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Anticancer therapies can induce cellular senescence or drug-tolerant persistence, two types of proliferative arrest that differ in their stability. While senescence is highly stable, persister cells efficiently resume proliferation upon therapy termination, resulting in tumor relapse. Here, we used an ATP-competitive mTOR inhibitor to induce and characterize persistence in human cancer cells of various origins. Using this model and previously described models of senescence, we compared the same cancer cell lines under the two types of proliferative arrest. Persister and senescent cancer cells shared an expanded lysosomal compartment and hypersensitivity to BCL-XL inhibition. However, persister cells lacked other features of senescence, such as loss of lamin B1, senescence-associated β-galactosidase activity, upregulation of MHC-I, and an inflammatory and secretory phenotype (senescence-associated secretory phenotype or SASP). A genome-wide CRISPR/Cas9 screening for genes required for the survival of persister cells revealed that they are hypersensitive to the inhibition of one-carbon (1C) metabolism, which was validated by the pharmacologic inhibition of serine hydroxymethyltransferase, a key enzyme that feeds methyl groups from serine into 1C metabolism. Investigation into the relationship between 1C metabolism and the epigenetic regulation of transcription uncovered the presence of the repressive heterochromatic mark H4K20me3 at the promoters of SASP and IFN response genes in persister cells, whereas it was absent in senescent cells. Moreover, persister cells overexpressed the H4K20 methyltransferases KMT5B/C, and their downregulation unleashed inflammatory programs and compromised the survival of persister cells. In summary, this study identifies distinctive features and actionable vulnerabilities of persister cancer cells and provides mechanistic insight into their low inflammatory activity. Significance: Cell persistence and senescence are distinct states of proliferative arrest induced by cancer therapy, with persister cells being characterized by the silencing of inflammatory genes through the heterochromatic mark H4K20me3. See related commentary by Schmitt, p. 7., (©2024 The Authors; Published by the American Association for Cancer Research.)

Senescent cells are commonly detected in tumors after chemo and radiotherapy, leading to a characteristic cellular phenotype that resists apoptotic cell death. In this study, we used multiple melanoma cell lines, molecular markers, and therapies to investigate the key role of the BCL-2 family proteins in the survival of senescent cells. We first used BH3 profiling to assess changes in apoptotic priming upon senescence induction. Unexpectedly, not all cell types analyzed showed a decrease in apoptotic priming, BIM was downregulated, there was variability in BAX expression and BAK remained constant or increased. Therefore, there was not a clear pattern for pro-survival adaptation. Many studies have been devoted to find ways to eliminate senescent cells, leading to one of the most studied senolytic agents: navitoclax, a promiscuous BH3 mimetic that inhibits BCL-2, BCL-xL and BCL-W. While it is known that the BCL-2 family of proteins is commonly upregulated in senescent cells, the complexity of the apoptotic network has not been fully explored. Interestingly, we found distinct protein expression changes always leading to a BCL-xL mediated pro-survival adaptation, as assessed by BH3 profiling. When analyzing potential therapeutic strategies, we observed a stronger senolytic activity in these melanoma cell lines when specifically targeting BCL-xL using A-1331852, navitoclax or the PROTAC BCL-xL degrader DT2216. We found that the sensitizer protein HRK was systematically downregulated when senescence was induced, leading to an increased availability of BCL-xL. Furthermore, we identified that the main apoptotic inhibition was shaped by BCL-xL and BAK binding increase that prevented mitochondrial permeabilization and apoptosis. To our knowledge, this is the first time that the molecular basis for BCL-xL anti-apoptotic adaptation in senescence is described, paving the way for the development of new molecules that either prevent HRK downregulation or displace BCL-xL binding to BAK to be used as senolytics., Competing Interests: Competing interests: JM is co-inventor of dynamic BH3 profiling (patented by Dana-Faber Cancer Institute, also BH3 profiling used in this study) and has received royalties. JM is a paid consultant for PEP-THERAPY, was a paid consultant for Oncoheroes Biosciences and Vivid Biosciences, is an unpaid board member for The Society for Functional Precision Medicine, and he is currently collaborating with AstraZeneca. MS is shareholder of Altos Labs, Inc., Senolytic Therapeutics, Inc., Life Biosciences, Inc., and Rejuveron Senescence Therapeutics, AG. No potential conflicts were disclosed by the other authors. Ethics approval and consent to participate: Patient samples were retrospectively included in our study with prior fully informed patient consent and approval from the Local Ethical Committee (HCB/2019/0995 V.4 11/12/2019). The study was conducted in accordance with the principles of the Declaration of Helsinki., (© 2024. The Author(s).)

Senescent cells play a causative role in many diseases, and their elimination is a promising therapeutic strategy. Here, through a genome-wide CRISPR/Cas9 screen, we identify the gene PPIF, encoding the mitochondrial protein cyclophilin D (CypD), as a novel senolytic target. Cyclophilin D promotes the transient opening of the mitochondrial permeability transition pore (mPTP), which serves as a failsafe mechanism for calcium efflux. We show that senescent cells exhibit a high frequency of transient CypD/mPTP opening events, known as 'flickering'. Inhibition of CypD using genetic or pharmacologic tools, including cyclosporin A, leads to the toxic accumulation of mitochondrial Ca 2+ and the death of senescent cells. Genetic or pharmacological inhibition of NCLX, another mitochondrial calcium efflux channel, also leads to senolysis, while inhibition of the main Ca 2+ influx channel, MCU, prevents senolysis induced by CypD inhibition. We conclude that senescent cells are highly vulnerable to elevated mitochondrial Ca 2+ ions, and that transient CypD/mPTP opening is a critical adaptation mechanism for the survival of senescent cells., Competing Interests: Disclosure and competing interests statement. MK has ongoing or completed research contracts with Galapagos NV, Rejuveron Senescence Therapeutics, and mesoestetic®. MS is shareholder of Senolytic Therapeutics, Inc., Life Biosciences, Inc., Rejuveron Senescence Therapeutics, AG, and Altos Labs, Inc. MS has been consultant, until the end of 2022, of Rejuveron Senescence Therapeutics, AG, and Altos Labs, Inc. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., (© 2024. The Author(s).)

Competing Interests: Competing interests: The authors declare no competing interests.

The escape of mitochondrial double-stranded dsRNA (mt-dsRNA) into the cytosol has been recently linked to a number of inflammatory diseases. Here, we report that the release of mt-dsRNA into the cytosol is a general feature of senescent cells and a critical driver of their inflammatory secretome, known as senescence-associated secretory phenotype (SASP). Inhibition of the mitochondrial RNA polymerase, the dsRNA sensors RIGI and MDA5, or the master inflammatory signaling protein MAVS, all result in reduced expression of the SASP, while broadly preserving other hallmarks of senescence. Moreover, senescent cells are hypersensitized to mt-dsRNA-driven inflammation due to their reduced levels of PNPT1 and ADAR1, two proteins critical for mitigating the accumulation of mt-dsRNA and the inflammatory potency of dsRNA, respectively. We find that mitofusin MFN1, but not MFN2, is important for the activation of the mt-dsRNA/MAVS/SASP axis and, accordingly, genetic or pharmacologic MFN1 inhibition attenuates the SASP. Finally, we report that senescent cells within fibrotic and aged tissues present dsRNA foci, and inhibition of mitochondrial RNA polymerase reduces systemic inflammation associated to senescence. In conclusion, we uncover the mt-dsRNA/MAVS/MFN1 axis as a key driver of the SASP and we identify novel therapeutic strategies for senescence-associated diseases., (© 2024. The Author(s).)

Purpose: We conducted research on CDK4/6 inhibitors (CDK4/6i) simultaneously in the preclinical and clinical spaces to gain a deeper understanding of how senescence influences tumor growth in humans., Patients and Methods: We coordinated a first-in-kind phase II clinical trial of the CDK4/6i abemaciclib for patients with progressive dedifferentiated liposarcoma (DDLS) with cellular studies interrogating the molecular basis of geroconversion., Results: Thirty patients with progressing DDLS enrolled and were treated with 200 mg of abemaciclib twice daily. The median progression-free survival was 33 weeks at the time of the data lock, with 23 of 30 progression-free at 12 weeks (76.7%, two-sided 95% CI, 57.7%-90.1%). No new safety signals were identified. Concurrent preclinical work in liposarcoma cell lines identified ANGPTL4 as a necessary late regulator of geroconversion, the pathway from reversible cell-cycle exit to a stably arrested inflammation-provoking senescent cell. Using this insight, we were able to identify patients in which abemaciclib induced tumor cell senescence. Senescence correlated with increased leukocyte infiltration, primarily CD4-positive cells, within a month of therapy. However, those individuals with both senescence and increased TILs were also more likely to acquire resistance later in therapy. These suggest that combining senolytics with abemaciclib in a subset of patients may improve the duration of response., Conclusions: Abemaciclib was well tolerated and showed promising activity in DDLS. The discovery of ANGPTL4 as a late regulator of geroconversion helped to define how CDK4/6i-induced cellular senescence modulates the immune tumor microenvironment and contributes to both positive and negative clinical outcomes. See related commentary by Weiss et al., p. 649., (©2023 The Authors; Published by the American Association for Cancer Research.)

Background: Vitamin B12 is an essential nutrient that is involved in numerous physiological processes, and its deficiency can lead to various complications, including neurological and haematological disorders. Some studies have suggested that vitamin B12 may have anti-inflammatory effects, but the mechanisms underlying this relationship are not yet fully understood. We investigated the relationship between circulating vitamin B12 and inflammatory markers interleukin (IL)-6 and C-reactive protein (CRP). The association of peripheral levels of vitamin B12 with IL-6 and CRP was assessed in 136 human samples from a high cardiovascular risk population. To corroborate the results from the human trial, the analysis was replicated in naturally aged mice., Results: Individuals with higher serum levels of vitamin B12 showed lower concentrations of IL-6 and CRP after adjustment for potential confounders, and an inverse association was also found between serum IL-6 and vitamin B12 levels in naturally aged mice., Conclusion: Circulating vitamin B12 was inversely associated with IL-6 and CRP in humans and with IL-6 in mice, suggesting that it may exert an anti-inflammatory effect through modulation of these pro-inflammatory molecules. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

Transient reprogramming by the expression of OCT4, SOX2, KLF4 and MYC (OSKM) is a therapeutic strategy for tissue regeneration and rejuvenation, but little is known about its metabolic requirements. Here we show that OSKM reprogramming in mice causes a global depletion of vitamin B 12 and molecular hallmarks of methionine starvation. Supplementation with vitamin B 12 increases the efficiency of reprogramming both in mice and in cultured cells, the latter indicating a cell-intrinsic effect. We show that the epigenetic mark H3K36me3, which prevents illegitimate initiation of transcription outside promoters (cryptic transcription), is sensitive to vitamin B 12 levels, providing evidence for a link between B 12 levels, H3K36 methylation, transcriptional fidelity and efficient reprogramming. Vitamin B 12 supplementation also accelerates tissue repair in a model of ulcerative colitis. We conclude that vitamin B 12 , through its key role in one-carbon metabolism and epigenetic dynamics, improves the efficiency of in vivo reprogramming and tissue repair., (© 2023. The Author(s).)

Differentiated cells can be converted into pluripotent stem cells by expressing the transcription factors OCT4, SOX2, KLF4, and MYC (OSKM) in a process known as reprogramming. Here, using single-cell RNA sequencing of pancreas undergoing reprogramming, we identify markers along the trajectory from acinar cell identity to pluripotency. These markers allow direct in situ visualization of cells undergoing dedifferentiation and acquiring features of early and advanced intermediate reprogramming. We also find that a fraction of cells do not dedifferentiate upon OSKM expression and are characterized by stress markers of the REG3 and AP-1 families. Importantly, most markers of intermediate reprogramming in the pancreas are also observed in stomach, colon, and cultured fibroblasts expressing OSKM. Among them is LY6A, a protein characteristic of progenitor cells and generally upregulated during tissue repair. Our roadmap defines intermediate reprogramming states that could be functionally relevant for tissue regeneration and rejuvenation., Competing Interests: Conflict of interests M.S. is shareholder and advisor of Senolytic Therapeutics, Inc., Life Biosciences, Inc, Rejuveron Senescence Therapeutics, AG, and Altos Labs, Inc. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

CDK4/6 inhibitors are among a new generation of therapeutics. Building upon the striking success of the combination of CDK4/6 inhibitors and the hormone receptor antagonist letrozole in breast cancer, many other combinations have recently entered clinical trials in multiple diseases. To achieve maximal benefit with CDK4/6 inhibitors it will be critical to understand the cellular mechanisms by which they act. Here we highlight the mechanisms by which CDK4/6 inhibitors can exert their anti-tumor activities beyond simply enforcing cytostatic growth arrest, and discuss how this knowledge may inform new combinations, improve outcomes, and modify dosing schedules in the future., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Senescence is an irreversible form of growth arrest and is generally considered a favorable outcome of cancer therapies, yet little is known about the molecular events that distinguish this state from readily reversible growth arrest (i.e. quiescence). Recently, we discovered that during therapy induced senescence the chromatin remodeling protein α-thalassemia, mental retardation, X-linked (ATRX) represses Harvey rat sarcoma viral oncogene homolog ( HRAS) , and repression of HRAS is necessary to establish senescence, suggesting how new clinical combinations might be used to achieve durable senescence.

Senescence is a state of stable cell cycle exit with important implications for development and disease. Here, we demonstrate that the chromatin remodeling enzyme ATRX is required for therapy-induced senescence. ATRX accumulates in nuclear foci and is required for therapy-induced senescence in multiple types of transformed cells exposed to either DNA damaging agents or CDK4 inhibitors. Mobilization into foci depends on the ability of ATRX to interact with H3K9me3 histone and HP1. Foci form soon after cells exit the cell cycle, before other hallmarks of senescence appear. Eliminating ATRX in senescent cells destabilizes the senescence-associated heterochromatic foci. Additionally, ATRX binds to and suppresses expression from the HRAS locus; repression of HRAS is sufficient to promote the transition of quiescent cells into senescence and preventing repression blocks progression into senescence. Thus ATRX is a critical regulator of therapy-induced senescence and acts in multiple ways to drive cells into this state.Therapy induced senescence (TIS) is a growth suppressive program activated by cytostatic agents in some cancer cells. Here the authors show that the chromatin remodeling enzyme ATRX is a regulator of TIS and drives cells into this state via multiple mechanisms.

CDK4 inhibitors (CDK4i) earned Breakthrough Therapy Designation from the FDA last year and are entering phase III clinical trials in several cancers. However, not all tumors respond favorably to these drugs. CDK4 activity is critical for progression through G1 phase and into the mitotic cell cycle. Inhibiting this kinase induces Rb-positive cells to exit the cell cycle into either a quiescent or senescent state. In this report, using well-differentiated and dedifferentiated liposarcoma (WD/DDLS) cell lines, we show that the proteolytic turnover of MDM2 is required for CDK4i-induced senescence. Failure to reduce MDM2 does not prevent CDK4i-induced withdrawal from the cell cycle but the cells remain in a reversible quiescent state. Reducing MDM2 in these cells drives them into the more stable senescent state. CDK4i-induced senescence associated with loss of MDM2 is also observed in some breast cancer, lung cancer and glioma cell lines indicating that this is not limited to WD/DDLS cells in which MDM2 is overexpressed or in cells that contain wild type p53. MDM2 turnover depends on its E3 ligase activity and expression of ATRX. Interestingly, in seven patients the changes in MDM2 expression were correlated with outcome. These insights identify MDM2 and ATRX as new regulators controlling geroconversion, the process by which quiescent cells become senescent, and this insight may be exploited to improve the activity of CDK4i in cancer therapy.