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There is an increasing need for biomarkers of senescent cell burden to facilitate the selection of participants for clinical trials. p16Ink4a is encoded by the CDKN2A locus, which produces five variant transcripts in humans, two of which encode homologous p16 proteins: p16Inka4a, encoded by p16_variant 1, and p16ɣ, encoded by p16_variant 5. While distinct quantitative polymerase chain reaction primers can be designed for p16_variant 5, primers for p16_variant 1 also measure p16_variant 5 (p16_variant 1 + 5). In a recent clinical trial evaluating the effects of the senolytic combination, dasatinib + quercetin (D + Q), on bone metabolism in postmenopausal women, we found that women in the highest tertile for T‐cell expression of p16_variant 5 had the most robust skeletal responses to D + Q. Importantly, the assessment of p16_variant 5 was more predictive of these responses than p16_variant 1 + 5. Here, we demonstrate that in vitro, p16_variant 1 + 5 increased rapidly (Week 1) following the induction of DNA damage, whereas p16_variant 5 increased later (Week 4), suggesting that p16_variant 5 becomes detectable only when the abundance of senescent cells reaches some threshold. Further analysis identified a SASP panel in plasma that performed as well in identifying postmenopausal women with a positive skeletal response to D + Q. Collectively, our findings provide further support for the T‐cell p16_variant 5 assay as a biomarker for selecting participants in clinical trials of senolytic interventions. In addition, our data indicate that correlated plasma SASP markers could be used in lieu of the more technically challenging T‐cell p16 assay. Trial Registration: ClinicalTrials.gov identifier: NCT04313634. Assessment of T‐cell p16_variant 5 expression may be useful for selecting participants in clinical trials of senolytics. We further characterize p16_variant 5 expression in the context of senescence and demonstrate that correlated plasma senescence‐associated secretory phenotype factors could be used in lieu of the technically challenging T‐cell p16_variant 5 assay.

Preclinical evidence demonstrates that senescent cells accumulate with aging and that senolytics delay multiple age-related morbidities, including bone loss. Thus, we conducted a phase 2 randomized controlled trial of intermittent administration of the senolytic combination dasatinib plus quercetin (D + Q) in postmenopausal women ( n  = 60 participants). The primary endpoint, percentage changes at 20 weeks in the bone resorption marker C-terminal telopeptide of type 1 collagen (CTx), did not differ between groups (median (interquartile range), D + Q −4.1% (−13.2, 2.6), control −7.7% (−20.1, 14.3); P  = 0.611). The secondary endpoint, percentage changes in the bone formation marker procollagen type 1 N-terminal propeptide (P1NP), increased significantly (relative to control) in the D + Q group at both 2 weeks (+16%, P  = 0.020) and 4 weeks (+16%, P  = 0.024), but was not different from control at 20 weeks (−9%, P  = 0.149). No serious adverse events were observed. In exploratory analyses, the skeletal response to D + Q was driven principally by women with a high senescent cell burden (highest tertile for T cell p16 (also known as CDKN2A ) mRNA levels) in which D + Q concomitantly increased P1NP (+34%, P  = 0.035) and reduced CTx (−11%, P  = 0.049) at 2 weeks, and increased radius bone mineral density (+2.7%, P  = 0.004) at 20 weeks. Thus, intermittent D + Q treatment did not reduce bone resorption in the overall group of postmenopausal women. However, our exploratory analyses indicate that further studies are needed testing the hypothesis that the underlying senescent cell burden may dictate the clinical response to senolytics. ClinicalTrials.gov identifier: NCT04313634 . In a phase 2 randomized control trial, intermittent senolytic therapy administered to postmenopausal women did not result in a reduction in the bone resorption marker, serum CTx, compared to control at 20 weeks.

Cellular senescence has been recently shown to have an important role in opposing tumour initiation and promotion. Senescence induced by oncogenes or by loss of tumour suppressor genes is thought to critically depend on induction of the p19 Arf –p53 pathway. The Skp2 E3-ubiquitin ligase can act as a proto-oncogene and its aberrant overexpression is frequently observed in human cancers. Here we show that although Skp2 inactivation on its own does not induce cellular senescence, aberrant proto-oncogenic signals as well as inactivation of tumour suppressor genes do trigger a potent, tumour-suppressive senescence response in mice and cells devoid of Skp2 . Notably, Skp2 inactivation and oncogenic-stress-driven senescence neither elicit activation of the p19 Arf –p53 pathway nor DNA damage, but instead depend on Atf4, p27 and p21. We further demonstrate that genetic Skp2 inactivation evokes cellular senescence even in oncogenic conditions in which the p19 Arf –p53 response is impaired, whereas a Skp2–SCF complex inhibitor can trigger cellular senescence in p53/Pten-deficient cells and tumour regression in preclinical studies. Our findings therefore provide proof-of-principle evidence that pharmacological inhibition of Skp2 may represent a general approach for cancer prevention and therapy. Senescence kills tumours Recent studies suggest that cellular senescence — an irreversible form of cell-cycle arrest — can halt tumour growth in vitro . Hui-Kuan Lin et al . now identify a previously unknown pathway that drives senescence without the involvement of most of the known mediators of senescence. Instead, it signals via the transcription factor Atf6, and the cyclin-dependent kinase inhibitors p27 and p21. The pathway is uncovered by inactivation of the proto-oncogene Skp2 , but only in the context of oncogenic signalling. Targeting the Skp2 complex pharmacologically restricts tumorigenesis by inducing cellular senescence, suggesting that such drugs may be effective in cancer prevention and therapy. Cellular senescence — an irreversible cell-cycle arrest — has been implicated in suppressing tumour formation or growth. A new cellular signalling pathway that drives senescence has now been identified. This pathway does not involve most known mediators of senescence, and instead signals via the proteins Atf4, p27 and p21. Inactivating the proto-oncogene Skp2 in the context of oncogenic signalling can induce senescence through this new pathway, indicating that drugs that target Skp2 might be useful in cancer treatment.

Senescent cells accumulate with age in vertebrates and promote aging largely through their senescence‐associated secretory phenotype (SASP). Many types of stress induce senescence, including genotoxic stress. ERCC1‐XPF is a DNA repair endonuclease required for multiple DNA repair mechanisms that protect the nuclear genome. Humans or mice with reduced expression of this enzyme age rapidly due to increased levels of spontaneous, genotoxic stress. Here, we asked whether this corresponds to an increased level of senescent cells. p16Ink4a and p21Cip1 mRNA were increased ~15‐fold in peripheral lymphocytes from 4‐ to 5‐month‐old Ercc1−/∆ and 2.5‐year‐old wild‐type (WT) mice, suggesting that these animals exhibit a similar biological age. p16Ink4a and p21Cip1 mRNA were elevated in 10 of 13 tissues analyzed from 4‐ to 5‐month‐old Ercc1−/∆ mice, indicating where endogenous DNA damage drives senescence in vivo. Aged WT mice had similar increases of p16Ink4a and p21Cip1 mRNA in the same 10 tissues as the mutant mice. Senescence‐associated β–galactosidase activity and p21Cip1 protein also were increased in tissues of the progeroid and aged mice, while Lamin B1 mRNA and protein levels were diminished. In Ercc1−/Δ mice with a p16Ink4a luciferase reporter, bioluminescence rose steadily with age, particularly in lung, thymus, and pancreas. These data illustrate where senescence occurs with natural and accelerated aging in mice and the relative extent of senescence among tissues. Interestingly, senescence was greater in male mice until the end of life. The similarities between Ercc1−/∆ and aged WT mice support the conclusion that the DNA repair‐deficient mice accurately model the age‐related accumulation of senescent cells, albeit six‐times faster. Senescent cells contribute to aging and its associated morbidities. Senescent cells accumulate in vertebrates with aging. Here, we survey where (in what tissues) senescence occurs with aging in mice, by measuring p16Ink4a and p21Cip1 mRNA. A similar survey in Ercc1−/Δ mice illustrates where (in what tissues) senescence occurs in vivo as a consequence of endogenous DNA damage.

A small-molecule inducer of apoptosis is able to kill senescent cells in the bone marrow of irradiated or aged mice, thereby improving hematopoietic stem cell function. Senescent cells (SCs) accumulate with age and after genotoxic stress, such as total-body irradiation (TBI) 1 , 2 , 3 , 4 , 5 , 6 . Clearance of SCs in a progeroid mouse model using a transgenic approach delays several age-associated disorders 7 , suggesting that SCs play a causative role in certain age-related pathologies. Thus, a 'senolytic' pharmacological agent that can selectively kill SCs holds promise for rejuvenating tissue stem cells and extending health span. To test this idea, we screened a collection of compounds and identified ABT263 (a specific inhibitor of the anti-apoptotic proteins BCL-2 and BCL-xL) as a potent senolytic drug. We show that ABT263 selectively kills SCs in culture in a cell type– and species-independent manner by inducing apoptosis. Oral administration of ABT263 to either sublethally irradiated or normally aged mice effectively depleted SCs, including senescent bone marrow hematopoietic stem cells (HSCs) and senescent muscle stem cells (MuSCs). Notably, this depletion mitigated TBI-induced premature aging of the hematopoietic system and rejuvenated the aged HSCs and MuSCs in normally aged mice. Our results demonstrate that selective clearance of SCs by a pharmacological agent is beneficial in part through its rejuvenation of aged tissue stem cells. Thus, senolytic drugs may represent a new class of radiation mitigators and anti-aging agents.

Background Frequent truncation mutations of the histone lysine N-methyltransferase KMT2C have been detected by whole exome sequencing studies in various cancers, including malignancies of the prostate. However, the biological consequences of these alterations in prostate cancer have not yet been elucidated. Methods To investigate the functional effects of these mutations, we deleted the C-terminal catalytic core motif of Kmt2c specifically in mouse prostate epithelium. We analysed the effect of Kmt2c SET domain deletion in a Pten -deficient PCa mouse model in vivo and of truncation mutations of KMT2C in a large number of prostate cancer patients. Results We show here for the first time that impaired KMT2C methyltransferase activity drives proliferation and PIN formation and, when combined with loss of the tumour suppressor PTEN , triggers loss of senescence, metastatic dissemination and dramatically reduces life expectancy. In Kmt2c -mutated tumours we show enrichment of proliferative MYC gene signatures and loss of expression of the cell cycle repressor p16 INK4A . In addition, we observe a striking reduction in disease-free survival of patients with KMT2C -mutated prostate cancer. Conclusions We identified truncating events of KMT2C as drivers of proliferation and PIN formation. Loss of PTEN and KMT2C in prostate cancer results in loss of senescence, metastatic dissemination and reduced life expectancy. Our data demonstrate the prognostic significance of KMT2C mutation status in prostate cancer patients. Inhibition of the MYC signalling axis may be a viable treatment option for patients with KMT2C truncations and therefore poor prognosis.

Cellular senescence, which is a major cause of tissue dysfunction with aging and multiple other conditions, is known to be triggered by p16Ink4a or p21Cip1, but the relative contributions of each pathway toward inducing senescence are unclear. Here, we directly addressed this issue by first developing and validating a p21‐ATTAC mouse with the p21Cip1 promoter driving a “suicide” transgene encoding an inducible caspase‐8 which, upon induction, selectively kills p21Cip1‐expressing senescent cells. Next, we used the p21‐ATTAC mouse and the established p16‐INK‐ATTAC mouse to directly compare the contributions of p21Cip1 versus p16Ink4a in driving cellular senescence in a condition where a tissue phenotype (bone loss and increased marrow adiposity) is clearly driven by cellular senescence—specifically, radiation‐induced osteoporosis. Using RNA in situ hybridization, we confirmed the reduction in radiation‐induced p21Cip1‐ or p16Ink4a‐driven transcripts following senescent cell clearance in both models. However, only clearance of p21Cip1+, but not p16Ink4a+, senescent cells prevented both radiation‐induced osteoporosis and increased marrow adiposity. Reduction in senescent cells with dysfunctional telomeres following clearance of p21Cip1+, but not p16Ink4a+, senescent cells also reduced several of the radiation‐induced pro‐inflammatory senescence‐associated secretory phenotype factors. Thus, by directly comparing senescent cell clearance using two parallel genetic models, we demonstrate that radiation‐induced osteoporosis is driven predominantly by p21Cip1‐ rather than p16Ink4a‐mediated cellular senescence. Further, this approach can be used to dissect the contributions of these pathways in other senescence‐associated conditions, including aging across tissues. We generated a new mouse model (p21‐ATTAC) for clearance of senescent cells expressing p21Cip1. Clearance of p21Cip1‐expressing senescent cells, but not of p16Ink4a‐expressing cells, prevented bone loss following focal radiation. The genetic approach described here can be used to dissect the contributions of p21Cip‐ versus p16Ink4a‐driven cellular senescence in other senescence‐associated conditions, including aging across tissues.

In spite of distinct clinical importance, the molecular mechanisms how Additional sex combs-like 1 ( ASXL1 ) mutation contributes to the pathogenesis of premalignant conditions are largely unknown. Here, with newly generated knock-in mice, we investigated the biological effects of the mutant. Asxl1 G643fs heterozygous ( Asxl1 G643fs/+ ) mice developed phenotypes recapitulating human low-risk myelodysplastic syndromes (MDS), and some of them developed MDS/myeloproliferative neoplasm-like disease after long latency. H2AK119ub1 level around the promoter region of p16Ink4a was significantly decreased in Asxl1 G643fs/+ hematopoietic stem cells (HSC), suggesting perturbation of Bmi1-driven H2AK119ub1 histone modification by mutated Asxl1. The mutant form of ASXL1 had no ability to interact with BMI1 as opposed to wild-type ASXL1 protein. Restoration of HSC pool and amelioration of increased apoptosis in hematopoietic stem and progenitor cells were obtained from Asxl1 G643fs/+ mice heterozygous for p16Ink4a . These results indicated that loss of protein interaction between Asxl1 mutant and Bmi1 affected the activity of PRC1, and subsequent derepression of p16Ink4a by aberrant histone ubiquitination could induce cellular senescence, resulting in low-risk MDS-like phenotypes in Asxl1 G643fs/+ mice. This model provides a useful platform to unveil the molecular basis for hematological disorders induced by ASXL1 mutation and to develop therapeutic strategies for these patients.

Regeneration of skeletal muscle depends on a population of adult stem cells (satellite cells) that remain quiescent throughout life. Satellite cell regenerative functions decline with ageing. Here we report that geriatric satellite cells are incapable of maintaining their normal quiescent state in muscle homeostatic conditions, and that this irreversibly affects their intrinsic regenerative and self-renewal capacities. In geriatric mice, resting satellite cells lose reversible quiescence by switching to an irreversible pre-senescence state, caused by derepression of p16 INK4a (also called Cdkn2a). On injury, these cells fail to activate and expand, undergoing accelerated entry into a full senescence state (geroconversion), even in a youthful environment. p16 INK4a silencing in geriatric satellite cells restores quiescence and muscle regenerative functions. Our results demonstrate that maintenance of quiescence in adult life depends on the active repression of senescence pathways. As p16 INK4a is dysregulated in human geriatric satellite cells, these findings provide the basis for stem-cell rejuvenation in sarcopenic muscles. This study shows that ageing satellite cells undergo an irreversible transition from a quiescent to a pre-senescent state that results in the loss of muscle regeneration in sarcopenia; furthermore, increased expression of p16 INK4a is identified as a common feature of senescent satellite cells. When muscles grow old One of the properties crucial to the function of adult mammalian stem cells is the capacity to remain in a quiescent state for prolonged periods — and to respond when the need to regenerate arises. Loss of skeletal muscle mass and function are common features of advanced ageing in humans, associated with a loss of regenerative capacity of the skeletal muscle stem cells, known as satellite cells. Pura Muñoz-Cánoves and colleagues show that ageing satellite cells undergo an irreversible transition from quiescence to a pre-senescence state associated with increased expression of p16 INK4a , a tumour-suppressor protein that has been identified as a marker for senescence. Repression of p16 INK4a during adult life is shown to maintain satellite cells in a reversible quiescence state that allows muscle regeneration; p16 INK4a is dysregulated in human geriatric satellite cells and the potential for muscle regeneration is lost.

The cell cycle regulator p16 is known as a biomarker and an effector of aging. However, its function in intervertebral disc degeneration (IVDD) is unclear. In this study, p16 expression levels were found to be positively correlated with the severity of human IVDD. In a mouse tail suspension (TS)-induced IVDD model, lumbar intervertebral disc height index and matrix protein expression levels were reduced significantly were largely rescued by p16 deletion. In TS mouse discs, reactive oxygen species levels, proportions of senescent cells, and the senescence-associated secretory phenotype (SASP) were all increased, cell cycling was delayed, and expression was downregulated for Sirt1, superoxide dismutase 1/2, cyclin-dependent kinases 4/6, phosphorylated retinoblastoma protein, and transcription factor E2F1/2. However, these effects were rescued by p16 deletion. Our results demonstrate that p16 plays an important role in IVDD pathogenesis and that its deletion attenuates IVDD by promoting cell cycle and inhibiting SASP, cell senescence, and oxidative stress. Neck and shoulder pain, lower back pain and leg numbness are conditions that many people will encounter as years go by. This is because intervertebral discs, the padding structures that fit between the bones in the spine, degenerate with age: their cells enter a ‘senescent’, inactive state, and stop multiplying. A protein known as p16, an important regulator of cell growth and division, is known to accumulate in senescent cells. In fact, in mouse fat tissue, muscles or eyes, removing the cells that contain high levels of p16 delays aging-associated disorders. However, it was still unknown whether deactivating the gene that codes p16 in senescent cells could delay disc degeneration. Here, Che, Li et al. discovered that p16 is highly present in the senescent cells of severely degenerated human intervertebral discs. The cells in the nucleus pulposus, the jelly-like and most critical tissue in the intervertebral discs, were extracted and grown in the lab under conditions that replicate the early stages of damage to the spine. Drugs and genetic manipulations were then used to decrease the amount of p16 in these cells. The experiments showed that reducing the levels of p16 results in the senescent cells multiplying more and showing fewer signs of damage and aging. In addition, the discs of mice in which the gene that codes for p16 had been deleted were less prone to degeneration compared to ‘normal’ mice in similar conditions. Overall, the work by Che, Li et al. shows that inhibiting p16 in disc cells delays the aging process and reduces the degeneration of intervertebral discs. These findings may one day be applicable to people with intervertebral disc diseases who, for example, could potentially benefit from a gene therapy targeting the cells which produce p16.