HRK downregulation and augmented BCL-xL binding to BAK confer apoptotic protection to therapy-induced senescent melanoma cells.

HRK downregulation and augmented BCL-xL binding to BAK confer apoptotic protection to therapy-induced senescent melanoma cells. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article HRK downregulation and augmented BCL-xL binding to BAK confer apoptotic protection to therapy-induced senescent melanoma cells. Joan Montero, Clara Alcon, Marta Kovatcheva, Paula Morales Sanchez, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4581617/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Dec, 2024 Read the published version in Cell Death & Differentiation → Version 1 posted 9 You are reading this latest preprint version Abstract 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 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 dependence in senescence is described, paving the way for molecules that either prevent HRK downregulation or displace BCL-xL binding to BAK to be used as senolytics. Health sciences/Diseases/Cancer Biological sciences/Cell biology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Cellular senescence is a stable form of cell cycle exit that can be induced by a variety of stressors including aberrant oncogene activation, oxidative stress, and chemotherapies. While senescence is thought to have evolved as a tumor-suppressive mechanism 1 , the inappropriate accumulation of senescent cells has pathogenic consequences, including risk of cancer relapse 2 , contributing to the long-term side effects of cancer therapy 3 , and acting as a driver of many age-associated illnesses, including fibrotic, cardiovascular, and neurologic diseases 4 , 5 . A recent field of study between cancer and senescence that is now extensively explored is therapy-induced senescence (TIS). TIS has been found in tumors after radiation or chemotherapy, but only in a subset of cells 6 . Commonly used treatments such as doxorubicin, paclitaxel, cisplatin… conventional chemotherapy and radiotherapy in general, but also targeted therapies and immunotherapies have been reported to induce TIS 7 , 8 . Thus, the selective elimination of senescent cells by a class of molecules known as senolytics has become a therapeutic objective 9 , 10 . While the therapeutic potential of senolytics in a wide swath of pathologies is clear, the identification and successful administration of these drugs has been proved challenging. Navitoclax (ABT-263) was identified as one of the first senolytic agents based on the transcriptional profiling of senescent cells, which revealed a key role of anti-apoptotic BCL-2 family proteins 11 , 12 . Resistance to apoptosis has become a so-called hallmark of senescent cells; paradoxically, the hyper-sensitivity of senescent cells to BH3 mimetic drugs indicates that they are simultaneously “primed” towards apoptosis 13 , 14 . Rationally increasing this apoptotic priming and inducing killing in senescent cells is a goal of senolytic therapeutics. While navitoclax is routinely used as a “gold standard” or proof of concept senolytic agent in preclinical studies, its therapeutic potential is confounded by dose-limiting toxicities largely based in thrombocytopenia. Navitoclax is a BH3 mimetic drug, whose mechanism of action is based on the inhibition of several anti-apoptotic BCL-2 family members, simultaneously targeting BCL-2, BCL-xL and BCL-W. Platelet toxicity has been attributed to BCL-xL inhibition, which was followed by second generation BH3 mimetics with enhanced specificity, such as venetoclax (ABT-199), which specifically targets BCL-2 15 , or DT2216, a proteolysis targeting chimera (PROTAC) that targets BCL-xL without causing thrombocytopenia 16 . Nevertheless, when and how such drugs may have efficacy as senolytics is unclear. The pro- and anti-apoptotic network is complex, and the expression levels of its constituent genes does not necessarily reflect their cellular activity 17 . Moreover, recent work has indicated that different cell types exhibit unique adaptations, such as the MCL-1-dependent resistance to navitoclax in melanocytes 18 . Given the relatively low predictive capacity of individual features of the BCL-2 family members, a new generation of functional biomarkers have been recently established to predict anticancer therapy induction of apoptosis and guide the use of BH3 mimetics. Directly exposing living cancer cells to therapeutic agents ex vivo to determine chemosensitivity has been historically explored, but the development of novel technologies have fostered functional precision medicine. Among them, BH3 profiling has demonstrated that it can rapidly identify anti-apoptotic dependencies in cancer cells 19 , 20 . This functional assay uses synthetic ̴ 20-mer BH3 peptides, mimicking BH3-only proteins, acting as a pro-death signal to induce mitochondrial outer membrane permeabilization (MOMP). By using different peptides, BH3 profiling can rapidly interrogate cancer cells and obtain precise information regarding the apoptotic status of the cell. For example, the BH3 peptides BIM and BID, that bind to all anti-apoptotic proteins and directly activate the effector proteins BAX/BAK and MOMP, measure overall apoptotic priming, or how close cancer cells are to the apoptotic threshold 2 1 , 2 2 . Further development of this technique led to dynamic BH3 profiling, which rapidly measures how much a given treatment primes cancer cells towards apoptosis (Δ% priming) by uniquely measuring early changes in the apoptotic signaling preceding frank cell death on the order of days or weeks 2 3 . In addition, by using peptides recapitulating the BH3 domain sequence of sensitizer proteins—such as BAD, HRK, or NOXA BH3 peptides— BH3 profiling can also identify changes in anti-apoptotic dependencies upon treatment and accurately identify BH3 mimetic combinations to enhance cytotoxicity 20, 2 4 , 2 5 , 2 6 , also to eliminate oncogene-induced senescent cells 2 7 . Nevertheless, to date BH3 profiling has not been fully validated on senescent cells. Given the hypersensitivity of senescent cells to BH3 mimetics, we reasoned that BH3 profiling could be a novel approach to dissect the complexity of anti-apoptotic networks in senescent cells, and with which to nominate novel senolytic therapeutic approaches. RESULTS Palbociclib-induced senescence distinctly affects apoptotic signaling in melanoma cells. We decided to focus in melanoma since senescence and apoptotic protection play a key role in this type of cancer development and therapy outcome 18 , 24 , 28 . We used three melanoma cell lines; SK-MEL-103 (NRAS mutant), SK-MEL-28 (BRAFV600E mutant) 28 , and the M16 (BRAFV600E mutant) that was derived from a patient continuously exposed to UV light as previously described 29 . To induce senescence, we chronically treated these cell lines with a CDK4/6 inhibitor for 7 days 8 , and, since this inhibitor does not uniformly induce senescence in all cancer cell lines 30 , we confirmed the phenotype using different well-established markers: SA-β−galactosidase, p21 and p16 31, 32 . As expected, we detected a significant increase in these markers in all three cell lines when treated with palbociclib, thus indicating therapy-induced senescence (TIS) (Fig. 1 A-B). To assess senescence morphologically, we examined by transmission electron microscopy (TEM) for potential phenotypic changes. Overall, we detected longer mitochondria (Fig. 1 C) and an increase in the number of lysosomes (Fig. 1 D) in palbociclib-treated cells, as previously described 5 , 33 . All these observations confirm that palbociclib effectively induces senescence in all three melanoma cell lines examined. Senescent cells often present resistance to die by apoptosis 2 . Therefore, we next sought to study how TIS modulates apoptosis using BH3 profiling. As mentioned above, senescent cells present longer mitochondria, and we reasoned that this could affect MOMP and cytochrome c release measurements, which are required for BH3 profiling analyses 22 , 23 , 24 . Thus, we first evaluated if these selected senescent melanoma cells presented a larger mitochondrial content compared to non-senescent cells. Using TOM20 as a mitochondrial marker, we observed a significant increase in its expression in palbociclib-induced senescent SK-MEL-28 cells compared to non-treated (proliferating) cells (Fig. 2 A). We also evaluated cytochrome c staining by flow cytometry and, correlating with TEM and fluorescence microscopy, we detected a significant increase in its intensity after senescence induction (Fig. 2 A). Therefore, for flow cytometry BH3 profiling analyses we adjusted the cytochrome c gating taking into consideration this variation in basal fluorescence (see the Methods section for more details). When comparing non-treated (proliferating) and palbociclib-treated melanoma cancer cells by BH3 profiling, we observed that these cell lines showed a different priming pattern when becoming senescent. If a given treatment or state sensitizes cells towards apoptosis, this would cause a leftward shift of the cytochrome c retained curve after BIM peptide exposure, since less amount of BIM is required to promote MOMP and cytochrome c release, indicating an increase in overall apoptotic priming (positive Δ% priming). In contrast, if a treatment or state renders cells more resistant to apoptosis, we would observe a shift of the curve to the right, indicating a decrease in priming (negative Δ% priming). Surprisingly, we observed that SK-MEL-103 cells treated with palbociclib became slightly primed for apoptosis, while the SK-MEL-28 and M16 cells clearly turn out to be less primed for apoptosis (Fig. 2 B). To further elucidate this distinct effect on apoptotic priming induced by palbociclib, we compared the expression of different pro-apoptotic members of the BCL-2 family of proteins between proliferating and senescent cells. For instance, in all cell lines we observed a significant reduction of the activator protein BIM (Fig. 2 C), that could partially explain why senescent cells exert resistance to cell death. When further analyzing the SK-MEL-103 cells, we identified that the expression of the effector proteins BAX and BAK significantly increased after palbociclib treatment, correlating with the observed increase in Δ% priming detected by BH3 profiling with the BIM BH3 peptide (Fig. 2 B). In fact, these results match previous mRNA expression data from the Serrano laboratory 34 (Suppl. Figure 1). However, in SK-MEL-28 and M16 cells we observed that BAK remained constant or increased while BAX expression decreased, again partially explaining the negative Δ% priming detected by BH3 profiling (Fig. 2 C). In summary, palbociclib promoted a decrease in the activator protein BIM, thus preventing apoptosis, and effectively induced senescence, assessed by SA-β-galactosidase, p21 and p16. Paradoxically one melanoma cell line became slightly primed for apoptosis (positive Δ% priming) while the other two showed protection towards this form of programmed cell death (negative Δ% priming) in part due to a distinct change in BAX expression after TIS. Therapy-induced senescence induces a BCL-xL dependent anti-apoptotic adaptation and can be targeted with specific BH3 mimetics or PROTAC. Another hallmark of senescent cells is their pro-survival adaptation through anti-apoptotic BCL-2 family proteins, and, non-surprisingly, one of the most studied senolytics has been the promiscuous BCL-2/BCL-xL/BCL-W inhibitor navitoclax 11 . We sought to elucidate if TIS melanoma cells also presented a similar adaptation and, if that was the case, how they prevented apoptosis. Thus, we performed BH3 profiling using specific BH3 peptides BAD (for BCL-2 and BCL-xL), HRK (for BCL-xL) and MS1 (for MCL-1), as previously described 24 , 25 , on the same cell lines after senescence induction using palbociclib, and γ-irradiation (which also increased SA-β-galactosidase activity in all three cell lines as shown in supplementary Fig. 2). In brief, a positive Δ% priming using these sensitizer BH3 peptides would point to a specific anti-apoptotic protein that senescent cells become addicted to. We found that SK-MEL-103, SK-MEL-28 and, to a lesser extent, M16 displayed a significant BCL-xL adaptation, as we detected an increase in the % of cytochrome c released with the HRK (and BAD, that binds to BCL-2 and BCL-xL) peptide (Fig. 3 A). We also detected a minor increase in priming with the MS1 BH3 peptide, indicating a partial adaptation through MCL-1 but to a lesser extent than BCL-xL (Fig. 3 A). We next explored anti-apoptotic inhibition potential to eliminate senescent cells. Using palbociclib and γ-irradiation, we generated senescent melanoma cells and tested BH3 mimetics’ senolytic activity. We exposed these cells to ABT-199 (venetoclax), the BCL-xL inhibitor A-1331852, the MCL-1 inhibitor S63845, ABT-263 (navitoclax) and the BCL-xL degrading PROTAC DT2216 16 . As anticipated by our BH3 profiling results, we observed significant senolytic activity in these melanoma cell lines when targeting BCL-xL using A-1331852, navitoclax or DT2216 (Fig. 3 B); and in SK-MEL-103 and SK-MEL-28 BCL-xL targeting therapies had a greater senolytic activity compared to M16. Furthermore, we detected a significant increase in cell death with the MCL-1 inhibitor S63845 in SK-MEL-28 suggesting an implication of this protein in apoptotic resistance in senescent cells harboring a BRAFV600 mutation 24 . These results indicate that TIS melanoma cells block apoptosis primarily through BCL-xL, and to a lesser extent using MCL-1. Consequently, inhibiting this anti-apoptotic protein with specific inhibitors - beyond navitoclax that also targets BCL-2 and BCL-W - such as the BH3 mimetic A-1331852 or a PROTACs like DT2216 that would not cause thrombocytopenia, due to the minor expression of E3 ligase in platelets 16 , appears as an effective therapeutic strategy to remove melanoma senescent cells. BCL-xL adaptation is mediated by HRK downregulation and changes in binding affinities to pro-apoptotic proteins. To elucidate how TIS melanoma cells become dependent on BCL-xL, we first analyzed the expression of this anti-apoptotic protein. We found in palbociclib-treated SK-MEL-103 and M16 cells that its expression increased. Surprisingly, SK-MEL-28 cells did not exert significant BCL-xL changes upon treatment, suggesting a potential role of another BCL-2 family member to regulate its availability (Fig. 4 A-C). In this regard, we analyzed several BCL-2 family proteins, and found that the sensitizer HRK, which specifically binds to BCL-xL 35 , was clearly downregulated in all three cell lines when induced into senescence. In other words, senescent inducers such as palbociclib and γ-irradiation decreased the expression of HRK, initially bound to BCL-xL, thus liberating this pro-survival protein to protect cells towards apoptosis. Similar results were observed in healthy donors comparing control skin with UV irradiated (Table 1), where the latter showed an increase trend in the senescence markers CDKN1A (p21) and CDKN2A (p16) (Supplementary Fig. 3), and a decrease trend in HRK mRNA expression (Fig. 4 D) pointing to a potential BCL-xL adaptation in senescent skin from patients. We previously showed that palbociclib treatment promoted a decrease in BIM, HRK and, in some cases, BAX expression, consequently preventing apoptosis. So, our next question was how BCL-xL inhibitors restored apoptotic cell death in senescent cells. The BCL-2 family of proteins represents a complex interactome regulated at multiple levels, including protein expression, posttranslational modifications, and dynamic binding affinities. To further elucidate the role of BCL-xL in TIS, we immunoprecipitated this anti-apoptotic protein with a high efficiency (Fig. 5 A) and analyzed its binding to BIM, BAX and BAK in SK-MEL-103 and SK-MEL-28. As shown in Fig. 2 , both cell lines exert distinct BAX/BAK expression changes when senescence is induced with palbociclib. However, in our immunoprecipitation analyses we observed a clear increase in binding between BCL-xL and BAK in senescent cells compared to control cells, while it decreased or remained similar for the other two proteins (Fig. 5 B). In summary, we found for the first time that palbociclib-induced senescence promotes different changes in the BCL-2 family proteins’ expression that lead to an increase in BCL-xL:BAK binding that prevents apoptosis and explains why specifically BCL-xL targeted agents are effective senolytics. DISCUSSION One of the hallmarks of senescent cells is their resistance to die by apoptosis 2 . Consequently, our first aim was to further study if senescent melanoma cells showed this resistance and specifically how apoptotic cell death was affected. We used three different melanoma cell lines: SK-MEL-103 that presents a mutation in NRAS, and SK-MEL-28 and M16 that have a BRAFV600E mutation 28 , 29 . We exposed all three cell types to different well-established senescence inducing factors, palbociclib and γ-irradiation, and positively detected an increase in p16, p21 and SA-β-galactosidase in all cases (Fig. 1 A-B). We confirmed senescence induction by electron microscopy where, beyond the increase in cellular size, we identified two commonly observed morphological changes: mitochondria elongation (Fig. 1 C, 2 A) and an increment in the number of lysosomes (Fig. 1 D) 33 , 34 . Since mitochondrial permeabilization is a key step in apoptosis induction and could be altered in senescent cells, we next evaluated potential changes in mitochondrial polarization and cytochrome c expression, and we clearly observed by confocal microscopy and flow cytometry an increased intensity on the latter with senescence (Fig. 2 A). Consequently, we had to adjust our flow cytometry settings when performing the BH3 profiling analyses on these cells 22 , 24 , 25 . Interestingly, we observed a different BH3 profiling trend when comparing the NRAS mutant cell line SK-MEL-103 with the BRAF mutant SK-MEL-28 and M16 cells. When measuring overall priming with the BIM BH3 peptide 14 , we observed that senescent SK-MEL-103 cells became slightly primed to apoptosis while SK-MEL-28 and M16 were less primed after palbociclib treatment. When we analyzed the expression of different pro-apoptotic BCL-2 family proteins, we detected that in all cases senescence induction caused a decrease in BIM (that would have a protective effect towards apoptosis), that BAK remained unchanged or increased, and that BAX decrease in the BRAF-mutant cells but increased in SK-MEL-103 (Fig. 2 C). Precisely the distinct changes in BAX expression between these melanoma cell lines, could partially explain the differences observed in BH3 profiling overall apoptotic priming with senescence. In summary, the studied melanoma cell lines presented variability in BAX protein expression and overall apoptotic priming when becoming senescent but in all cases, BIM was downregulated and importantly BAK expression remained constant or increased. The cellular process of senescence has been related to ageing 5 and cancer cells’ survival to therapy 3 , 36 . However, how therapy-induced senescent cells avoid apoptosis is not fully understood. Several studies previously described that senescent cells utilize anti-apoptotic BCL-2 family proteins to resist cell death and, because of that, they are particularly sensitive to BH3 mimetics such as navitoclax, that blocks BCL-2, BCL-xL and BCL-W 7 , 11 . Thus, we next sought to further understand how this anti-apoptotic adaptation occurs using BH3 profiling 14 , 20 , 24 , 25 , 26 . We identified that when exposed to palbociclib or γ-irradiation, melanoma cells clearly became BCL-xL dependent (Fig. 3 A). In other words, TIS in these cells promoted a pro-survival adaptation mostly through BCL-xL to prevent cell death. Other anti-apoptotic proteins such as MCL-1 were identified by BH3 profiling but to a lesser extent, indicating a secondary role in TIS pro-survival adaptation. We next used different anti-apoptotic inhibitors or degraders as senolytics to test if we could specifically eliminate these senescent melanoma cells. As anticipated by BH3 profiling, those molecules that specifically inhibited BCL-xL (A-1331852 and navitoclax) or the PROTAC DT2216 showed a significant cytotoxic effect on senescent cells in all cases (Fig. 3 B). Other BH3 mimetics directed towards BCL-2 or specially MCL-1 had a minor effect eliminating these cells. These results suggest that despite the observed variability in overall apoptotic priming and BCL-2 family protein expression (Figs. 1 and 2 ), TIS in melanoma leads to a marked BCL-xL dependence in all cases, and its inhibition or degradation efficiently eliminates senescent cells. After identifying the key role of BCL-xL in melanoma cells survival upon senescence induction, we next sought to study how precisely this anti-apoptotic protein impedes cell death. We first analyzed its expression when TIS was induced and observed that it increased in SK-MEL-103 and M16, or remained unchanged in SK-MEL-28 (Fig. 4 A). We were particularly intrigued by the latter since in these cells we also observed a BCL-xL adaptation. We then analyzed other BCL-2 family members, particularly the sensitizer proteins, and found that HRK, that specifically binds and blocks BCL-xL 35 , protein expression decreased with TIS in all cases. Furthermore, we observed in photodamaged skin from donors that senescence markers tend to increase while HRK expression tend to decrease (Fig. 4 D), correlating with our in vitro observations. In other words, senescence induction, both in cell lines and in UV-damaged skin from donors, promotes a downregulation of HRK that frees BCL-xL to block MOMP and apoptotic cell death (Fig. 6 ). But at that point a key question was still unanswered: which specific pro-apoptotic proteins are inhibited by BCL-xL and how is mitochondrial permeabilization prevented? To answer this question, we immunoprecipitated BCL-xL and analyzed by Western Blot which proteins were bound to it. Importantly, we found in SK-MEL-103 and SK-MEL-28 cells that TIS promoted an increase in BAK binding to BCL-xL that blocked MOMP and apoptosis (Figs. 5 and 6 ). In summary, TIS is emerging as a key determinant in cancer cells survival after anticancer treatment and could explain why some melanoma patients’ relapse. Senescence has been extensively studied, in part because of its implications in age-related diseases, and navitoclax has been for years one of the most studied senolytics through its capacity to simultaneously inhibit three important anti-apoptotic proteins: BCL-2, BCL-xL and BCL-W 5 , 11 , 37 . Nevertheless, there is emerging evidence that cell-type specific features contribute to the efficacy and specificity of senolytic treatments, and that navitoclax is not effective in all cases 3 , 18 , 38 . Thus, a mechanistic and functional understanding of the anti-apoptotic network in senescent cells will be necessary to effectively include senolytic agents in treatment regimens. In this study, we use a panel of melanoma cell lines to demonstrate the value of BH3 profiling in uncovering specific senolytic vulnerabilities. We here report that TIS in melanoma promotes a BCL-xL mediated pro-survival adaptation– distinct from what has been reported in untransformed melanocytes 29 – that could be prevented by using specific inhibitors (also navitoclax) or a PROTAC degrader against BCL-xL. We also show that targeting other anti-apoptotic proteins such as BCL-2, BCL-W or MCL-1 have a minor senolytic effect, and its concomitant inhibition would only lead to undesired toxicities. We further investigated how this adaptation occurred and found that TIS promoted a consistent decrease of the sensitizer protein HRK that liberated BCL-xL, and that the latter specifically blocked the effector BCL-2 family protein BAK to prevent MOMP and apoptosis. To our knowledge this is the first time that this senescence pro-survival mechanism is described and could help develop new effective senolytics directed to avoid HRK downregulation or disrupt the BCL-xL:BAK binding. We believe that this new senolytic strategy guided by BH3 profiling could prevent cancer therapy endurance and potentially avoid some melanoma relapse cases in the future. MATERIALS AND METHODS Cell lines and treatments Melanoma cell line SK-MEL-28 was purchased at ATCC (ATCC® CRL-1772™, ATCC, Manassas, VI, USA) and SK-MEL-103 cell line was provided by Dr. Marisol Soengas. Patient-derived cell line M16 was kindly provided by Dr. Susana Puig from Hospital Clínic de Barcelona. SK-MEL-28 cell line was cultured in RPMI 1640 medium (31870, Thermo Fisher, Gibco, Paisley, Scotland) supplemented with 10% heat-inactivated fetal bovine serum (10270, Thermo Fisher, Gibco), 1% of l-glutamine (25030, Thermo Fisher, Gibco), and 1% of penicillin and streptomycin (15140, Thermo Fisher, Gibco). SK-MEL-103 and M16 cells were cultured in DMEM high glucose medium (41965, Thermo Fisher, Gibco) supplemented with 10% heat-inactivated fetal bovine serum, 1% of penicillin and streptomycin and 1% of l-glutamine. All cells were tested for mycoplasma and maintained at 37°C in a humidified atmosphere of 5% CO 2 . BH3 mimetics ABT-199 (venetoclax), A-1331852 (A-133), S63845 and ABT-263 (navitoclax) were purchased at Selleckchem (Munich, Germany) and the PROTAC BCL-xL degrader DT2216 was purchased at AbMole BioScience (Houston, Texas, USA). Treatments were performed directly in the culture media at the indicated concentrations. Palbociclib-induced senescence For palbociclib-induced senescence, cells were seeded in a 6 well-plate and treated with palbociclib (S1116, Selleckchem, Munich, Germany) at 1µM dose. After 3 days cells were replenished with fresh media and palbociclib. Seven days after the treatment, senescent cells were used for further analysis. Irradiation-induced senescence For irradiated induced senescence, cells were seeded in a 6 well-plate and irradiated with a gamma irradiator MARK I 30 (J.L. Shepherd & Associates, San Fernando, CA, USA) at 10Gy. After 3 days, the media was changed and cells were maintained in culture till day 7 after irradiation and used for further analysis. Senescence associated β-galactosidase staining Cells were stained using CellEvent™ Senescence Green Detection Kit (C10851, Thermo Fisher). Briefly, cells were washed with PBS, tripsinized, fixed with 4% formaldehyde during 10 minutes at room temperature, washed with PBS + 1% BSA and incubated with the working solution at 37ºC without CO 2 during 2 hours. After incubation, cells were washed with PBS and then imaged using a spectral flow cytometry Cytek AURORA instrument (Cytek Biosciences, California, USA) from the Scientific and Technological Centers of the University of Barcelona. BH3 profiling BH3 profiling was developed at the Letai laboratory, and the experiments were performed as previously described 20 , 22 , 25 . After inducing senescence for 7 days cells were stained with the viability marker Zombie Violet (423113, BioLegend, Koblenz, Germany) and then washed with PBS and resuspended in MEB (150 mM mannitol, 10 mM hepes–KOH pH 7.5, 150 mM KCl, 1 mM EGTA, 1 mM EDTA, 0.1% BSA, 5 mM succinate) in a final volume of 25 µL. Afterwards, cells were exposed to the following peptides: 25 µL of BIM BH3 peptide (final concentration of 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 µM), 25 µL of BAD BH3 peptide (final concentration of 10 µM), 25 µL of HRK BH3 peptide (final concentration of 100 µM), and 25 µL of MS1 BH3 peptide (final concentration of 10 µM) in MEB with 0.002% digitonin in a 96-well plate (3795, Corning, Madrid, Spain) for 1h following fixation with formaldehyde. Cells were finally stained with cytochrome c antibody (Alexa Fluor® 647 anti-Cytochrome c—6H2.B4, 612310, BioLegend), p21 antibody (sc-6246 PE, Santa Cruz Biotechnology, Texas, USA) and p16 antibody (Alexa Fluor® 488 CDKN2A/p16INK4a, bs-4592R-A488, Bioss Antibodies, Woburn, Massachusetts, USA). Individual BH3 profiling analyses were performed using triplicates for DMSO, alamethecin (BML-A150-0005, Enzo Life Sciences, Lorrach, Germany), the different BIM BH3 concentrations used, BAD, HRK, and MS1 BH3 peptides. The expressed values stand for the average of three different readings performed with a high-throughput spectral flow cytometry Cytek AURORA instrument (Cytek Biosciences, California, USA) from the Scientific and Technological Centers of the University of Barcelona. % of cytochrome c released is used to calculate Δ% priming which represents the difference between treated cells minus non-treated cells for a given peptide. Flow cytometry analysis Due to the difference of mitochondrial content between proliferating and senescence cells and therefore a major cytochrome c retained signalling was detected in senescent cells versus control cells, we adjusted the gating for cytochrome c retention with the control condition of each experimental setting (proliferating and senescent). Analyses were performed using FlowJo software. Cell death assay Cells were trypsinized and stained with fluorescent conjugates of Annexin V (Alexa Fluor® 647 Annexin V, 640912, BioLegend) and DAPI (62248, Thermo Fisher) and analyzed on a flow cytometry Gallios instrument (Beckman Coulter, Nyon, Switzerland) from the Scientific and Technological Centers of the University of Barcelona. Viable cells are Annexin V DAPI negative, and cell death is expressed as 100%-viable cells. Protein extraction and quantification Cells were lysed using RIPA buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris–HCl pH = 8, 1% Triton X-100, 0.1% SDS, EDTA-free Protease Inhibitor Cocktail (4693159001 Roche, Mannkin, Germany)) for 30 min at 4°C followed by a centrifugation at 16,100 × g for 10 min. The supernatant was stored at − 20°C for protein quantification performed using PierceTM BCA Protein Assay Kit (23227, Thermo Fisher). Immunoprecipitation Cells were lysed using Immunoprecipitation buffer (150 mM NaCl, 10 mM Hepes, 2 mM EDTA, 1% Triton, 1.5 mM MgCl2, 10% glycerol, and EDTA-free Protease Inhibitor Cocktail (4693159001 Roche)) and centrifuged at 16,100 × g , 15 min at 4°C. Supernatants were incubated with magnetic beads (Dynabeads 10003D, Thermo Fisher) conjugated to 5 µg of rabbit anti-BCL-xL antibody (CST2764, Cell Signaling, Leiden, The Netherlands) or 5 µg of rabbit IgG antibody (CST2729, Cell Signaling) at 4°C overnight. After magnetization, a part of the supernatant was mixed with half volume of 4× SDS–PAGE sample buffer, heated at 96°C for 5 min and stored at − 80°C as unbound fractions. The rest of the supernatant was discarded. The resulting pellet was washed and mixed with 60 µL 4× SDS–PAGE sample buffer and heated for 10 min at 70°C following by magnetization and collection of the supernatant for further immunoblotting analyses. Immunoblotting Proteins were separated by SDS–PAGE (Mini-Protean TGX Precast Gel 12%, 456–1045, Bio-Rad) and transferred to PVDF membranes (10600023, Amersham Hybond, Pittsburgh, PA, USA). Membranes were blocked with dry milk dissolved in Tris buffer saline with 1% Tween 20 (TBST) for 1 h and probed overnight at 4°C with the primary antibodies of interest directed against: rabbit anti-BCL-xL (CST2764, Cell Signaling), rabbit anti-BIM (CST2933, Cell Signaling), rabbit anti-BAK (CST12105, Cell Signaling), rabbit anti-BAX (CST2772, Cell Signaling), rabbit anti-HRK (PRS3771, Sigma-Aldrich, San Luis, Missouri, USA), mouse anti-γ−tubulin (T6557, Sigma-Aldrich) followed by anti-rabbit or anti-mouse IgG HRP-linked secondary antibody (CST7074 or CST7076 Cell Signaling) in 3% BSA in TBST for 1 h at room temperature. Immunoblots were developed using Clarity ECL Western substrate (1705060, Bio-Rad), visualized with LAS4000 imager (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and ImageJ was then used to measure the integrated optical density of bands. Transmission electron microscopy procedure for flat embedding preparation of cell monolayers For transmission electron microscopy (TEM) preparation and analysis of melanoma cells (proliferating and senescence) growing on coverslips, the cells were first washed with 0.1 M phosphate buffer (PB) to remove the excess of culture medium and fixed with a freshly made 3% glutaraldehyde solution in 0.1M PB for 1 hour at room temperature. Subsequently, the fixative was removed, and the samples were maintained at 4º C in a fresh fixative solution until processing. The post-fixation procedure was conducted using 1% OsO4 for a period of 90 minutes at 4°C. Then, samples were dehydrated in increasing ethanol solutions (in accordance with the standard procedures). Coverslips with cells were then inverted (with the cells facing downwards) and placed on top of BEEM® capsules filled with Spurr resin (Electron Microscopy Sciences, Hatfield, PA, USA). The polymerization of the resin was carried out at a temperature of 60 ºC in a stove for a period of three days. At this point, the glass is removed by thermal contrast switch while the cell monolayer remains at the top of the polymerized block. A Leica ultramicrotome EM UC7 (Leica Microsystems, Wetzlar, Germany) was employed for sectioning. Ultra-thin sections (60–70 nm) were mounted on cooper grids and stained with 2% Uranyl-less solution for 10 minutes and with a lead-staining solution for 5 minutes. The sections were observed using a JEOL JEM-1010 transmission electron microscope (JEOL Ltd., Akishima, Tokyo, Japan) coupled with an Orius SC1000 CCD camera (model 832) (Gatan Inc., Pleasanton, CA, USA) at the Unit of Electron Microscopy, Scientific and Technological Centers of the University of Barcelona, School of Medicine and Health Sciences (Barcelona, Spain). Immunofluorescence Cells were gently washed once with cold PBS and then fixed for 20 minutes with 4% paraformaldehyde in PBS. Cells were then washed three times with PBS and permeabilized for 10 minutes with 0.1% saponin in PBS. Blocking was performed for 5 minutes with 0.02% saponin and 1% BSA in PBS. For primary antibody incubation, cells were incubated with anti-Tom20 (ab186734, Abcam, Cambridge, UK) diluted in 0.02% saponin and 0.1% BSA solution for 1 hour at room temperature. After primary antibody incubation, cells were washed three times with PBS. Secondary antibody incubation was carried out by incubating cells with anti-Mouse IgG (H + L), Superclonal™ Recombinant Secondary Antibody Alexa Fluor™ 488 (A28175, Thermo Fisher) and anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 (A21428, Thermo Fisher) for 45 minutes in 0.02% saponin and 0.1% BSA solution at RT. For DAPI staining, cells were incubated with diluted DAPI stock solution (10374168, Thermo Fisher) to a final concentration of 300 nM in PBS for 5 minutes. Following staining, cells were washed three times with PBS, rinsed in water and mounted in Mowiol ® 4–88 (17951-500, Polysciences, Warrington, PA, USA). Bioinformatic analysis of SK-MEL-103 data In the present study, GEO2R ( www.ncbi.nlm.nih.gov/geo/geo2r/ ) was utilized to download the raw counts of the GSE246690 series to identify genes differentially expressed across experimental conditions of the SK-MEL-103 cell line (control and Palbociclib) 34 . The data was analyzed using the edgeR (v.4.0.16) package from Bioconductor in the R environment (v.4.3.3) 39 . The sva (v. 3.50.0) package was used for removing batch effects and other unwanted variation 40 . Subsequently, differential expression analysis was conducted using the quasi-likelihood negative binomial generalized log-linear model (GLM) functions provided by the edgeR package. For visualization purposes Log2CPM expression values were converted to Z-scores and statistical significance was represented as Fold Discovery Rate (FDR) < 0.05. Patients and samples for molecular validation Fourteen healthy subjects, comprising 10 female and 4 male, mean age 52.6 years (range 41–66) were recruited in Hospital Clínic de Barcelona (Table 1). Two distinct areas on the forearm were identified for biopsy: one area of photodamaged skin (L01) and one area of less sun-damaged (naturally protected) skin on the inner forearm (L02). Subsequently, two 3-mm punch biopsies were taken on L01 and L02 areas. Each biopsy was included in RNAlater (RNAprotect Tissue Reagent, Qiagen) and frozen to extract RNA and perform sequencing. RNA extraction and RNA-sequencing analysis Biopsies in RNAlater were frozen to extract RNA for sequencing. Total RNA was isolated from fresh tissue with Trizol, and purified with RNeasy Minikit (Trizol-chloroform + RNeasy MinElute Cleanup Kit, Qiagen). RNA quantification was performed using Qubit RNA BR Assay Kit. Integrity was measured via Bioanalyzer RNA 6000 NanoKit (Agilent Technologies, Santa Clara, CA). RNA samples were sent to “Centro Nacional de Análisis Genómico” to conduct the stranded mRNA library preparation and sequencing on HiSeq 4000 (Illumina). RNA-Seq libraries were prepared from total RNA using TruSeq Stranded mRNA LibraryPrep Kit (Illumina). mRNA was enriched with oligo-dT magnetic beads from the total RNA (500ng) and fragmented to 80-450nt. The second strand cDNA synthesis was performed in the presence of dUTP to achieve strand specificity. The blunt-ended double-stranded cDNA was 3´adenylated and Illumina platform-compatible adaptors with unique dual indexes and unique molecular identifiers (Integrated DNA Technologies) were ligated. The ligation product was enriched with 15 PCR cycles. The final library was validated on a Bioanalyzer DNA 7500 assay. The libraries were sequenced on NovaSeq6000 (Illumina) in paired-end mode with a read length of 2x101bp, following the manufacturer’s protocol for dual indexing. Image analysis, base calling and quality scoring of the run were processed using the manufacturer’s software Real-Time Analysis (RTA 3.4.4) and followed by the generation of FASTQ sequence files. Statistical analysis Data was analyzed using GraphPad Prism v.9.3.0 software and was represented as mean ± SEM of independent biological replicates. Statistical significance of the results was analyzed using Student’s t -tail test. * p < 0.05 and ** p < 0.01 were considered significant. Specifically, for RNA-Seq data analysis of patient samples, the R package LIMMA was used for data normalization, specifically employing the Variance Modeling at the Observational Leve (VOOM). After normalization of expression data, the genes of interest for validation (CDKN2A, CDKN1A, and HRK) were selected. Global results of the RNA-sequencing analysis in under revision for their publication. Comparisons between the expression between L01 and L02 samples from the same patient were conducted using a paired Student's t-test. Assumptions of normality and homogeneity of variances were checked and appropriately addressed. All analyses were performed using R version 3.3.0. Declarations Acknowledgments We would also like to thank the Cytometry Facility and the Irradiation Service from the Scientific and Technological Centers of the University of Barcelona (CCITUB) for their assistance with experiments. Conflict of interest J. M. is co-inventor of dynamic BH3 profiling (patented by Dana-Faber Cancer Institute) and has received royalties. J.M. 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. No potential conflicts of interest were disclosed by the other authors. M.S. is shareholder of Altos Labs, Inc., Senolytic Therapeutics, Inc., Life Biosciences, Inc., and Rejuveron Senescence Therapeutics, AG. Author contributions C. Alcon performed and analyzed all the experiments. C. Alcon and J. Montero supported the conceptualization of this work. P. Morales-Sánchez and T. Torres performed the bioinformatic analyses. C. Enrich performed the transmission electron microscopy analyses. C. Alcon, M. Kovatcheva and J. Montero wrote the manuscript. J. Montero supervised the work. Ethics statement 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. Funding statement We would like to acknowledge Ministerio de Ciencia, Innovación y Universidades grant RTI2018-094533-A-I00; Ministerio de Ciencia e Innovación, Proy. de Generación del Conocimiento 2021 grant PID2021-128644OB-I0; CELLEX foundation; José Carreras Leukämie-Stiftung grant DJCLS 19R/2022; and Agència de Gestió d'Ajuts Universitaris i de Recerca-Generalitat de Catalunya 2021-SGR-00284. JS acknowledges “Plan Complementario de Biotecnología aplicada a la Salud”, coordinado por el Institut de Bioenginyeria de Catalunya (IBEC) en el marco del Plan de Recuperación, Transformación y Resiliencia (C17.I1) ‐ Financiado por la Unión Europea – NextGenerationEU”. Also, Department of Research and Universities of the Generalitat de Catalunya (2021 SGR 01545). CERCA Programme / Generalitat de Catalunya and Networking Biomedical Research Center (CIBER) of Spain. CIBER is an initiative funded by the VI National R&D&i Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and the Instituto de Salud Carlos III (RD16/0006/0012), with the support of the European Regional Development Fund (ERDF). References Campisi J. Cellular senescence as a tumor-suppressor mechanism. Trends Cell Biol 2001, 11(11): S27-31. Schmitt CA, Wang B, Demaria M. Senescence and cancer - role and therapeutic opportunities. Nat Rev Clin Oncol 2022, 19(10): 619–636. Demaria M, O'Leary MN, Chang J, Shao L, Liu S, Alimirah F, et al. Cellular Senescence Promotes Adverse Effects of Chemotherapy and Cancer Relapse. Cancer Discov 2017, 7(2): 165–176. Zhang L, Pitcher LE, Yousefzadeh MJ, Niedernhofer LJ, Robbins PD, Zhu Y. Cellular senescence: a key therapeutic target in aging and diseases. J Clin Invest 2022, 132(15). Munoz-Espin D, Serrano M. Cellular senescence: from physiology to pathology. Nat Rev Mol Cell Biol 2014, 15(7): 482–496. Ewald JA, Desotelle JA, Wilding G, Jarrard DF. Therapy-induced senescence in cancer. J Natl Cancer Inst 2010, 102(20): 1536–1546. Maggiorani D, Le O, Lisi V, Landais S, Moquin-Beaudry G, Lavallee VP, et al. Senescence drives immunotherapy resistance by inducing an immunosuppressive tumor microenvironment. Nat Commun 2024, 15(1): 2435. Watt AC, Cejas P, DeCristo MJ, Metzger-Filho O, Lam EYN, Qiu X, et al. CDK4/6 inhibition reprograms the breast cancer enhancer landscape by stimulating AP-1 transcriptional activity. Nat Cancer 2021, 2(1): 34–48. Chaib S, Tchkonia T, Kirkland JL. Cellular senescence and senolytics: the path to the clinic. Nat Med 2022, 28(8): 1556–1568. Gasek NS, Kuchel GA, Kirkland JL, Xu M. Strategies for Targeting Senescent Cells in Human Disease. Nat Aging 2021, 1(10): 870–879. Zhu Y, Tchkonia T, Fuhrmann-Stroissnigg H, Dai HM, Ling YY, Stout MB, et al. Identification of a novel senolytic agent, navitoclax, targeting the Bcl-2 family of anti-apoptotic factors. Aging Cell 2016, 15(3): 428–435. Zhu Y, Tchkonia T, Pirtskhalava T, Gower AC, Ding H, Giorgadze N, et al. The Achilles' heel of senescent cells: from transcriptome to senolytic drugs. Aging Cell 2015, 14(4): 644–658. Childs BG, Baker DJ, Kirkland JL, Campisi J, van Deursen JM. Senescence and apoptosis: dueling or complementary cell fates? EMBO Rep 2014, 15(11): 1139–1153. Montero J, Haq R. Adapted to Survive: Targeting Cancer Cells with BH3 Mimetics. Cancer Discov 2022, 12(5): 1217–1232. Debrincat MA, Pleines I, Lebois M, Lane RM, Holmes ML, Corbin J, et al. BCL-2 is dispensable for thrombopoiesis and platelet survival. Cell Death Dis 2015, 6(4): e1721. Khan S, Zhang X, Lv D, Zhang Q, He Y, Zhang P, et al. A selective BCL-X(L) PROTAC degrader achieves safe and potent antitumor activity. Nat Med 2019, 25(12): 1938–1947. Singh R, Letai A, Sarosiek K. Regulation of apoptosis in health and disease: the balancing act of BCL-2 family proteins. Nat Rev Mol Cell Biol 2019, 20(3): 175–193. Kohli J, Ge C, Fitsiou E, Doepner M, Brandenburg SM, Faller WJ, et al. Targeting anti-apoptotic pathways eliminates senescent melanocytes and leads to nevi regression. Nat Commun 2022, 13(1): 7923. Certo M, Del Gaizo Moore V, Nishino M, Wei G, Korsmeyer S, Armstrong SA, et al. Mitochondria primed by death signals determine cellular addiction to antiapoptotic BCL-2 family members. Cancer Cell 2006, 9(5): 351–365. Ryan J, Letai A. BH3 profiling in whole cells by fluorimeter or FACS. Methods 2013, 61(2): 156–164. Ni Chonghaile T, Sarosiek KA, Vo TT, Ryan JA, Tammareddi A, Moore Vdel G, et al. Pretreatment mitochondrial priming correlates with clinical response to cytotoxic chemotherapy. Science 2011, 334(6059): 1129–1133. Ryan J, Montero J, Rocco J, Letai A. iBH3: simple, fixable BH3 profiling to determine apoptotic priming in primary tissue by flow cytometry. Biol Chem 2016, 397(7): 671–678. Montero J, Sarosiek KA, DeAngelo JD, Maertens O, Ryan J, Ercan D, et al. Drug-induced death signaling strategy rapidly predicts cancer response to chemotherapy. Cell 2015, 160(5): 977–989. Montero J, Gstalder C, Kim DJ, Sadowicz D, Miles W, Manos M, et al. Destabilization of NOXA mRNA as a common resistance mechanism to targeted therapies. Nat Commun 2019, 10(1): 5157. Alcon C, Manzano-Munoz A, Prada E, Mora J, Soriano A, Guillen G, et al. Sequential combinations of chemotherapeutic agents with BH3 mimetics to treat rhabdomyosarcoma and avoid resistance. Cell Death Dis 2020, 11(8): 634. Montero J, Letai A. Why do BCL-2 inhibitors work and where should we use them in the clinic? Cell Death Differ 2018, 25(1): 56–64. Selt F, Sigaud R, Valinciute G, Sievers P, Zaman J, Alcon C, et al. BH3 mimetics targeting BCL-XL impact the senescent compartment of pilocytic astrocytoma. Neuro Oncol 2023, 25(4): 735–747. Verhaegen M, Bauer JA, Martin de la Vega C, Wang G, Wolter KG, Brenner JC, et al. A novel BH3 mimetic reveals a mitogen-activated protein kinase-dependent mechanism of melanoma cell death controlled by p53 and reactive oxygen species. Cancer Res 2006, 66(23): 11348–11359. Vilella R, Benitez D, Mila J, Vilalta A, Rull R, Cuellar F, et al. Treatment of patients with progressive unresectable metastatic melanoma with a heterologous polyvalent melanoma whole cell vaccine. Int J Cancer 2003, 106(4): 626–631. Kovatcheva M, Liu DD, Dickson MA, Klein ME, O'Connor R, Wilder FO, et al. MDM2 turnover and expression of ATRX determine the choice between quiescence and senescence in response to CDK4 inhibition. Oncotarget 2015, 6(10): 8226–8243. Hernandez-Segura A, Rubingh R, Demaria M. Identification of stable senescence-associated reference genes. Aging Cell 2019, 18(2): e12911. Freedberg DE, Rigas SH, Russak J, Gai W, Kaplow M, Osman I, et al. Frequent p16-independent inactivation of p14ARF in human melanoma. J Natl Cancer Inst 2008, 100(11): 784–795. Sebastian D, Palacin M, Zorzano A. Mitochondrial Dynamics: Coupling Mitochondrial Fitness with Healthy Aging. Trends Mol Med 2017, 23(3): 201–215. Rovira M, Sereda R, Pladevall-Morera D, Ramponi V, Marin I, Maus M, et al. The lysosomal proteome of senescent cells contributes to the senescence secretome. Aging Cell 2022, 21(10): e13707. Chen L, Willis SN, Wei A, Smith BJ, Fletcher JI, Hinds MG, et al. Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function. Mol Cell 2005, 17(3): 393–403. Saleh T, Tyutyunyk-Massey L, Gewirtz DA. Tumor Cell Escape from Therapy-Induced Senescence as a Model of Disease Recurrence after Dormancy. Cancer Res 2019, 79(6): 1044–1046. Campisi J. Aging, cellular senescence, and cancer. Annu Rev Physiol 2013, 75: 685–705. Jochems F, Thijssen B, De Conti G, Jansen R, Pogacar Z, Groot K, et al. The Cancer SENESCopedia: A delineation of cancer cell senescence. Cell Rep 2021, 36(4): 109441. Robinson MD, McCarthy DJ, Smyth GK. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 2010, 26(1): 139–140. Robinson MD, Oshlack A. A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol 2010, 11(3): R25. Tables Table 1. Description of subjects included in the analysis shown in Figure 4D and their main characteristics. Volunteer Age Phototype Sex P002 57 2 Male P003 55 3 Male P005 49 3 Female P006 52 3 Female P007 50 3 Female P013 47 2 Male P014 41 3 Female P015 54 3 Female P016 46 3 Female P017 52 2 Female P018 50 3 Male P020 66 3 Female P021 60 2 Female P022 58 3 Female Additional Declarations There is no duality of interest Supplementary Files AlconetalCDDiff2024SUPPLEMENTARYINFORMATION.docx Cite Share Download PDF Status: Published Journal Publication published 03 Dec, 2024 Read the published version in Cell Death & Differentiation → Version 1 posted Editorial decision: revise 08 Jul, 2024 Review # 1 received at journal 06 Jul, 2024 Review # 2 received at journal 02 Jul, 2024 Reviewer # 2 agreed at journal 18 Jun, 2024 Reviewer # 1 agreed at journal 18 Jun, 2024 Reviewers invited by journal 18 Jun, 2024 Submission checks completed at journal 18 Jun, 2024 Editor assigned by journal 14 Jun, 2024 First submitted to journal 14 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4581617","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":315842892,"identity":"620724ff-3d36-4f93-a4b8-e824d8b0d5a8","order_by":0,"name":"Joan Montero","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsUlEQVRIiWNgGAWjYBACxgYog5+BB0iyEavlABBLNhCrBQxAWgwOEKuFedrhY48/VNyTM76Re4DhQ9lhIhw2Oy3d4MCZYmOzG3kJjDPOEaUlx0ziYFtC4rYbOQbMvG1Ea/mXkLh5BlDLX+K1NCQkbpAAamEkTktamsSZYwnGEmfeJRzsOZdOWIvh7ORjEhU1CXL87bkHH/wosyZCSwMS5wBh9UAgT5SqUTAKRsEoGNkAAKW3PJHWhN96AAAAAElFTkSuQmCC","orcid":"","institution":"University of Barcelona","correspondingAuthor":true,"prefix":"","firstName":"Joan","middleName":"","lastName":"Montero","suffix":""},{"id":315842893,"identity":"c3b3a7e9-d59a-4d7d-8b7f-929ad16b3929","order_by":1,"name":"Clara Alcon","email":"","orcid":"https://orcid.org/0000-0003-3243-9440","institution":"University of Barcelona","correspondingAuthor":false,"prefix":"","firstName":"Clara","middleName":"","lastName":"Alcon","suffix":""},{"id":315842894,"identity":"ccd2f708-6c16-4001-a66e-e6cd292e139c","order_by":2,"name":"Marta Kovatcheva","email":"","orcid":"https://orcid.org/0000-0002-2536-1043","institution":"IFOM ETS - The AIRC Institute of Molecular Oncology","correspondingAuthor":false,"prefix":"","firstName":"Marta","middleName":"","lastName":"Kovatcheva","suffix":""},{"id":315842895,"identity":"1e270f10-7692-4834-91e3-bd7dd3fab127","order_by":3,"name":"Paula Morales Sanchez","email":"","orcid":"https://orcid.org/0000-0002-9563-668X","institution":"University of Barcelona","correspondingAuthor":false,"prefix":"","firstName":"Paula","middleName":"Morales","lastName":"Sanchez","suffix":""},{"id":315842896,"identity":"21a22526-0c8a-4a73-b07e-2f4297929594","order_by":4,"name":"Teresa Torres","email":"","orcid":"","institution":"Hospital Clinic and Fundació Clínic per la Recerca Biomèdica","correspondingAuthor":false,"prefix":"","firstName":"Teresa","middleName":"","lastName":"Torres","suffix":""},{"id":315842897,"identity":"e6da55df-3f4f-46c3-b600-486d1adfb845","order_by":5,"name":"Susana Puig","email":"","orcid":"","institution":"Hospital Clinic and Fundació Clínic per la Recerca Biomèdica","correspondingAuthor":false,"prefix":"","firstName":"Susana","middleName":"","lastName":"Puig","suffix":""},{"id":315842898,"identity":"235de7d5-4227-4c0f-b0aa-a687c7dd7a36","order_by":6,"name":"Josep Samitier","email":"","orcid":"https://orcid.org/0000-0002-1140-3679","institution":"Institute for Bioengineering of Catalonia (IBEC), Barcelona Institute of Science and Technology (BIST)","correspondingAuthor":false,"prefix":"","firstName":"Josep","middleName":"","lastName":"Samitier","suffix":""},{"id":315842899,"identity":"8982cd8d-911e-4afa-a613-9d61bc9deb45","order_by":7,"name":"Carlos Enrich","email":"","orcid":"","institution":"University of Barcelona","correspondingAuthor":false,"prefix":"","firstName":"Carlos","middleName":"","lastName":"Enrich","suffix":""},{"id":315842900,"identity":"7a4ce7be-eaec-425c-b7fc-21d774713952","order_by":8,"name":"Manuel Serrano","email":"","orcid":"https://orcid.org/0000-0001-7177-9312","institution":"Cambridge Institute of Science, Altos Labs","correspondingAuthor":false,"prefix":"","firstName":"Manuel","middleName":"","lastName":"Serrano","suffix":""}],"badges":[],"createdAt":"2024-06-14 11:05:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4581617/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4581617/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41418-024-01417-z","type":"published","date":"2024-12-03T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":59591995,"identity":"3e8062fa-72c9-4f4b-8211-a8eea45c25a7","added_by":"auto","created_at":"2024-07-03 14:57:50","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":375934,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of palbociclib-induced senescent melanoma cells. \u003c/strong\u003eA: Quantification of SA-β-galactosidase in proliferating and palbociclib-induced senescent melanoma cells (senescent-P) by flow cytometry. B. % of p21 and p16 in proliferating and palbociclib-induced senescent (senescent-P) melanoma cells measured by flow cytometry. C and D: Representative images of transmission electron microscopy from proliferating and palbociclib-induced senescent (senescent-P) melanoma cells. ER, endoplasmic reticulum; ly, lysosome; mit, mitochondria; LD, lipid droplet; g, Golgi complex; Nu, nucleus. Scale bar: 1mm. Values indicate mean values ± SEM from at least three independent experiments. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 compared to control.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4581617/v1/c86dfc53bd99f9c1569fc709.jpg"},{"id":59591992,"identity":"00044b90-3004-4083-9b43-b7afffc37ce1","added_by":"auto","created_at":"2024-07-03 14:57:49","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":364241,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePalbociclib-induced senescence distinctly affects apoptotic priming and protein expression in melanoma cells. \u003c/strong\u003e\u0026nbsp;A. Left panel: Confocal images of proliferating and palbociclib-induced senescent SK-MEL-28 cells (senescent-P)stained with the mitochondrial marker TOM20 (red) and DAPI (blue). Right panel: Representative histograms of cytochrome c signal detected by flow cytometry in proliferating and palbociclib-induced senescent (senescent-P) SK-MEL-28 cells. B. Upper panel: % of cytochrome c retained inside the mitochondria with increasing concentrations of BIM peptide in control and palbociclib-induced senescent SK-MEL-103, SK-MEL-28 and M16 cells (senescent-P). Lower panel: Maximum Δ% priming BIM peptide in the three palbociclib-induced cell lines. C. Representative western blot images and quantification of the optical density of each protein and normalized with tubulin. Results expressed as fold increase represents the increase in optical density compared to proliferating cells. Values indicate mean values ± SEM from at least three independent experiments. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 compared to control.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4581617/v1/18ce56f5a6508e29c5b7d91c.jpg"},{"id":59591994,"identity":"3971e854-6209-43a0-9a7f-3119ca60bb87","added_by":"auto","created_at":"2024-07-03 14:57:49","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":281010,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSenescent melanoma cells acquire BCL-xL dependence. \u003c/strong\u003eA: % cytochrome c release after the incubation with sensitizer peptides (BAD 10µM, HRK 100µM and MS1 10µM) in proliferating, palbociclib-induced (senescent-P) and irradiation-induced senescent (senescent-IR) melanoma cells measured by flow cytometry. B: % cell death assessed by annexin V and DAPI staining and flow cytometry analysis in proliferating, palbociclib-induced (senescent-P) and irradiated-induced senescent (senescent-IR) melanoma cells after the incubation with the treatments for 48 hours in SK-MEL-103 and SK-MEL-28 and 96 hours in M16 cells. Values indicate mean values ± SEM from at least three independent experiments. ** p\u0026lt;0.01, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 compared to control.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4581617/v1/e8f92c1b08b5e5808a571262.jpg"},{"id":59592425,"identity":"570dc6dd-2551-431a-88ac-a8f1f2550a24","added_by":"auto","created_at":"2024-07-03 15:05:50","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":165207,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBCLxL dependence in senescent melanoma cells is mediated through \u0026nbsp;HRK downregulation.\u003c/strong\u003e A: Representative western blot images and optical density quantification for BCL-xL and HRK of proliferating, palbociclib-induced (senescent-P) and irradiated-induced senescent (senescent-IR) SK-MEL-103 cells. B: Representative western blot images and optical density quantification for BCL-xL and HRK of proliferating, palbociclib-induced (senescent-P) and irradiated-induced senescent (senescent-IR) SK-MEL-28 cells. C: Representative western blot images and optical density quantification for BCL-xL and HRK of proliferating and palbociclib-induced senescent M16 cells (senescent-P). Results expressed as fold increase represents the increase in optical density compared to proliferating cells. \u0026nbsp;D: mRNA expression of \u003cem\u003eHRK\u003c/em\u003e in control and lesioned skin from healthy donors. Values indicate mean values ± SEM from at least three independent experiments. ** p\u0026lt;0.01, *p \u0026lt; 0.05 compared to control.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4581617/v1/0dc79714daa395265f093c52.jpg"},{"id":59591991,"identity":"c708d23f-bf20-41e2-9e02-1950b9928d10","added_by":"auto","created_at":"2024-07-03 14:57:49","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":133824,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBCL-xL presents higher binding affinity for BAK in senescent melanoma cells. \u003c/strong\u003eA: Representative western blot image from unbound fractions after RabIgG or BCL-xL immunoprecipitation. B: Representative western blot images from the co-immunoprecipitation of BCL-xL with BAK, BIM and BAX in proliferating and palbociclib-induced senescent (senescent-P) SK-MEL-28 and SK-MEL-103 cell lysates. Values indicate mean values ± SEM from at least three independent experiments.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4581617/v1/091a295938694d3be6ecb92e.jpg"},{"id":59591990,"identity":"eb4a5b79-5321-41b6-8268-42af2cd7e1bd","added_by":"auto","created_at":"2024-07-03 14:57:49","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":107751,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMechanism of apoptotic protection in senescent melanoma cells. \u003c/strong\u003eSchematic representation of BCL-2 family proteins interaction in senescent melanoma cells. Therapy-induced senescence with palbociclib or γ-irradiation reduces HRK levels which allow the binding of available BCL-xL to BAK and confers protection against apoptosis induction. When BCL-xL is targeted, BAK could be displaced and apoptosis could be restored.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4581617/v1/465b470a17fc213890434e46.jpg"},{"id":70540126,"identity":"fe990ab2-614e-4265-b154-da0878832434","added_by":"auto","created_at":"2024-12-04 08:07:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3499237,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4581617/v1/84495fe2-5aaa-4eb7-b913-03a25f71f454.pdf"},{"id":59592424,"identity":"01d858aa-049a-45d2-8559-f32a4ddcabdf","added_by":"auto","created_at":"2024-07-03 15:05:49","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":409463,"visible":true,"origin":"","legend":"","description":"","filename":"AlconetalCDDiff2024SUPPLEMENTARYINFORMATION.docx","url":"https://assets-eu.researchsquare.com/files/rs-4581617/v1/6e3f67229becfd3b082f7dd9.docx"}],"financialInterests":"There is no duality of interest","formattedTitle":"HRK downregulation and augmented BCL-xL binding to BAK confer apoptotic protection to therapy-induced senescent melanoma cells.","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eCellular senescence is a stable form of cell cycle exit that can be induced by a variety of stressors including aberrant oncogene activation, oxidative stress, and chemotherapies. While senescence is thought to have evolved as a tumor-suppressive mechanism \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, the inappropriate accumulation of senescent cells has pathogenic consequences, including risk of cancer relapse \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, contributing to the long-term side effects of cancer therapy \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, and acting as a driver of many age-associated illnesses, including fibrotic, cardiovascular, and neurologic diseases \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. A recent field of study between cancer and senescence that is now extensively explored is therapy-induced senescence (TIS). TIS has been found in tumors after radiation or chemotherapy, but only in a subset of cells \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Commonly used treatments such as doxorubicin, paclitaxel, cisplatin\u0026hellip; conventional chemotherapy and radiotherapy in general, but also targeted therapies and immunotherapies have been reported to induce TIS \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Thus, the selective elimination of senescent cells by a class of molecules known as senolytics has become a therapeutic objective \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWhile the therapeutic potential of senolytics in a wide swath of pathologies is clear, the identification and successful administration of these drugs has been proved challenging. Navitoclax (ABT-263) was identified as one of the first senolytic agents based on the transcriptional profiling of senescent cells, which revealed a key role of anti-apoptotic BCL-2 family proteins \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Resistance to apoptosis has become a so-called hallmark of senescent cells; paradoxically, the hyper-sensitivity of senescent cells to BH3 mimetic drugs indicates that they are simultaneously \u0026ldquo;primed\u0026rdquo; towards apoptosis \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Rationally increasing this apoptotic priming and inducing killing in senescent cells is a goal of senolytic therapeutics.\u003c/p\u003e \u003cp\u003eWhile navitoclax is routinely used as a \u0026ldquo;gold standard\u0026rdquo; or proof of concept senolytic agent in preclinical studies, its therapeutic potential is confounded by dose-limiting toxicities largely based in thrombocytopenia. Navitoclax is a BH3 mimetic drug, whose mechanism of action is based on the inhibition of several anti-apoptotic BCL-2 family members, simultaneously targeting BCL-2, BCL-xL and BCL-W. Platelet toxicity has been attributed to BCL-xL inhibition, which was followed by second generation BH3 mimetics with enhanced specificity, such as venetoclax (ABT-199), which specifically targets BCL-2 \u003csup\u003e15\u003c/sup\u003e, or DT2216, a proteolysis targeting chimera (PROTAC) that targets BCL-xL without causing thrombocytopenia \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNevertheless, when and how such drugs may have efficacy as senolytics is unclear. The pro- and anti-apoptotic network is complex, and the expression levels of its constituent genes does not necessarily reflect their cellular activity \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Moreover, recent work has indicated that different cell types exhibit unique adaptations, such as the MCL-1-dependent resistance to navitoclax in melanocytes \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Given the relatively low predictive capacity of individual features of the BCL-2 family members, a new generation of functional biomarkers have been recently established to predict anticancer therapy induction of apoptosis and guide the use of BH3 mimetics. Directly exposing living cancer cells to therapeutic agents \u003cem\u003eex vivo\u003c/em\u003e to determine chemosensitivity has been historically explored, but the development of novel technologies have fostered functional precision medicine. Among them, BH3 profiling has demonstrated that it can rapidly identify anti-apoptotic dependencies in cancer cells \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. This functional assay uses synthetic ̴ 20-mer BH3 peptides, mimicking BH3-only proteins, acting as a pro-death signal to induce mitochondrial outer membrane permeabilization (MOMP). By using different peptides, BH3 profiling can rapidly interrogate cancer cells and obtain precise information regarding the apoptotic status of the cell. For example, the BH3 peptides BIM and BID, that bind to all anti-apoptotic proteins and directly activate the effector proteins BAX/BAK and MOMP, measure overall apoptotic priming, or how close cancer cells are to the apoptotic threshold \u003csup\u003e2\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, 2\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Further development of this technique led to dynamic BH3 profiling, which rapidly measures how much a given treatment primes cancer cells towards apoptosis (Δ% priming) by uniquely measuring early changes in the apoptotic signaling preceding frank cell death on the order of days or weeks \u003csup\u003e2\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In addition, by using peptides recapitulating the BH3 domain sequence of sensitizer proteins\u0026mdash;such as BAD, HRK, or NOXA BH3 peptides\u0026mdash; BH3 profiling can also identify changes in anti-apoptotic dependencies upon treatment and accurately identify BH3 mimetic combinations to enhance cytotoxicity \u003csup\u003e20, 2\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, 2\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, 2\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, also to eliminate oncogene-induced senescent cells \u003csup\u003e2\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Nevertheless, to date BH3 profiling has not been fully validated on senescent cells. Given the hypersensitivity of senescent cells to BH3 mimetics, we reasoned that BH3 profiling could be a novel approach to dissect the complexity of anti-apoptotic networks in senescent cells, and with which to nominate novel senolytic therapeutic approaches.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003ePalbociclib-induced senescence distinctly affects apoptotic signaling in melanoma cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe decided to focus in melanoma since senescence and apoptotic protection play a key role in this type of cancer development and therapy outcome\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. We used three melanoma cell lines; SK-MEL-103 (NRAS mutant), SK-MEL-28 (BRAFV600E mutant)\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, and the M16 (BRAFV600E mutant) that was derived from a patient continuously exposed to UV light as previously described \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. To induce senescence, we chronically treated these cell lines with a CDK4/6 inhibitor for 7 days \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, and, since this inhibitor does not uniformly induce senescence in all cancer cell lines \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, we confirmed the phenotype using different well-established markers: SA-β\u0026minus;galactosidase, p21 and p16 \u003csup\u003e31, 32\u003c/sup\u003e. As expected, we detected a significant increase in these markers in all three cell lines when treated with palbociclib, thus indicating therapy-induced senescence (TIS) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B). To assess senescence morphologically, we examined by transmission electron microscopy (TEM) for potential phenotypic changes. Overall, we detected longer mitochondria (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) and an increase in the number of lysosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) in palbociclib-treated cells, as previously described\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. All these observations confirm that palbociclib effectively induces senescence in all three melanoma cell lines examined.\u003c/p\u003e\u003cp\u003eSenescent cells often present resistance to die by apoptosis \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Therefore, we next sought to study how TIS modulates apoptosis using BH3 profiling. As mentioned above, senescent cells present longer mitochondria, and we reasoned that this could affect MOMP and cytochrome c release measurements, which are required for BH3 profiling analyses \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Thus, we first evaluated if these selected senescent melanoma cells presented a larger mitochondrial content compared to non-senescent cells. Using TOM20 as a mitochondrial marker, we observed a significant increase in its expression in palbociclib-induced senescent SK-MEL-28 cells compared to non-treated (proliferating) cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). We also evaluated cytochrome c staining by flow cytometry and, correlating with TEM and fluorescence microscopy, we detected a significant increase in its intensity after senescence induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Therefore, for flow cytometry BH3 profiling analyses we adjusted the cytochrome c gating taking into consideration this variation in basal fluorescence (see the Methods section for more details). When comparing non-treated (proliferating) and palbociclib-treated melanoma cancer cells by BH3 profiling, we observed that these cell lines showed a different priming pattern when becoming senescent. If a given treatment or state sensitizes cells towards apoptosis, this would cause a leftward shift of the cytochrome c retained curve after BIM peptide exposure, since less amount of BIM is required to promote MOMP and cytochrome c release, indicating an increase in overall apoptotic priming (positive Δ% priming). In contrast, if a treatment or state renders cells more resistant to apoptosis, we would observe a shift of the curve to the right, indicating a decrease in priming (negative Δ% priming). Surprisingly, we observed that SK-MEL-103 cells treated with palbociclib became slightly primed for apoptosis, while the SK-MEL-28 and M16 cells clearly turn out to be less primed for apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eTo further elucidate this distinct effect on apoptotic priming induced by palbociclib, we compared the expression of different pro-apoptotic members of the BCL-2 family of proteins between proliferating and senescent cells. For instance, in all cell lines we observed a significant reduction of the activator protein BIM (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), that could partially explain why senescent cells exert resistance to cell death. When further analyzing the SK-MEL-103 cells, we identified that the expression of the effector proteins BAX and BAK significantly increased after palbociclib treatment, correlating with the observed increase in Δ% priming detected by BH3 profiling with the BIM BH3 peptide (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). In fact, these results match previous mRNA expression data from the Serrano laboratory \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e (Suppl. Figure\u0026nbsp;1). However, in SK-MEL-28 and M16 cells we observed that BAK remained constant or increased while BAX expression decreased, again partially explaining the negative Δ% priming detected by BH3 profiling (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eIn summary, palbociclib promoted a decrease in the activator protein BIM, thus preventing apoptosis, and effectively induced senescence, assessed by SA-β-galactosidase, p21 and p16. Paradoxically one melanoma cell line became slightly primed for apoptosis (positive Δ% priming) while the other two showed protection towards this form of programmed cell death (negative Δ% priming) in part due to a distinct change in BAX expression after TIS.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTherapy-induced senescence induces a BCL-xL dependent anti-apoptotic adaptation and can be targeted with specific BH3 mimetics or PROTAC.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAnother hallmark of senescent cells is their pro-survival adaptation through anti-apoptotic BCL-2 family proteins, and, non-surprisingly, one of the most studied senolytics has been the promiscuous BCL-2/BCL-xL/BCL-W inhibitor navitoclax \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. We sought to elucidate if TIS melanoma cells also presented a similar adaptation and, if that was the case, how they prevented apoptosis. Thus, we performed BH3 profiling using specific BH3 peptides BAD (for BCL-2 and BCL-xL), HRK (for BCL-xL) and MS1 (for MCL-1), as previously described \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, on the same cell lines after senescence induction using palbociclib, and γ-irradiation (which also increased SA-β-galactosidase activity in all three cell lines as shown in supplementary Fig.\u0026nbsp;2). In brief, a positive Δ% priming using these sensitizer BH3 peptides would point to a specific anti-apoptotic protein that senescent cells become addicted to. We found that SK-MEL-103, SK-MEL-28 and, to a lesser extent, M16 displayed a significant BCL-xL adaptation, as we detected an increase in the % of cytochrome c released with the HRK (and BAD, that binds to BCL-2 and BCL-xL) peptide (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). We also detected a minor increase in priming with the MS1 BH3 peptide, indicating a partial adaptation through MCL-1 but to a lesser extent than BCL-xL (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). We next explored anti-apoptotic inhibition potential to eliminate senescent cells. Using palbociclib and γ-irradiation, we generated senescent melanoma cells and tested BH3 mimetics\u0026rsquo; senolytic activity. We exposed these cells to ABT-199 (venetoclax), the BCL-xL inhibitor A-1331852, the MCL-1 inhibitor S63845, ABT-263 (navitoclax) and the BCL-xL degrading PROTAC DT2216 \u003csup\u003e16\u003c/sup\u003e. As anticipated by our BH3 profiling results, we observed significant senolytic activity in these melanoma cell lines when targeting BCL-xL using A-1331852, navitoclax or DT2216 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB); and in SK-MEL-103 and SK-MEL-28 BCL-xL targeting therapies had a greater senolytic activity compared to M16. Furthermore, we detected a significant increase in cell death with the MCL-1 inhibitor S63845 in SK-MEL-28 suggesting an implication of this protein in apoptotic resistance in senescent cells harboring a BRAFV600 mutation\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. These results indicate that TIS melanoma cells block apoptosis primarily through BCL-xL, and to a lesser extent using MCL-1. Consequently, inhibiting this anti-apoptotic protein with specific inhibitors - beyond navitoclax that also targets BCL-2 and BCL-W - such as the BH3 mimetic A-1331852 or a PROTACs like DT2216 that would not cause thrombocytopenia, due to the minor expression of E3 ligase in platelets\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, appears as an effective therapeutic strategy to remove melanoma senescent cells.\u003c/p\u003e\u003cp\u003e \u003cb\u003eBCL-xL adaptation is mediated by HRK downregulation and changes in binding affinities to pro-apoptotic proteins.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo elucidate how TIS melanoma cells become dependent on BCL-xL, we first analyzed the expression of this anti-apoptotic protein. We found in palbociclib-treated SK-MEL-103 and M16 cells that its expression increased. Surprisingly, SK-MEL-28 cells did not exert significant BCL-xL changes upon treatment, suggesting a potential role of another BCL-2 family member to regulate its availability (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C). In this regard, we analyzed several BCL-2 family proteins, and found that the sensitizer HRK, which specifically binds to BCL-xL \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, was clearly downregulated in all three cell lines when induced into senescence. In other words, senescent inducers such as palbociclib and γ-irradiation decreased the expression of HRK, initially bound to BCL-xL, thus liberating this pro-survival protein to protect cells towards apoptosis. Similar results were observed in healthy donors comparing control skin with UV irradiated (Table\u0026nbsp;1), where the latter showed an increase trend in the senescence markers \u003cem\u003eCDKN1A\u003c/em\u003e (p21) and \u003cem\u003eCDKN2A\u003c/em\u003e (p16) (Supplementary Fig.\u0026nbsp;3), and a decrease trend in \u003cem\u003eHRK\u003c/em\u003e mRNA expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) pointing to a potential BCL-xL adaptation in senescent skin from patients.\u003c/p\u003e \u003cp\u003eWe previously showed that palbociclib treatment promoted a decrease in BIM, HRK and, in some cases, BAX expression, consequently preventing apoptosis. So, our next question was how BCL-xL inhibitors restored apoptotic cell death in senescent cells. The BCL-2 family of proteins represents a complex interactome regulated at multiple levels, including protein expression, posttranslational modifications, and dynamic binding affinities. To further elucidate the role of BCL-xL in TIS, we immunoprecipitated this anti-apoptotic protein with a high efficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) and analyzed its binding to BIM, BAX and BAK in SK-MEL-103 and SK-MEL-28. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, both cell lines exert distinct BAX/BAK expression changes when senescence is induced with palbociclib. However, in our immunoprecipitation analyses we observed a clear increase in binding between BCL-xL and BAK in senescent cells compared to control cells, while it decreased or remained similar for the other two proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). In summary, we found for the first time that palbociclib-induced senescence promotes different changes in the BCL-2 family proteins\u0026rsquo; expression that lead to an increase in BCL-xL:BAK binding that prevents apoptosis and explains why specifically BCL-xL targeted agents are effective senolytics.\u003c/p\u003e "},{"header":"DISCUSSION","content":"\u003cp\u003eOne of the hallmarks of senescent cells is their resistance to die by apoptosis \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Consequently, our first aim was to further study if senescent melanoma cells showed this resistance and specifically how apoptotic cell death was affected. We used three different melanoma cell lines: SK-MEL-103 that presents a mutation in NRAS, and SK-MEL-28 and M16 that have a BRAFV600E mutation \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. We exposed all three cell types to different well-established senescence inducing factors, palbociclib and γ-irradiation, and positively detected an increase in p16, p21 and SA-β-galactosidase in all cases (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B). We confirmed senescence induction by electron microscopy where, beyond the increase in cellular size, we identified two commonly observed morphological changes: mitochondria elongation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and an increment in the number of lysosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Since mitochondrial permeabilization is a key step in apoptosis induction and could be altered in senescent cells, we next evaluated potential changes in mitochondrial polarization and cytochrome c expression, and we clearly observed by confocal microscopy and flow cytometry an increased intensity on the latter with senescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Consequently, we had to adjust our flow cytometry settings when performing the BH3 profiling analyses on these cells \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Interestingly, we observed a different BH3 profiling trend when comparing the NRAS mutant cell line SK-MEL-103 with the BRAF mutant SK-MEL-28 and M16 cells. When measuring overall priming with the BIM BH3 peptide \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, we observed that senescent SK-MEL-103 cells became slightly primed to apoptosis while SK-MEL-28 and M16 were less primed after palbociclib treatment. When we analyzed the expression of different pro-apoptotic BCL-2 family proteins, we detected that in all cases senescence induction caused a decrease in BIM (that would have a protective effect towards apoptosis), that BAK remained unchanged or increased, and that BAX decrease in the BRAF-mutant cells but increased in SK-MEL-103 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Precisely the distinct changes in BAX expression between these melanoma cell lines, could partially explain the differences observed in BH3 profiling overall apoptotic priming with senescence. In summary, the studied melanoma cell lines presented variability in BAX protein expression and overall apoptotic priming when becoming senescent but in all cases, BIM was downregulated and importantly BAK expression remained constant or increased.\u003c/p\u003e \u003cp\u003eThe cellular process of senescence has been related to ageing \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e and cancer cells\u0026rsquo; survival to therapy \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. However, how therapy-induced senescent cells avoid apoptosis is not fully understood. Several studies previously described that senescent cells utilize anti-apoptotic BCL-2 family proteins to resist cell death and, because of that, they are particularly sensitive to BH3 mimetics such as navitoclax, that blocks BCL-2, BCL-xL and BCL-W \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Thus, we next sought to further understand how this anti-apoptotic adaptation occurs using BH3 profiling \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. We identified that when exposed to palbociclib or γ-irradiation, melanoma cells clearly became BCL-xL dependent (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In other words, TIS in these cells promoted a pro-survival adaptation mostly through BCL-xL to prevent cell death. Other anti-apoptotic proteins such as MCL-1 were identified by BH3 profiling but to a lesser extent, indicating a secondary role in TIS pro-survival adaptation. We next used different anti-apoptotic inhibitors or degraders as senolytics to test if we could specifically eliminate these senescent melanoma cells. As anticipated by BH3 profiling, those molecules that specifically inhibited BCL-xL (A-1331852 and navitoclax) or the PROTAC DT2216 showed a significant cytotoxic effect on senescent cells in all cases (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Other BH3 mimetics directed towards BCL-2 or specially MCL-1 had a minor effect eliminating these cells. These results suggest that despite the observed variability in overall apoptotic priming and BCL-2 family protein expression (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), TIS in melanoma leads to a marked BCL-xL dependence in all cases, and its inhibition or degradation efficiently eliminates senescent cells.\u003c/p\u003e \u003cp\u003eAfter identifying the key role of BCL-xL in melanoma cells survival upon senescence induction, we next sought to study how precisely this anti-apoptotic protein impedes cell death. We first analyzed its expression when TIS was induced and observed that it increased in SK-MEL-103 and M16, or remained unchanged in SK-MEL-28 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). We were particularly intrigued by the latter since in these cells we also observed a BCL-xL adaptation. We then analyzed other BCL-2 family members, particularly the sensitizer proteins, and found that HRK, that specifically binds and blocks BCL-xL\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, protein expression decreased with TIS in all cases. Furthermore, we observed in photodamaged skin from donors that senescence markers tend to increase while HRK expression tend to decrease (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), correlating with our \u003cem\u003ein vitro\u003c/em\u003e observations. In other words, senescence induction, both in cell lines and in UV-damaged skin from donors, promotes a downregulation of HRK that frees BCL-xL to block MOMP and apoptotic cell death (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). But at that point a key question was still unanswered: which specific pro-apoptotic proteins are inhibited by BCL-xL and how is mitochondrial permeabilization prevented? To answer this question, we immunoprecipitated BCL-xL and analyzed by Western Blot which proteins were bound to it. Importantly, we found in SK-MEL-103 and SK-MEL-28 cells that TIS promoted an increase in BAK binding to BCL-xL that blocked MOMP and apoptosis (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn summary, TIS is emerging as a key determinant in cancer cells survival after anticancer treatment and could explain why some melanoma patients\u0026rsquo; relapse. Senescence has been extensively studied, in part because of its implications in age-related diseases, and navitoclax has been for years one of the most studied senolytics through its capacity to simultaneously inhibit three important anti-apoptotic proteins: BCL-2, BCL-xL and BCL-W \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Nevertheless, there is emerging evidence that cell-type specific features contribute to the efficacy and specificity of senolytic treatments, and that navitoclax is not effective in all cases \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Thus, a mechanistic and functional understanding of the anti-apoptotic network in senescent cells will be necessary to effectively include senolytic agents in treatment regimens. In this study, we use a panel of melanoma cell lines to demonstrate the value of BH3 profiling in uncovering specific senolytic vulnerabilities. We here report that TIS in melanoma promotes a BCL-xL mediated pro-survival adaptation\u0026ndash; distinct from what has been reported in untransformed melanocytes \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e \u0026ndash; that could be prevented by using specific inhibitors (also navitoclax) or a PROTAC degrader against BCL-xL. We also show that targeting other anti-apoptotic proteins such as BCL-2, BCL-W or MCL-1 have a minor senolytic effect, and its concomitant inhibition would only lead to undesired toxicities. We further investigated how this adaptation occurred and found that TIS promoted a consistent decrease of the sensitizer protein HRK that liberated BCL-xL, and that the latter specifically blocked the effector BCL-2 family protein BAK to prevent MOMP and apoptosis. To our knowledge this is the first time that this senescence pro-survival mechanism is described and could help develop new effective senolytics directed to avoid HRK downregulation or disrupt the BCL-xL:BAK binding. We believe that this new senolytic strategy guided by BH3 profiling could prevent cancer therapy endurance and potentially avoid some melanoma relapse cases in the future.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCell lines and treatments\u003c/h2\u003e \u003cp\u003eMelanoma cell line SK-MEL-28 was purchased at ATCC (ATCC\u0026reg; CRL-1772\u0026trade;, ATCC, Manassas, VI, USA) and SK-MEL-103 cell line was provided by Dr. Marisol Soengas. Patient-derived cell line M16 was kindly provided by Dr. Susana Puig from Hospital Cl\u0026iacute;nic de Barcelona. SK-MEL-28 cell line was cultured in RPMI 1640 medium (31870, Thermo Fisher, Gibco, Paisley, Scotland) supplemented with 10% heat-inactivated fetal bovine serum (10270, Thermo Fisher, Gibco), 1% of l-glutamine (25030, Thermo Fisher, Gibco), and 1% of penicillin and streptomycin (15140, Thermo Fisher, Gibco). SK-MEL-103 and M16 cells were cultured in DMEM high glucose medium (41965, Thermo Fisher, Gibco) supplemented with 10% heat-inactivated fetal bovine serum, 1% of penicillin and streptomycin and 1% of l-glutamine. All cells were tested for mycoplasma and maintained at 37\u0026deg;C in a humidified atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eBH3 mimetics ABT-199 (venetoclax), A-1331852 (A-133), S63845 and ABT-263 (navitoclax) were purchased at Selleckchem (Munich, Germany) and the PROTAC BCL-xL degrader DT2216 was purchased at AbMole BioScience (Houston, Texas, USA). Treatments were performed directly in the culture media at the indicated concentrations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePalbociclib-induced senescence\u003c/h2\u003e \u003cp\u003eFor palbociclib-induced senescence, cells were seeded in a 6 well-plate and treated with palbociclib (S1116, Selleckchem, Munich, Germany) at 1\u0026micro;M dose. After 3 days cells were replenished with fresh media and palbociclib. Seven days after the treatment, senescent cells were used for further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eIrradiation-induced senescence\u003c/h2\u003e \u003cp\u003eFor irradiated induced senescence, cells were seeded in a 6 well-plate and irradiated with a gamma irradiator MARK I 30 (J.L. Shepherd \u0026amp; Associates, San Fernando, CA, USA) at 10Gy. After 3 days, the media was changed and cells were maintained in culture till day 7 after irradiation and used for further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSenescence associated β-galactosidase staining\u003c/h2\u003e \u003cp\u003eCells were stained using CellEvent\u0026trade; Senescence Green Detection Kit (C10851, Thermo Fisher). Briefly, cells were washed with PBS, tripsinized, fixed with 4% formaldehyde during 10 minutes at room temperature, washed with PBS\u0026thinsp;+\u0026thinsp;1% BSA and incubated with the working solution at 37\u0026ordm;C without CO\u003csub\u003e2\u003c/sub\u003e during 2 hours. After incubation, cells were washed with PBS and then imaged using a spectral flow cytometry Cytek AURORA instrument (Cytek Biosciences, California, USA) from the Scientific and Technological Centers of the University of Barcelona.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eBH3 profiling\u003c/h2\u003e \u003cp\u003eBH3 profiling was developed at the Letai laboratory, and the experiments were performed as previously described \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. After inducing senescence for 7 days cells were stained with the viability marker Zombie Violet (423113, BioLegend, Koblenz, Germany) and then washed with PBS and resuspended in MEB (150 mM mannitol, 10 mM hepes\u0026ndash;KOH pH 7.5, 150 mM KCl, 1 mM EGTA, 1 mM EDTA, 0.1% BSA, 5 mM succinate) in a final volume of 25 \u0026micro;L. Afterwards, cells were exposed to the following peptides: 25 \u0026micro;L of BIM BH3 peptide (final concentration of 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 \u0026micro;M), 25 \u0026micro;L of BAD BH3 peptide (final concentration of 10 \u0026micro;M), 25 \u0026micro;L of HRK BH3 peptide (final concentration of 100 \u0026micro;M), and 25 \u0026micro;L of MS1 BH3 peptide (final concentration of 10 \u0026micro;M) in MEB with 0.002% digitonin in a 96-well plate (3795, Corning, Madrid, Spain) for 1h following fixation with formaldehyde. Cells were finally stained with cytochrome c antibody (Alexa Fluor\u0026reg; 647 anti-Cytochrome c\u0026mdash;6H2.B4, 612310, BioLegend), p21 antibody (sc-6246 PE, Santa Cruz Biotechnology, Texas, USA) and p16 antibody (Alexa Fluor\u0026reg; 488 CDKN2A/p16INK4a, bs-4592R-A488, Bioss Antibodies, Woburn, Massachusetts, USA). Individual BH3 profiling analyses were performed using triplicates for DMSO, alamethecin (BML-A150-0005, Enzo Life Sciences, Lorrach, Germany), the different BIM BH3 concentrations used, BAD, HRK, and MS1 BH3 peptides. The expressed values stand for the average of three different readings performed with a high-throughput spectral flow cytometry Cytek AURORA instrument (Cytek Biosciences, California, USA) from the Scientific and Technological Centers of the University of Barcelona. % of cytochrome c released is used to calculate Δ% priming which represents the difference between treated cells minus non-treated cells for a given peptide.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry analysis\u003c/h2\u003e \u003cp\u003eDue to the difference of mitochondrial content between proliferating and senescence cells and therefore a major cytochrome c retained signalling was detected in senescent cells versus control cells, we adjusted the gating for cytochrome c retention with the control condition of each experimental setting (proliferating and senescent). Analyses were performed using FlowJo software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell death assay\u003c/h2\u003e \u003cp\u003eCells were trypsinized and stained with fluorescent conjugates of Annexin V (Alexa Fluor\u0026reg; 647 Annexin V, 640912, BioLegend) and DAPI (62248, Thermo Fisher) and analyzed on a flow cytometry Gallios instrument (Beckman Coulter, Nyon, Switzerland) from the Scientific and Technological Centers of the University of Barcelona. Viable cells are Annexin V DAPI negative, and cell death is expressed as 100%-viable cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eProtein extraction and quantification\u003c/h2\u003e \u003cp\u003eCells were lysed using RIPA buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris\u0026ndash;HCl pH\u0026thinsp;=\u0026thinsp;8, 1% Triton X-100, 0.1% SDS, EDTA-free Protease Inhibitor Cocktail (4693159001 Roche, Mannkin, Germany)) for 30 min at 4\u0026deg;C followed by a centrifugation at 16,100 \u0026times; \u003cem\u003eg\u003c/em\u003e for 10 min. The supernatant was stored at \u0026minus;\u0026thinsp;20\u0026deg;C for protein quantification performed using PierceTM BCA Protein Assay Kit (23227, Thermo Fisher).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eImmunoprecipitation\u003c/h2\u003e \u003cp\u003eCells were lysed using Immunoprecipitation buffer (150 mM NaCl, 10 mM Hepes, 2 mM EDTA, 1% Triton, 1.5 mM MgCl2, 10% glycerol, and EDTA-free Protease Inhibitor Cocktail (4693159001 Roche)) and centrifuged at 16,100 \u0026times; \u003cem\u003eg\u003c/em\u003e, 15 min at 4\u0026deg;C. Supernatants were incubated with magnetic beads (Dynabeads 10003D, Thermo Fisher) conjugated to 5 \u0026micro;g of rabbit anti-BCL-xL antibody (CST2764, Cell Signaling, Leiden, The Netherlands) or 5 \u0026micro;g of rabbit IgG antibody (CST2729, Cell Signaling) at 4\u0026deg;C overnight. After magnetization, a part of the supernatant was mixed with half volume of 4\u0026times; SDS\u0026ndash;PAGE sample buffer, heated at 96\u0026deg;C for 5 min and stored at \u0026minus;\u0026thinsp;80\u0026deg;C as unbound fractions. The rest of the supernatant was discarded. The resulting pellet was washed and mixed with 60 \u0026micro;L 4\u0026times; SDS\u0026ndash;PAGE sample buffer and heated for 10 min at 70\u0026deg;C following by magnetization and collection of the supernatant for further immunoblotting analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eImmunoblotting\u003c/h2\u003e \u003cp\u003eProteins were separated by SDS\u0026ndash;PAGE (Mini-Protean TGX Precast Gel 12%, 456\u0026ndash;1045, Bio-Rad) and transferred to PVDF membranes (10600023, Amersham Hybond, Pittsburgh, PA, USA). Membranes were blocked with dry milk dissolved in Tris buffer saline with 1% Tween 20 (TBST) for 1 h and probed overnight at 4\u0026deg;C with the primary antibodies of interest directed against: rabbit anti-BCL-xL (CST2764, Cell Signaling), rabbit anti-BIM (CST2933, Cell Signaling), rabbit anti-BAK (CST12105, Cell Signaling), rabbit anti-BAX (CST2772, Cell Signaling), rabbit anti-HRK (PRS3771, Sigma-Aldrich, San Luis, Missouri, USA), mouse anti-γ\u0026minus;tubulin (T6557, Sigma-Aldrich) followed by anti-rabbit or anti-mouse IgG HRP-linked secondary antibody (CST7074 or CST7076 Cell Signaling) in 3% BSA in TBST for 1 h at room temperature. Immunoblots were developed using Clarity ECL Western substrate (1705060, Bio-Rad), visualized with LAS4000 imager (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and ImageJ was then used to measure the integrated optical density of bands.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eTransmission electron microscopy procedure for flat embedding preparation of cell monolayers\u003c/h2\u003e \u003cp\u003eFor transmission electron microscopy (TEM) preparation and analysis of melanoma cells (proliferating and senescence) growing on coverslips, the cells were first washed with 0.1 M phosphate buffer (PB) to remove the excess of culture medium and fixed with a freshly made 3% glutaraldehyde solution in 0.1M PB for 1 hour at room temperature. Subsequently, the fixative was removed, and the samples were maintained at 4\u0026ordm; C in a fresh fixative solution until processing.\u003c/p\u003e \u003cp\u003eThe post-fixation procedure was conducted using 1% OsO4 for a period of 90 minutes at 4\u0026deg;C. Then, samples were dehydrated in increasing ethanol solutions (in accordance with the standard procedures). Coverslips with cells were then inverted (with the cells facing downwards) and placed on top of BEEM\u0026reg; capsules filled with Spurr resin (Electron Microscopy Sciences, Hatfield, PA, USA). The polymerization of the resin was carried out at a temperature of 60 \u0026ordm;C in a stove for a period of three days. At this point, the glass is removed by thermal contrast switch while the cell monolayer remains at the top of the polymerized block. A Leica ultramicrotome EM UC7 (Leica Microsystems, Wetzlar, Germany) was employed for sectioning. Ultra-thin sections (60\u0026ndash;70 nm) were mounted on cooper grids and stained with 2% Uranyl-less solution for 10 minutes and with a lead-staining solution for 5 minutes. The sections were observed using a JEOL JEM-1010 transmission electron microscope (JEOL Ltd., Akishima, Tokyo, Japan) coupled with an Orius SC1000 CCD camera (model 832) (Gatan Inc., Pleasanton, CA, USA) at the Unit of Electron Microscopy, Scientific and Technological Centers of the University of Barcelona, School of Medicine and Health Sciences (Barcelona, Spain).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003eCells were gently washed once with cold PBS and then fixed for 20 minutes with 4% paraformaldehyde in PBS. Cells were then washed three times with PBS and permeabilized for 10 minutes with 0.1% saponin in PBS. Blocking was performed for 5 minutes with 0.02% saponin and 1% BSA in PBS. For primary antibody incubation, cells were incubated with anti-Tom20 (ab186734, Abcam, Cambridge, UK) diluted in 0.02% saponin and 0.1% BSA solution for 1 hour at room temperature. After primary antibody incubation, cells were washed three times with PBS. Secondary antibody incubation was carried out by incubating cells with anti-Mouse IgG (H\u0026thinsp;+\u0026thinsp;L), Superclonal\u0026trade; Recombinant Secondary Antibody Alexa Fluor\u0026trade; 488 (A28175, Thermo Fisher) and anti-Rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 (A21428, Thermo Fisher) for 45 minutes in 0.02% saponin and 0.1% BSA solution at RT. For DAPI staining, cells were incubated with diluted DAPI stock solution (10374168, Thermo Fisher) to a final concentration of 300 nM in PBS for 5 minutes. Following staining, cells were washed three times with PBS, rinsed in water and mounted in Mowiol\u003csup\u003e\u0026reg;\u003c/sup\u003e 4\u0026ndash;88 (17951-500, Polysciences, Warrington, PA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eBioinformatic analysis of SK-MEL-103 data\u003c/h2\u003e \u003cp\u003eIn the present study, GEO2R (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.ncbi.nlm.nih.gov/geo/geo2r/\" target=\"_blank\"\u003ewww.ncbi.nlm.nih.gov/geo/geo2r/\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/geo/geo2r/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was utilized to download the raw counts of the GSE246690 series to identify genes differentially expressed across experimental conditions of the SK-MEL-103 cell line (control and Palbociclib)\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The data was analyzed using the edgeR (v.4.0.16) package from Bioconductor in the R environment (v.4.3.3) \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. The sva (v. 3.50.0) package was used for removing batch effects and other unwanted variation \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Subsequently, differential expression analysis was conducted using the quasi-likelihood negative binomial generalized log-linear model (GLM) functions provided by the edgeR package. For visualization purposes Log2CPM expression values were converted to Z-scores and statistical significance was represented as Fold Discovery Rate (FDR)\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003ePatients and samples for molecular validation\u003c/h2\u003e \u003cp\u003eFourteen healthy subjects, comprising 10 female and 4 male, mean age 52.6 years (range 41\u0026ndash;66) were recruited in Hospital Cl\u0026iacute;nic de Barcelona (Table\u0026nbsp;1). Two distinct areas on the forearm were identified for biopsy: one area of photodamaged skin (L01) and one area of less sun-damaged (naturally protected) skin on the inner forearm (L02).\u003c/p\u003e \u003cp\u003eSubsequently, two 3-mm punch biopsies were taken on L01 and L02 areas. Each biopsy was included in RNAlater (RNAprotect Tissue Reagent, Qiagen) and frozen to extract RNA and perform sequencing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and RNA-sequencing analysis\u003c/h2\u003e \u003cp\u003eBiopsies in RNAlater were frozen to extract RNA for sequencing. Total RNA was isolated from fresh tissue with Trizol, and purified with RNeasy Minikit (Trizol-chloroform\u0026thinsp;+\u0026thinsp;RNeasy MinElute Cleanup Kit, Qiagen). RNA quantification was performed using Qubit RNA BR Assay Kit. Integrity was measured via Bioanalyzer RNA 6000 NanoKit (Agilent Technologies, Santa Clara, CA).\u003c/p\u003e \u003cp\u003eRNA samples were sent to \u0026ldquo;Centro Nacional de An\u0026aacute;lisis Gen\u0026oacute;mico\u0026rdquo; to conduct the stranded mRNA library preparation and sequencing on HiSeq 4000 (Illumina).\u003c/p\u003e \u003cp\u003eRNA-Seq libraries were prepared from total RNA using TruSeq Stranded mRNA LibraryPrep Kit (Illumina). mRNA was enriched with oligo-dT magnetic beads from the total RNA (500ng) and fragmented to 80-450nt. The second strand cDNA synthesis was performed in the presence of dUTP to achieve strand specificity. The blunt-ended double-stranded cDNA was 3\u0026acute;adenylated and Illumina platform-compatible adaptors with unique dual indexes and unique molecular identifiers (Integrated DNA Technologies) were ligated. The ligation product was enriched with 15 PCR cycles. The final library was validated on a Bioanalyzer DNA 7500 assay. The libraries were sequenced on NovaSeq6000 (Illumina) in paired-end mode with a read length of 2x101bp, following the manufacturer\u0026rsquo;s protocol for dual indexing. Image analysis, base calling and quality scoring of the run were processed using the manufacturer\u0026rsquo;s software Real-Time Analysis (RTA 3.4.4) and followed by the generation of FASTQ sequence files.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData was analyzed using GraphPad Prism v.9.3.0 software and was represented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM of independent biological replicates. Statistical significance of the results was analyzed using Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-tail test. *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 were considered significant.\u003c/p\u003e \u003cp\u003eSpecifically, for RNA-Seq data analysis of patient samples, the R package LIMMA was used for data normalization, specifically employing the Variance Modeling at the Observational Leve (VOOM). After normalization of expression data, the genes of interest for validation (CDKN2A, CDKN1A, and HRK) were selected. Global results of the RNA-sequencing analysis in under revision for their publication. Comparisons between the expression between L01 and L02 samples from the same patient were conducted using a paired Student's t-test. Assumptions of normality and homogeneity of variances were checked and appropriately addressed. All analyses were performed using R version 3.3.0.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would also like to thank the Cytometry Facility and the Irradiation Service from the\u0026nbsp;Scientific and Technological Centers of the University of Barcelona\u0026nbsp;(CCITUB) for their assistance with experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ. M. is co-inventor of dynamic BH3 profiling (patented by Dana-Faber Cancer Institute) and has received royalties. J.M. 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. No potential conflicts of interest were disclosed by the other authors. M.S. is shareholder of Altos Labs, Inc., Senolytic Therapeutics, Inc., Life Biosciences, Inc., and Rejuveron\u0026nbsp;Senescence Therapeutics, AG.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC. Alcon performed and analyzed all the experiments. C. Alcon and J. Montero supported the conceptualization of this work. P. Morales-S\u0026aacute;nchez and T. Torres performed the bioinformatic analyses. C. Enrich performed the transmission electron microscopy analyses. C. Alcon, M. Kovatcheva and J. Montero wrote the manuscript. J. Montero supervised the work.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePatient 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to acknowledge Ministerio de Ciencia, Innovaci\u0026oacute;n y Universidades grant RTI2018-094533-A-I00; Ministerio de Ciencia e Innovaci\u0026oacute;n, Proy. de Generaci\u0026oacute;n del Conocimiento 2021 grant PID2021-128644OB-I0; CELLEX foundation; Jos\u0026eacute; Carreras Leuk\u0026auml;mie-Stiftung grant DJCLS 19R/2022; and Ag\u0026egrave;ncia de Gesti\u0026oacute; d\u0026apos;Ajuts Universitaris i de Recerca-Generalitat de Catalunya 2021-SGR-00284.\u003c/p\u003e\n\u003cp\u003eJS acknowledges \u0026ldquo;Plan Complementario de Biotecnolog\u0026iacute;a aplicada a la Salud\u0026rdquo;, coordinado por el Institut de Bioenginyeria de Catalunya (IBEC) en el marco del Plan de Recuperaci\u0026oacute;n, Transformaci\u0026oacute;n y Resiliencia (C17.I1) ‐ Financiado por la Uni\u0026oacute;n Europea \u0026ndash; NextGenerationEU\u0026rdquo;. \u0026nbsp;Also, Department of Research and Universities of the Generalitat de Catalunya (2021 SGR 01545). CERCA Programme / Generalitat de Catalunya and Networking Biomedical Research Center (CIBER) of Spain. CIBER is an initiative funded by the VI National R\u0026amp;D\u0026amp;i Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and the Instituto de Salud Carlos III (RD16/0006/0012), with the support of the European Regional Development Fund (ERDF).\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCampisi J. Cellular senescence as a tumor-suppressor mechanism. Trends Cell Biol 2001, 11(11): S27-31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchmitt CA, Wang B, Demaria M. Senescence and cancer - role and therapeutic opportunities. Nat Rev Clin Oncol 2022, 19(10): 619\u0026ndash;636.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDemaria M, O'Leary MN, Chang J, Shao L, Liu S, Alimirah F, \u003cem\u003eet al.\u003c/em\u003e Cellular Senescence Promotes Adverse Effects of Chemotherapy and Cancer Relapse. Cancer Discov 2017, 7(2): 165\u0026ndash;176.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang L, Pitcher LE, Yousefzadeh MJ, Niedernhofer LJ, Robbins PD, Zhu Y. Cellular senescence: a key therapeutic target in aging and diseases. J Clin Invest 2022, 132(15).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMunoz-Espin D, Serrano M. Cellular senescence: from physiology to pathology. Nat Rev Mol Cell Biol 2014, 15(7): 482\u0026ndash;496.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEwald JA, Desotelle JA, Wilding G, Jarrard DF. Therapy-induced senescence in cancer. J Natl Cancer Inst 2010, 102(20): 1536\u0026ndash;1546.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaggiorani D, Le O, Lisi V, Landais S, Moquin-Beaudry G, Lavallee VP, \u003cem\u003eet al.\u003c/em\u003e Senescence drives immunotherapy resistance by inducing an immunosuppressive tumor microenvironment. Nat Commun 2024, 15(1): 2435.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWatt AC, Cejas P, DeCristo MJ, Metzger-Filho O, Lam EYN, Qiu X, \u003cem\u003eet al.\u003c/em\u003e CDK4/6 inhibition reprograms the breast cancer enhancer landscape by stimulating AP-1 transcriptional activity. Nat Cancer 2021, 2(1): 34\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChaib S, Tchkonia T, Kirkland JL. Cellular senescence and senolytics: the path to the clinic. Nat Med 2022, 28(8): 1556\u0026ndash;1568.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGasek NS, Kuchel GA, Kirkland JL, Xu M. Strategies for Targeting Senescent Cells in Human Disease. Nat Aging 2021, 1(10): 870\u0026ndash;879.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu Y, Tchkonia T, Fuhrmann-Stroissnigg H, Dai HM, Ling YY, Stout MB, \u003cem\u003eet al.\u003c/em\u003e Identification of a novel senolytic agent, navitoclax, targeting the Bcl-2 family of anti-apoptotic factors. Aging Cell 2016, 15(3): 428\u0026ndash;435.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu Y, Tchkonia T, Pirtskhalava T, Gower AC, Ding H, Giorgadze N, \u003cem\u003eet al.\u003c/em\u003e The Achilles' heel of senescent cells: from transcriptome to senolytic drugs. Aging Cell 2015, 14(4): 644\u0026ndash;658.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChilds BG, Baker DJ, Kirkland JL, Campisi J, van Deursen JM. Senescence and apoptosis: dueling or complementary cell fates? EMBO Rep 2014, 15(11): 1139\u0026ndash;1153.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMontero J, Haq R. Adapted to Survive: Targeting Cancer Cells with BH3 Mimetics. Cancer Discov 2022, 12(5): 1217\u0026ndash;1232.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDebrincat MA, Pleines I, Lebois M, Lane RM, Holmes ML, Corbin J, \u003cem\u003eet al.\u003c/em\u003e BCL-2 is dispensable for thrombopoiesis and platelet survival. Cell Death Dis 2015, 6(4): e1721.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhan S, Zhang X, Lv D, Zhang Q, He Y, Zhang P, \u003cem\u003eet al.\u003c/em\u003e A selective BCL-X(L) PROTAC degrader achieves safe and potent antitumor activity. Nat Med 2019, 25(12): 1938\u0026ndash;1947.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh R, Letai A, Sarosiek K. Regulation of apoptosis in health and disease: the balancing act of BCL-2 family proteins. Nat Rev Mol Cell Biol 2019, 20(3): 175\u0026ndash;193.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKohli J, Ge C, Fitsiou E, Doepner M, Brandenburg SM, Faller WJ, \u003cem\u003eet al.\u003c/em\u003e Targeting anti-apoptotic pathways eliminates senescent melanocytes and leads to nevi regression. Nat Commun 2022, 13(1): 7923.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCerto M, Del Gaizo Moore V, Nishino M, Wei G, Korsmeyer S, Armstrong SA, \u003cem\u003eet al.\u003c/em\u003e Mitochondria primed by death signals determine cellular addiction to antiapoptotic BCL-2 family members. Cancer Cell 2006, 9(5): 351\u0026ndash;365.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRyan J, Letai A. BH3 profiling in whole cells by fluorimeter or FACS. Methods 2013, 61(2): 156\u0026ndash;164.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNi Chonghaile T, Sarosiek KA, Vo TT, Ryan JA, Tammareddi A, Moore Vdel G, \u003cem\u003eet al.\u003c/em\u003e Pretreatment mitochondrial priming correlates with clinical response to cytotoxic chemotherapy. Science 2011, 334(6059): 1129\u0026ndash;1133.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRyan J, Montero J, Rocco J, Letai A. iBH3: simple, fixable BH3 profiling to determine apoptotic priming in primary tissue by flow cytometry. Biol Chem 2016, 397(7): 671\u0026ndash;678.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMontero J, Sarosiek KA, DeAngelo JD, Maertens O, Ryan J, Ercan D, \u003cem\u003eet al.\u003c/em\u003e Drug-induced death signaling strategy rapidly predicts cancer response to chemotherapy. Cell 2015, 160(5): 977\u0026ndash;989.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMontero J, Gstalder C, Kim DJ, Sadowicz D, Miles W, Manos M, \u003cem\u003eet al.\u003c/em\u003e Destabilization of NOXA mRNA as a common resistance mechanism to targeted therapies. Nat Commun 2019, 10(1): 5157.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlcon C, Manzano-Munoz A, Prada E, Mora J, Soriano A, Guillen G, \u003cem\u003eet al.\u003c/em\u003e Sequential combinations of chemotherapeutic agents with BH3 mimetics to treat rhabdomyosarcoma and avoid resistance. Cell Death Dis 2020, 11(8): 634.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMontero J, Letai A. Why do BCL-2 inhibitors work and where should we use them in the clinic? Cell Death Differ 2018, 25(1): 56\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSelt F, Sigaud R, Valinciute G, Sievers P, Zaman J, Alcon C, \u003cem\u003eet al.\u003c/em\u003e BH3 mimetics targeting BCL-XL impact the senescent compartment of pilocytic astrocytoma. Neuro Oncol 2023, 25(4): 735\u0026ndash;747.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVerhaegen M, Bauer JA, Martin de la Vega C, Wang G, Wolter KG, Brenner JC, \u003cem\u003eet al.\u003c/em\u003e A novel BH3 mimetic reveals a mitogen-activated protein kinase-dependent mechanism of melanoma cell death controlled by p53 and reactive oxygen species. Cancer Res 2006, 66(23): 11348\u0026ndash;11359.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVilella R, Benitez D, Mila J, Vilalta A, Rull R, Cuellar F, \u003cem\u003eet al.\u003c/em\u003e Treatment of patients with progressive unresectable metastatic melanoma with a heterologous polyvalent melanoma whole cell vaccine. Int J Cancer 2003, 106(4): 626\u0026ndash;631.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKovatcheva M, Liu DD, Dickson MA, Klein ME, O'Connor R, Wilder FO, \u003cem\u003eet al.\u003c/em\u003e MDM2 turnover and expression of ATRX determine the choice between quiescence and senescence in response to CDK4 inhibition. Oncotarget 2015, 6(10): 8226\u0026ndash;8243.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHernandez-Segura A, Rubingh R, Demaria M. Identification of stable senescence-associated reference genes. Aging Cell 2019, 18(2): e12911.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFreedberg DE, Rigas SH, Russak J, Gai W, Kaplow M, Osman I, \u003cem\u003eet al.\u003c/em\u003e Frequent p16-independent inactivation of p14ARF in human melanoma. J Natl Cancer Inst 2008, 100(11): 784\u0026ndash;795.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSebastian D, Palacin M, Zorzano A. Mitochondrial Dynamics: Coupling Mitochondrial Fitness with Healthy Aging. Trends Mol Med 2017, 23(3): 201\u0026ndash;215.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRovira M, Sereda R, Pladevall-Morera D, Ramponi V, Marin I, Maus M, \u003cem\u003eet al.\u003c/em\u003e The lysosomal proteome of senescent cells contributes to the senescence secretome. Aging Cell 2022, 21(10): e13707.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen L, Willis SN, Wei A, Smith BJ, Fletcher JI, Hinds MG, \u003cem\u003eet al.\u003c/em\u003e Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function. Mol Cell 2005, 17(3): 393\u0026ndash;403.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaleh T, Tyutyunyk-Massey L, Gewirtz DA. Tumor Cell Escape from Therapy-Induced Senescence as a Model of Disease Recurrence after Dormancy. Cancer Res 2019, 79(6): 1044\u0026ndash;1046.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCampisi J. Aging, cellular senescence, and cancer. Annu Rev Physiol 2013, 75: 685\u0026ndash;705.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJochems F, Thijssen B, De Conti G, Jansen R, Pogacar Z, Groot K, \u003cem\u003eet al.\u003c/em\u003e The Cancer SENESCopedia: A delineation of cancer cell senescence. Cell Rep 2021, 36(4): 109441.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRobinson MD, McCarthy DJ, Smyth GK. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 2010, 26(1): 139\u0026ndash;140.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRobinson MD, Oshlack A. A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol 2010, 11(3): R25.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Description of subjects included in the analysis shown in Figure 4D and their main characteristics.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"569\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eVolunteer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eAge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003ePhototype\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eSex\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eP002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eP003\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eP005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eP006\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eP007\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eP013\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eP014\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eP015\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eP016\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eP017\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eP018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eP020\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eP021\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eP022\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-differentiation","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cdd","sideBox":"Learn more about [Cell Death \u0026 Differentiation](http://www.nature.com/cdd/)","snPcode":"41418","submissionUrl":"https://mts-cdd.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Differentiation","twitterHandle":"@cddpress","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4581617/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4581617/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSenescent 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 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 dependence in senescence is described, paving the way for molecules that either prevent HRK downregulation or displace BCL-xL binding to BAK to be used as senolytics.\u003c/p\u003e","manuscriptTitle":"HRK downregulation and augmented BCL-xL binding to BAK confer apoptotic protection to therapy-induced senescent melanoma cells.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-03 14:57:44","doi":"10.21203/rs.3.rs-4581617/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2024-07-08T13:53:31+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-07-06T19:29:19+00:00","index":1,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-07-02T20:23:30+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-06-18T19:09:26+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-06-18T11:26:11+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-06-18T10:05:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-18T04:51:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-14T11:00:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Differentiation","date":"2024-06-14T11:00:04+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-differentiation","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cdd","sideBox":"Learn more about [Cell Death \u0026 Differentiation](http://www.nature.com/cdd/)","snPcode":"41418","submissionUrl":"https://mts-cdd.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Differentiation","twitterHandle":"@cddpress","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"df3ac0a6-8ae7-42c7-aed7-aeae3db437a1","owner":[],"postedDate":"July 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":33399508,"name":"Health sciences/Diseases/Cancer"},{"id":33399509,"name":"Biological sciences/Cell biology"}],"tags":[],"updatedAt":"2024-12-04T08:07:04+00:00","versionOfRecord":{"articleIdentity":"rs-4581617","link":"https://doi.org/10.1038/s41418-024-01417-z","journal":{"identity":"cell-death-and-differentiation","isVorOnly":false,"title":"Cell Death \u0026 Differentiation"},"publishedOn":"2024-12-03 05:00:00","publishedOnDateReadable":"December 3rd, 2024"},"versionCreatedAt":"2024-07-03 14:57:44","video":"","vorDoi":"10.1038/s41418-024-01417-z","vorDoiUrl":"https://doi.org/10.1038/s41418-024-01417-z","workflowStages":[]},"version":"v1","identity":"rs-4581617","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4581617","identity":"rs-4581617","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}