Senotherapeutics for Healthy Ageing of Its Broad Efficacy

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careful consideration when defining a senolytic or senomorphic drug and the likelihood Senotherapeutics for healthy ageing of its broad efficacy. Once evidence is found for senolytic activ- Laura J. Niedernhofer and Paul D. Robbins ity of a compound in vitro, it is even more 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 vivo4. 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 treating 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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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. ink4a cent cell has high p16 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: [email protected]; [email protected] 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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Competing interests 3. Zhu, Y. et al. New agents that target senescent cells: the 7. Jeon, O. H. et al. Local clearance of senescent cells The authors declare no competing interests. flavone, fisetin, and the BCL-XL inhibitors, A1331852 attenuates the development of post-traumatic and A1155463. Aging 9, 955–963 (2017). osteoarthritis and creates a pro-regenerative Supplementary information 4. Fuhrmann-Stroissnigg, H. et al. Identification of environment. Nat. Med. 23, 775–781 (2017). Supplementary information is available for this paper at HSP90 inhibitors as a novel class of senolytics. 8. Baker, D. J. et al. Clearance of p16Ink4a-positive https://doi.org/nrd.2018.44 Nat. Commun. 8, 422 (2017). senescent cells delays ageing-associated disorders. 5. Tilstra, J. S. et al. NF-kappaB inhibition delays DNA Nature 479, 232–236 (2011). 1. Childs, B. G. et al. Senescent cells: an emerging target damage-induced senescence and aging in mice. 9. Schafer, M. J. et al. Cellular senescence mediates fibrotic for diseases of ageing. Nat. Rev. Drug Discov. 16, J. Clin. Invest. 122, 2601–2612 (2012). pulmonary disease. Nat. Commun. 8, 14532 (2017). 718–735 (2017). 6. Xu, M. et al. JAK inhibition alleviates the cellular 10. Hall, B. M. et al. p16(Ink4a) and senescence- 2. Zhu, Y. et al. The Achilles’ heel of senescent cells: senescence-associated secretory phenotype and associated beta-galactosidase can be induced in from transcriptome to senolytic drugs. Aging Cell 14, frailty in old age. Proc. Natl Acad. Sci. USA 112, macrophages as part of a reversible response to 644–658 (2015). E6301–E6310 (2015). physiological stimuli. Aging 9, 1867–1884 (2017).