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CORRESPONDENCE L I N K T O O R I G I N A L A RT I C L E

careful consideration when defining a seno-

Senotherapeutics for healthy ageing


lytic or senomorphic drug and the likelihood
of its broad efficacy.
Once evidence is found for senolytic activ-
ity of a compound in vitro, it is even more
Laura J. Niedernhofer and Paul D. Robbins
difficult to document potential senolytic
activity in vivo (TABLE 1). Demonstrating that
The recent manuscript by Childs et al. in The natural compounds fisetin3, a quercetin- a compound reduces the number of SA-ß-gal+
Nature Reviews Drug Discovery1 thoroughly related flavonoid, and piperlongumine also cells or the level of p16ink4a expression, such as
reviewed the important role senescent cells exhibit evidence of senolytic or senomorphic through the use of p16ink4a–luciferase reporter
play in driving ageing and age-related dis- activity in certain cell types in vitro. Clinically mice, does not definitively demonstrate kill-
eases. The review also highlighted the clinical used compounds targeting the co-chaperone ing, let alone selective killing, of senescent
importance of developing senotherapeutic heat shock protein 90 (HSP90) were also iden- cells. Also, since senolytics are cell type-spe-
approaches to selectively kill senescent cells tified as a novel class of potential senolytics, cific, the lack of an effect on a specific tissue
(senolytics) or to suppress the senescence- able to induce apoptosis of senescent murine doesn’t preclude senolytic activity in other
associated secretory phenotype (SASP) that and human cells in vitro and improve health- tissues. Even more rigorous approaches, such
drives sterile inflammation associated with span in vivo 4. Finally, the FDA-approved as the injection of labelled senescent cells fol-
ageing (senomorphics), in order to extend histone deacetylase inhibitor panobinostat lowed by drug treatment, only serve to docu-
healthspan and potentially lifespan. Clearly, induces apoptosis of senescent tumour cells ment that the putative senolytic is functioning
senotherapeutic approaches can revolution- in vitro. Clearly, there are multiple SCAP tar- in vivo similar to in vitro, that is, by killing
ize how age-related diseases and ultimately gets and thus it is highly likely that additional that specific type of transplanted senescent
how ageing itself can be treated. Given how classes of potential senolytics will be identi- cell. Eventually, improved methods for colo-
quickly the field of senotherapeutics is mov- fied through bioinformatic analyses and drug- calization of apoptosis and senescence in vivo
ing towards clinical trials, we would like to screening approaches. at the single cell level, such as through the use
expand upon several important issues regard- Several classes of senomorphics — drugs of cytometry by time-of-flight (CyTOF) or
ing the current and future development of that suppress markers of senescence or their dual fluorescent reporters, will be needed to
senotherapeutics. This is particularly impor- secretory phenotype without inducing apopto- define a compound as senolytic.
tant since the nascent field of senotherapeu- sis — have also been identified. Senomorphics One current tactic to provide evidence
tics is faced with the challenge of establishing include inhibitors of IkB kinase (IKK) and that a compound has senolytic activity is the
important standards and criteria for docu- nuclear factor (NF)-kB5, free radical scaven- ‘hit-and-run’ approach, for example, the one-
menting a compound’s or a combination of gers and Janus kinase (JAK) pathway inhibi- time treatment of mice with dasatinib and
compounds’ function as advertised, that is, as tors6. Even rapamycin acts as a senomorphic quercetin following hind-leg irradiation3.
senotherapeutics. by reducing the SASP. Some compounds, for Here, a single administration of dasatinib
It is established that senescent cells play a example fisetin, have senomorphic effects on plus quercetin yielded a therapeutic benefit
causative role in ageing and age-related dis- some cell types while having senolytic activity that endured for months in terms of tread-
ease. Therefore, the development of drugs that on others, at least in vitro3. mill performance, consistent with a senolytic
specifically kill senescent cells is envisioned to It is important to note that definitively mechanism in which disease-causing cells are
have significant therapeutic effects on slow- demonstrating that a drug is senolytic or killed. In general, if a short course of treat-
ing ageing phenotypes, treating age-related senomorphic is challenging and should be ment yields a sustained reduction in senes-
comorbidities and improving resiliency. approached critically. Simply measuring cence, it is likely acting in a senolytic manner.
However, not all senescent cells are the same, a reduction in senescence markers (such In contrast, if chronic treatment is needed to
expressing different senescence markers, as the expression of p16INK4A or p21CIP1 or suppress senescence markers or prevent sec-
secreting different SASP factors and, more SASP factors) or in senescence-associated ondary senescence, this is more consistent
importantly, using different senescent cell β-galactosidase (SA-β-gal) activity is inad- with senomorphic activity. However, more
anti-apoptotic pathways (SCAPs) to resist equate to distinguish between the two mech- refined approaches are needed to rapidly
apoptosis. The elimination of senescent cells anisms of action. Careful determination of demonstrate senolytic activity in vivo. Likely
from multiple tissues or even a single tissue whether there is selective loss of senescent combinations of approaches may be neces-
will probably require the combination of mul- cells is required. Notably, the assay selected to sary. For example, if a drug kills senescent
tiple senotherapeutic drugs2. test potential therapeutics influences the out- endothelial cells, it could yield a health divi-
To date, seven classes of compound with come. For example, the cell type selected and dend, but it will be very difficult to document
some evidence of senolytic activity have the method used to induce senescence influ- a reduction in the expression of senescence
been reported (see Supplementary Table 1), ences which SCAPs are expressed and there- markers specifically in the endothelium with-
including the combination of dasatinib and fore whether a particular class of drugs (for out sophisticated reporter systems.
quercetin, as well as BCL2 family inhibitors, example, BCL2 inhibitors) will be effective in Another current approach to provide
identified using a bioinformatics approach killing cells. Also, the end points measured evidence that a compound potentially has
for SCAPs2. In addition, a forkhead box pro- will influence interpretation (for example, senolytic activity in vivo is to compare the
tein O4 (FOXO4)-interacting peptide, which senescence markers, SASP, number of viable therapeutic efficacy of the drug to outcomes
blocks the association of FOXO4 with p53, cells and/or apoptosis markers) as well as what yielded by genetic ablation of senescent cells
induces apoptosis of senescent human cells in control cells are used (for example, proliferat- in transgenic INK-ATTAC or p16-3MR
vitro and reduces the expression of senescence ing or quiescent). Thus, the assays used and mice7,8. For example, in the bleomycin mouse
markers while extending healthspan in vivo. end points measured should be taken into model of idiopathic pulmonary fibrosis, the

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CORRESPONDENCE

Table 1 | Approaches for demonstrating the mechanism of action of senotherapeutics.


System for testing Class of drug New methodologies
Senolytic Senomorphic
p16-reporter mice and/or Short-term administration of a Chronic treatment with a • Additional transgenic reporters for
measuring senescence in senolytic must yield a sustained senomorphic is required for markers of senescence such as p21
multiple tissues using multiple reduction in senescence signala a sustained suppression in • Dual reporters for apoptosis and
end points (expression of p16, senescence signal senescence
p21 and SASP, and/or decreased • Analysis of activity in
SA-ß-gal activity) immune-deficient mice
Compare pharmacological A short-term or intermittent Chronic treatment with a • Additional genetic ablation models
ablation of senescent course of a senolytic yields the senomorphic is required to get the such as p21-ATTAC
cells to genetic ablation same outcome as using a drug same outcome as genetic ablation • Analysis of activity in
(p16-INK-ATTAC or p16-3MR to activate a transgene to ablate of senescent cells immune-deficient mice
mice)b senescent cells
Human tissue explants Drug treatment yields a Drug treatment yields a suppression CyTOF, in situ hybridization and
harbouring senescent cells reduction in senescence markers in senescence markers (expression related approaches to colocalize
(expression of p16, p21 and of p16, p21 and SASP, and/or downregulation of senescence
SASP, and/or decreased SA-ß-gal decreased SA-ß-gal activity) with no with upregulation of apoptosis
activity) and an increase in evidence of cell death markers as well as cell identity
apoptosis markers markers
Transplantation of labelled Short-term or intermittent Chronic administration of a • Transplantation of cells dually
senescent cells into a host administration of a senolytic must senomorphic will suppress labelled with markers expressed
organism; the method of yield a long-term attenuation senescence end points but not from senescence-dependent
labelling should be independent of labelled cells, as well as attenuate the signal from the and senescence-independent
of senescence (e.g. a ubiquitous expression of senescence end labelled cells promoters
rather than a p16 promoter) points • Monitor both markers to
distinguish between senolytics
and senomorphics
Measure therapeutic efficacy in Short-term or intermittent Chronic administration of a Coordinate analysis of therapeutic
aged or diseased organisms intervention with a senolytic must senomorphic is required to yield a efficacy and a reduction of
yield a sustained health benefit in sustained health benefit in old or senescence using bioluminescent
old or diseased organisms diseased organisms reporters or serum markers with
short or intermittent treatment in
models of natural and accelerated
ageing
Multiple systems are currently being used to test if a drug specifically targets senescent cells in vivo. Often what is reported is a reduction in senescence markers.
To document that an intervention is truly senolytic will require additional lines of evidence including the development of new methods. These are outlined for
several experimental systems. CyTOF, cytometry by time-of-flight; SA-β-gal, senescence-associated β-galactosidase; SASP, senescence-associated secretory
phenotype. aDrugs that improve immune function can also reduce senescence by improving immune-mediated clearance. bSince senolytics may target senescent
cells that are p16-negative, the results obtained with a putative senolytic may differ from the results achieved with p16-INK-ATTAC or p16-3MR mice.

therapeutic benefit of treatment with dasatinib attenuate senescence markers while induc- the SASP might improve immune clearance
and quercetin is comparable to clearing senes- ing apoptosis. However, while this approach of senescent cells. Thus, demonstrating that
cent cells in INK-ATTAC mice9. However, it documents senolytic activity of a compound, a drug directly targets senescent cells must
will still be important to determine if the same it does not identify the types of cell undergo- address the possibility that the drug improves
cell types are targeted in the INK-ATTAC and ing cell death and whether it is only a sub- host clearance of senescent cells.
the dasatinib and quercetin-treated mice. In set of senescent cells. Additional methods, All in all, there is reason to be tremen-
fact, the mechanism of cell clearance in INK- including flow cytometry and CyTOF-based dously excited about the health and economic
ATTAC mice differs fundamentally from mice approaches, are needed to define the cell impact of senotherapeutic drugs. However, as
treated with senolytic agents. In the transgenic types within a tissue explant that are targeted noted, caution is warranted in interpreting the
models, cells with high p16ink4a promoter activ- by senotherapeutics, as well as to colocalize mechanism of action and relative specificity,
ity are targeted, while senolytics act by killing markers of senescence and apoptosis. selectivity and efficacy. Furthermore, there is
cells expressing specific pro-survival SCAPs. Proving that a compound exhibits in vivo still much to learn about how best to identify,
Importantly, not every cell with high p16ink4a senolytic activity is further complicated by characterize and apply these drugs to improve
expression is senescent and not every senes- potential indirect effects on the immune human health.
cent cell has high p16ink4a expression. For system. Functional immune cells, including Laura J. Niedernhofer and Paul D. Robbins are at the
example, p16INK4A and SA-β-gal activity are natural killer and T lymphocytes, can remove Department of Molecular Medicine and The Center on
induced in macrophages as part of a reversible senescent cells. However, as the immune sys- Aging, The Scripps Research Institute, Jupiter, FL, USA.
response to physiological stimuli even though tem ages, the ability to clear senescent cells e-mail: lniedern@scripps.edu; probbins@scripps.edu
these cells are not senescent10. wanes, contributing to the accumulation of doi:10.1038/nrd.2018.44
An additional approach to provide evi- senescent cells. It is possible that some drugs, Published online 13 Apr 2018
dence of senolytic activity in vivo involves for example, rapamycin, reduce the burden Acknowledgements
This work was supported by National Institutes of Health
the use of human tissue explants harbouring of senescent cells indirectly by improving grants P01-AG043376, U19-AG056278 and AG044376
senescent cells7. A putative senolytic should immune cell function. Similarly, inhibiting and by the Glenn Foundation.

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CORRESPONDENCE

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