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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.

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.

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.

Palbociclib (PD-0332991) is an oral, small-molecule inhibitor of cyclin-dependent kinases (CDKs) 4 and 6 with preclinical evidence of growth-inhibitory activity in oestrogen receptor-positive breast cancer cells and synergy with anti-oestrogens. We aimed to assess the safety and efficacy of palbociclib in combination with letrozole as first-line treatment of patients with advanced, oestrogen receptor-positive, HER2-negative breast cancer. In this open-label, randomised phase 2 study, postmenopausal women with advanced oestrogen receptor-positive and HER2-negative breast cancer who had not received any systemic treatment for their advanced disease were eligible to participate. Patients were enrolled in two separate cohorts that accrued sequentially: in cohort 1, patients were enrolled on the basis of their oestrogen receptor-positive and HER2-negative biomarker status alone, whereas in cohort 2 they were also required to have cancers with amplification of cyclin D1 (CCND1), loss of p16 (INK4A or CDKN2A), or both. In both cohorts, patients were randomly assigned 1:1 via an interactive web-based randomisation system, stratified by disease site and disease-free interval, to receive continuous oral letrozole 2·5 mg daily or continuous oral letrozole 2·5 mg daily plus oral palbociclib 125 mg, given once daily for 3 weeks followed by 1 week off over 28-day cycles. The primary endpoint was investigator-assessed progression-free survival in the intention-to-treat population. Accrual to cohort 2 was stopped after an unplanned interim analysis of cohort 1 and the statistical analysis plan for the primary endpoint was amended to a combined analysis of cohorts 1 and 2 (instead of cohort 2 alone). The study is ongoing but closed to accrual; these are the results of the final analysis of progression-free survival. The study is registered with the ClinicalTrials.gov, number NCT00721409. Between Dec 22, 2009, and May 12, 2012, we randomly assigned 165 patients, 84 to palbociclib plus letrozole and 81 to letrozole alone. At the time of the final analysis for progression-free survival (median follow-up 29·6 months [95% CI 27·9–36·0] for the palbociclib plus letrozole group and 27·9 months [25·5–31·1] for the letrozole group), 41 progression-free survival events had occurred in the palbociclib plus letrozole group and 59 in the letrozole group. Median progression-free survival was 10·2 months (95% CI 5·7–12·6) for the letrozole group and 20·2 months (13·8–27·5) for the palbociclib plus letrozole group (HR 0·488, 95% CI 0·319–0·748; one-sided p=0·0004). In cohort 1 (n=66), median progression-free survival was 5·7 months (2·6–10·5) for the letrozole group and 26·1 months (11·2–not estimable) for the palbociclib plus letrozole group (HR 0·299, 0·156–0·572; one-sided p<0·0001); in cohort 2 (n=99), median progression-free survival was 11·1 months (7·1–16·4) for the letrozole group and 18·1 months (13·1–27·5) for the palbociclib plus letrozole group (HR 0·508, 0·303–0·853; one-sided p=0·0046). Grade 3–4 neutropenia was reported in 45 (54%) of 83 patients in the palbociclib plus letrozole group versus one (1%) of 77 patients in the letrozole group, leucopenia in 16 (19%) versus none, and fatigue in four (4%) versus one (1%). Serious adverse events that occurred in more than one patient in the palbociclib plus letrozole group were pulmonary embolism (three [4%] patients), back pain (two [2%]), and diarrhoea (two [2%]). No cases of febrile neutropenia or neutropenia-related infections were reported during the study. 11 (13%) patients in the palbociclib plus letrozole group and two (2%) in the letrozole group discontinued the study because of adverse events. The addition of palbociclib to letrozole in this phase 2 study significantly improved progression-free survival in women with advanced oestrogen receptor-positive and HER2-negative breast cancer. A phase 3 trial is currently underway. Pfizer.

Human-papillomavirus (HPV) testing is more sensitive, but less specific, than conventional cytology for detecting high-grade cervical intraepithelial neoplasia (CIN). We assessed whether HPV testing with triage by p16-INK4A overexpression can increase specificity while maintaining sensitivity. HPV-positive women were enrolled between June 10, 2003, and Dec 31, 2004 in a multicentre randomised controlled trial, which compared stand-alone HPV testing by Hybrid Capture 2 (experimental group) with conventional cytology, were referred for colposcopy. In seven of nine centres, cytospin preparations from these women were tested for p16-INK4A overexpression by immunostaining. The sensitivity and specificity for CIN grade 2 or more, determined at blind review of histology, were calculated for these women. We also estimated the relative sensitivity and relative referral to colposcopy that would have been obtained by HPV testing with p16-INK4A triage compared with conventional cytology. This trial is registered as a Standard Randomised Controlled Trial, number ISRCTN81678807. 24 661 women were randomly assigned to the experimental group. 1137 women (74% of those undergoing colposcopy in relevant centres), including 50 with CIN2 and 42 with CIN3 or cancer, had valid p16-INK4A immunostaining. For the endpoint of CIN2+, sensitivity and specificity of p16-INK4A (deemed positive with any number of stained cells—except endocervical, metaplastic, and atrophic cells if morphologically normal) in HPV-positive women of any age were 88% (81 of 92; 95% CI 80–94) and 61% (633 of 1045; 57–64), respectively. In the 35–60-year age group, the relative sensitivity of HPV testing and p16-INK4A triage versus conventional cytology for CIN2+ was 1·53 (95% CI 1·15–2·02) and relative referral was 1·08 (0·96–1·21). In the 25–34-year age group, relative sensitivity was 3·01 (1·82–5·17) and relative referral was 1·15 (0·96–1·37). In the latter age group, when 5% or more stained cells were deemed positive, the corresponding values were 2·06 (1·20–3·68) and 0·58 (0·46–0·73), respectively. HPV testing with p16-INK4A triage produces a significant increase in sensitivity compared with conventional cytology, with no substantial increase in referral to colposcopy. European Union, Italian Ministry of Health, Regional Health Administrations of Piemonte, Tuscany, Veneto, and Emilia-Romagna, and Public Health Agency of Lazio Region.

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.

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.

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.

T-helper-1-cell cytokines tumour necrosis factor and interferon-γ are shown to drive tumour cells into senescence in a mouse model of β-cell carcinoma and human carcinoma cells. Adaptive immunity tags cancer cells for senescence Well-defined cell-death mechanisms such as cytolysis and apoptosis are known to be involved in the destruction and clearance of cancer cells, yet how the immune system actually arrests cancer cell proliferation remains unclear. Using mice with Tag-induced cancers, in which T-helper-1 (T H 1) cell immunity doubles the lifespan, Martin Röcken and colleagues demonstrate that adaptive immunity mediated by the T H 1 cytokines interferon-γ and tumour necrosis factor (TNF) directly induces senescence in cancers. Interferon- and TNF-induced senescence protects even against endogenous cancers that develop through transgenic expression of an oncogene, suggesting that it may be of broad relevance for cancer control. Cancer control by adaptive immunity involves a number of defined death 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 and clearance 9 , 10 , 11 mechanisms. However, efficient inhibition of exponential cancer growth by T cells and interferon-γ (IFN-γ) requires additional undefined mechanisms that arrest cancer cell proliferation 1 , 2 , 3 , 4 , 5 , 12 , 13 . Here we show that the combined action of the T-helper-1-cell cytokines IFN-γ and tumour necrosis factor (TNF) directly induces permanent growth arrest in cancers. To safely separate senescence induced by tumour immunity from oncogene-induced senescence 9 , 10 , 11 , 14 , 15 , 16 , 17 , we used a mouse model in which the Simian virus 40 large T antigen (Tag) expressed under the control of the rat insulin promoter creates tumours by attenuating p53- and Rb-mediated cell cycle control 18 , 19 . When combined, IFN-γ and TNF drive Tag-expressing cancers into senescence by inducing permanent growth arrest in G1/G0, activation of p16INK4a (also known as CDKN2A), and downstream Rb hypophosphorylation at serine 795. This cytokine-induced senescence strictly requires STAT1 and TNFR1 (also known as TNFRSF1A) signalling in addition to p16INK4a. In vivo , Tag-specific T-helper 1 cells permanently arrest Tag-expressing cancers by inducing IFN-γ- and TNFR1-dependent senescence. Conversely, Tnfr1 −/− Tag-expressing cancers resist cytokine-induced senescence and grow aggressively, even in TNFR1-expressing hosts. Finally, as IFN-γ and TNF induce senescence in numerous murine and human cancers, this may be a general mechanism for arresting cancer progression.

The INK4/ARF locus encodes three tumor suppressor genes (p15(Ink4b), Arf and p16(Ink4a)) and is frequently inactivated in a large number of human cancers. Mechanisms regulating INK4/ARF expression are not fully characterized. Here we show that in young proliferating embryonic fibroblasts (MEFs) the Polycomb Repressive Complex 2 (PRC2) member EZH2 together with PRC1 members BMI1 and M33 are strongly expressed and localized at the INK4/ARF regulatory domain (RD) identified as a DNA replication origin. When cells enter senescence the binding to RD of both PRC1 and PRC2 complexes is lost leading to a decreased level of histone H3K27 trimethylation (H3K27me3). This loss is accompanied with an increased expression of the histone demethylase Jmjd3 and with the recruitment of the MLL1 protein, and correlates with the expression of the Ink4a/Arf genes. Moreover, we show that the Polycomb protein BMI1 interacts with CDC6, an essential regulator of DNA replication in eukaryotic cells. Finally, we demonstrate that Polycomb proteins and associated epigenetic marks are crucial for the control of the replication timing of the INK4a/ARF locus during senescence. We identified the replication licencing factor CDC6 as a new partner of the Polycomb group member BMI1. Our results suggest that in young cells Polycomb proteins are recruited to the INK4/ARF locus through CDC6 and the resulting silent locus is replicated during late S-phase. Upon senescence, Jmjd3 is overexpressed and the MLL1 protein is recruited to the locus provoking the dissociation of Polycomb from the INK4/ARF locus, its transcriptional activation and its replication during early S-phase. Together, these results provide a unified model that integrates replication, transcription and epigenetics at the INK4/ARF locus.