitrogen) according to manufacturer’s instructions.
Drug response assays were performed by seeding 5,000 cells overnight, treating
the following day with increasing drug concentrations, and reading by Alamar
Blue absorption following 48 h of treatment. Trametinib (GSK1120212) was
purchased from Selleck Chemicals (Cat#S2673).
Plasmids and plasmid contruction. Full length SH3PXD2A-HTRA1 fusion
cDNA was synthesized by IDT and also amplified from a fusion-positive sam-
ple with sequence encoding an N-terminal HA tag using the SuperScript VILO
RT-PCR Kit (Life Technologies, #210210, primers available upon request). The
fusion cDNA was cloned into a Gateway-compatible vector under the control of
a CAG promoter (PB-CAG-IRES-PURO) vector with a puromycin-selectable
marker using the Gateway Cloning system. Stable cell lines expressing the
Nature Genetics
fusion were generated as follows. Briefly, 2 µg of fusion vector or empty
control vector were transfected into HEI-193 or immortalized Schwann
cells in 10-cm dishes both with a puromycin-selectable marker using PEI
transfection reagent (Polyethylenimine, from Polysciences, #23966-2).
Puromycin-resistant clones were selected 14 d later and fusion-positive clones
were confirmed by HA protein blotting. Full-length NF2 cDNA was cloned into
the pcDNA3.1/nFLAG-DEST vector. NF2 short hairpin (sh)RNA vectors were
obtained from the GIPZ Lentiviral shRNA collection also from the Hospital
for Sick Children SPARC facility. HTRA1 Trilencer-27 siRNA (#SR303800)
and SH3PXD2A Trilencer-27 siRNA (# SR306419) was purchased from
Origene. Briefly, 10 nM of control siRNA or SH3PXD2A or HTRA1 siRNAs
were transfected with SiTran1.0: Transfection reagent as per manufacturer’s
instructions (#TT300001, Origene).
Animal xenograft studies. All animal procedures were carried out according
to animal use protocols approved ethically by Institutional Animal Care
Committee under the guidelines of the Canadian Council on Animal Care.
Twenty 4-6 week old non-obese diabetic severe combined immune deficiency
spontaneous male mice (NOD-SCID-Prkdcscid) were randomly separated into
two groups and injected with 2 × 106 HEI-193 schwannoma cells expressing
empty vector or SH3PXD2A-HTRA1. The technician was blinded as to which
cells were injected into the two groups of mice. Cells were resuspended in
10 µl of PBS before injection. Immortalized Schwann cells expressing empty
vector control or fusion cells were injected at 2 × 106 cells as described above.
Mice were monitored for tumor growth, and three months was selected as an
endpoint. Mice were killed at endpoint, and flank tumors were extracted and
fixed in 4% PFA.
Anoikis and invasion assays. Invasion assays were performed using the
Trevigen 24-Well Collagen I cell invasion assay (#3457-024-K) according
to manufacturer’s protocol. Anoikis assays were performed as previously
described11. Cells were transferred from standard adhesive plates to ultralow
cluster plates (Corning) and incubated for 48 h at a density of 1 × 106 cells/ml.
Cells were then collected and allowed to reattach on regular tissue culture
plates. Viability was measured by using Trypan blue on the Beckman Coulter
Vi-CELL (12-Sample Carousel) Cell Viability Analyzer (Beckman Coulter).
Protein blot analysis. Cells or frozen patient samples were lysed in stand-
ard PLC lysis buffer containing protease and phosphatase inhibitors (Roche
Inc.). Protein from cell lysate was quantified using the bicinchoninic acid
(BCA) assay (Pierce Chemical Co.). 30 µg total lysate protein was loaded
onto 10% SDS-PAGE gels and electrophoresed. Proteins were next transferred
onto PVDF membranes (NEN Research Products) and were blocked for 1 h
and probed for varying proteins overnight in 5% non-fat milk or 5% BSA in
Tris Buffered Saline Solution, 0.5% Tween-20 (TBST) or Phospho Buffered
Saline Solution with 0.5% Tween-20 (PBST). Membranes were next washed
for 5 min with PBST or TBST (3×) and incubated with horseradish-
peroxidase-conjugated antibodies specific for the primary antibody (BioRad
Laboratories, Inc.). Binding was detected using Chemiluminescence Reagent
Plus (PerkinElmer Inc.). Antibodies were used at the following dilutions:
beta-actin (Sigma-Aldrich Inc., A2228, 1:10,000), cleaved PARP (ASN214)
(Cell Signaling #9546, 1:1,000), HTRA1 C-term (1:500, Santa Cruz, H-95, sc-
50335), HTRA1-N-term (1:1,000, Sigma-Aldrich, HPA036655), SH3PXD2A
(1:100, GeneTex, #GTX50879), HA tag (1:100, Cell Signaling #C29F4), MYC
tag (1:1,000, Myc-Tag (9B11), #2276), phospho-p44/42 MAPK (Erk1/2)
(Thr202/Tyr204) (1:500, Cell Signaling #4370), Phospho-p70S6 kinase
(1:5,000, Cell Signaling #108D2), p44/42 MAPK (Erk1/2) (1:2,000,Cell
Signaling #9102), p70 S6 kinase (1:2,000, Cell Signaling #2708), GAPDH
(1:10,000, Cell Signaling, #5174), phospho-MEK1/2 (Ser217/221) (1:2,000,
Cell Signaling #9121), MEK1/2 (1:2,000, Cell Signaling #4694(L38C12)), anti-
histone H3 (tri methyl K27, abcam #ab6002).
Immunohistochemistry and immunofluorescence. Paraffin-embedded
blocks were cut in 5 µm sections. Slides were processed as follows: they
were dewaxed in xylene followed by rehydration in a standard alcohol series.
Antigen retrieval was done by pressure-cooking for 20 min in citrate buffer
(pH 6.0), followed by blocking of endogenous peroxidase in 0.3% H2O2.
doi:10.1038/ng.3688
 The antibodies were added and incubated overnight at 4 °C. Antibodies were
detected using a secondary HRP-labeled mouse or rabbit antibody detection
system (Dako EnVision+ System-HRP #k4401, #k4403) followed by addi-
tion 3,3′-diaminobenzidine (DAB) chromagen (Vector Labs) for visualiza-
tion. Sections were counterstained with hematoxylin (Fisher Scientific Inc.)
and slides were dehydrated in 70, 80 and 100% ethanol and xylene. Slides
were covered by coverslips and mounted in Permount (Fisher Scientific Inc.).
Antibodies and concentrations for IHC were as follows: HA tag (1:500, Cell
Signaling #C29F4), Ki67 (1:100, Dako #F7268), S100B (1:100, Dako #Z0311),
phospho-p70S6 kinase (1:500, Cell Signaling #108D2), phospho-p44/42 MAPK
(Erk1/2) (Thr202/Tyr204) (1:500 Cell Signaling cat#4370), ARID1A (1:100,
Cell Signaling #12354), ARID1B (1:100, Abnova # H00057492-M01). For
immunofluorescence, 1 × 105 cells were grown on coverslips in 6-well media
plates overnight. Cells were then fixed using 4% paraformaldehyde (PFA)
for 10 min (Pierce Chemical Co.) and permeabilized with 0.5% Triton X for
15 min. After removal of Triton X, cells were incubated with primary antibod-
ies and detected using secondary antibodies labeled with Alexa Fluor 488 or
Alexa Fluor 594 (Invitrogen Inc.). Cells were mounted with DAPI (Vectashield
#H-1200). Antibodies for immunofluorescence analysis were as follows: HA
© 2016 Nature America, Inc., part of Springer Nature. All rights reserved.
tag (1:100, Cell Signaling #C29F4), EEA1 (1:100, BD Biosciences #610457),
N-term HTRA1 (1:100, Sigma-Aldrich #HPA036655), SH3PXD2A (1:100,
GeneTex #GTX50879). All images were captured on an Olympus IX73 fluores-
cence microscope system and analyzed using CellSens Dimension software.
Statistical analysis. All in vitro experiments were performed in triplicate
unless specifically stated in figure legends, with mean and s.e.m. reported.
Analysis of variance (ANOVA) was conducted for multigroup comparisons
followed by a post-hoc Tukey’s test (to identify differences among subgroups).
For direct comparisons, an unpaired two-tailed Student’s t-test was used.
Groups being compared had similar variance, and significance was estab-
lished as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 unless specifically
stated in figure legends.
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