Human melanoma cancer cell lines used in this study: Mel-SK23 which expresses wild-type BRAF, Mel-1102 carrying NRASQ61 K mutation, and two cell lines with BRAFV600E mutation (Mel-624.38 and Mel-526). These cell lines were originally obtained from the National Cancer Institute (NCI), NIH, Bethesda, MD United States (Topalian et al., 1989) and were generously provided by Dr. Réjean Lapointe at the Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM), Canada. All cell lines were routinely screened and confirmed to be negative for mycoplasma. Cells were cultured in RPMI medium with 8% fetal bovine serum, 100 I.U ml^-1 penicillin, 100 μg mL^-1 streptomycin, and 2 mM ml^-1 L-glutamine (all obtained from Wisent, QC, Canada).

H2B-GFP lentiviruses were produced as described previously (Fleury et al., 2019) and viral titers were adjusted to achieve ∼90% infectivity (Rodier et al., 2011). Infections were followed 48 h later by hygromycin selection (200 μg/mL for 6 days) and stable cells were used in experiments. The generation of H2B-GFP-infected cells and related methodologies can be found in our previous publication (Fleury et al., 2019).

ABT-263 (Navitoclax) was from APExBIO (Houston, TX, United States of America). A-1155463 (S7800) was from Selleckchem (Houston, TX, United States of America). Piperlongumine (1919) was from BioVision (Milpitas, CA, United States of America). ABT-199 (Venetoclax) and Dabrafenib were from Cayman Chemical (Ann Arbor, MI, United States of America). Trametinib was from Toronto Research Chemicals (North York, ON, CA). Carboplatin and paclitaxel were from Accord Healthcare (Kirkland, QC, CA). Drugs were first dissolved in 100% dimethyl sulfoxide (DMSO) and then further diluted in complete culture media for each experiment. For treatments with senolytic, we first conducted a dose-response assay to determine the half-maximal inhibitory concentration (IC50) for each senolytic in each melanoma cell line (Supplementary Figure S1A). The final concentrations used in subsequent assays were selected based on this dosage combined to established precedents (Fleury et al., 2019; Malaquin et al., 2020). The drugs were added to the cell culture 24 h after seeding, and BMi treatment was refreshed every 3 days throughout all experiments. The selected concentrations for both BMi (50 nM and 5 nM), or carboplatin (10 µM) and paclitaxel (30 nM) in combination treatment (administered over a period of 24 h), were determined based on clinically relevant doses that are reflective of plasma levels observed in treated patients (Huizing et al., 1997; Ozols et al., 2003; Puszkiel et al., 2019; Goldwirt et al., 2021). After treatment, cells were washed with PBS (twice) and provided with fresh complete culture media. Irradiation was performed using Gammacell^® 3000 irradiator Elan at a dose rate of 0.75 Gy/min for a total dose of 10 Gy followed by a fresh media change.

For live cell proliferation assessment in 96-well, 1000 cells/well were seeded for Mel-SK23, 3000 cells/well were seeded for Mel-1102, and 1500 cells/well were seeded for Mel-624.38 and Mel-526 (all expressing H2B-GFP). Cells were either irradiated (10 Gy) before seeding, or incubated with DMSO (control), or Braf and Mek inhibitors (Dabrafenib + Trametinb at 50nM+5 nM) or carboplatin and paclitaxel (10µM + 30 nM) at different times. IncuCyte^™ Live-Cell Imaging System (IncuCyte HD) was used to image cell number by phase contrast and fluorescence. Frames were captured every 8 h (×10 objective). Proliferation data were analyzed by using IncuCyte^™ S3 software based on green element count (H2B-GFP cell nuclei) or cell confluency and proliferation curves were plotted by GraphPad Prism 9.0 software (GraphPad Inc., San Diego, CA). Each experiment was performed in triplicate and repeated three times.

Propidium iodide (PI) fluorescently label dead cells based on the loss of cell membrane integrity (PI is normally a cell impermeant DNA binding dye (Belloc et al., 1994). PI (Sigma Aldrich, Saint-Louis, United States) was added to the culture media at a concentration of 0,5 μg/mL, and cells imaged as for the real-time proliferation assay. Frames were captured at 8 h intervals from two separate regions per well using ×10 objective. The IncuCyte^™ S3 software was used to quantify the percent of dead cells by scoring PI positive fluorescently labeled red nuclei against total green H2B-GFP cell nuclei. Graph were plotted using GraphPad Prism 9 (GraphPad Inc., San Diego, CA). For quantification of specific death caused by senolytic combinations we adapted a chromium-51 release assay formula (Wallace et al., 2004). We normalized the death data using two strategies, first by removing the baseline level of cell death in each condition (from the matched control), second by creating a 100% death measurement using complete cell lysis with a short Triton treatment at concentration of 0,01% in the culture media (TritonTM X-100 solution, Sigma Aldrich, Saint-Louis, United States) (Cummings and Schnellmann, 2004). To further ascertain that we measured the additional cell death caused by the senolytic treatment, prior to senolytics drugs and PI addition, melanoma cells (treated or untreated) were washed with PBS to remove floating cells. Each experimental condition was performed in triplicate and repeated at least two times. S p e c i f i c   d e a t h   p e r c e n t = E x p e r i m e n t a l   d e a t h − S p o n t a n e o u s   d e a t h M a x i m u m   d e a t h −   S p o n t a n e o u s   d e a t h X   100 Experimental death = (PI+/H2B-GFP+)/(H2B-GFP+) In each experimental condition.Spontaneous death = (PI+/H2B-GFP+)/(H2B-GFP+) In control (DMSO) condition.Maximum death = (PI+/H2B-GFP+)/(H2B-GFP+) In Triton treated condition.Each experiment was performed in triplicate and repeated at least 2 times.

Cells were seeded in a 6-well plate at a density of 500 cells (Mel-SK23), 2000 cells (Mel-1102) or 1000 cells (Mel-624.38 and Mel-526) per well. The media was removed and replaced with complete media (RPMI 8%FBS) containing Braf and Mek inhibitors alone or in combination at indicated concentrations. Cells were treated for 15 days and analyzed or released for an additional 15 days in a drug-free medium and analyzed. Cells were fixed and colored with a mix of 50% v/v methanol and 0.5% m/v of crystal violet (Sigma-Aldrich Inc., St. Louis, MO). Colonies were counted under a stereomicroscope at a ×2 magnification and reported as a percentage of control. Each experiment was performed in duplicate and repeated 3 times.

Cells were seeded in 8-wells chamber slides (Life Sciences, Corning, NY, United States) and allowed to adhere for 24 h before exposition to treatments. Cells were fixed for 5 min in formalin at room temperature (RT) and permeabilized in 0.25% Triton in phosphate-buffered saline (PBS) for 10 min. Slides were blocked for 1 h in PBS containing 1% bovine serum albumin (BSA) and 4% donkey serum. Primary antibodies (γH2AX and 53BP1) diluted (1/2500) in blocking buffer were added in each well and slides were incubated overnight at 4 °C. Cells were washed (PBS) and incubated with secondary antibodies (dilution 1/5000) for 1 h at RT, then washed again.

To detect DNA synthesis EdU (5-ethynyl-2′-deoxyuridine; 10 μM, Invitrogen) was added to the medium and incubated for 24 h from days 8–9 post-treatment. Cells were washed three times with TBS and fixed with 10% formalin for 5 min. EdU fluorescence staining was assessed using the Click-iT^® EdU Alexa Fluor^® 488 Imaging Kit (Invitrogen).

For immunofluorescence and EDU coverslips were mounted onto slides using Prolong^® Gold antifade reagent with DAPI (Life Technologies Inc.). Images were obtained using a Zeiss microscope (Zeiss AxioObserver Z1, Carl Zeiss, Jena, Germany). An automated analysis software from Zeiss (AxioVision^™, Carl Zeiss) was used to count DNA damage foci to calculate the average number of foci per nucleus. The fold change was calculated as the ratio between percentages of γH2AX or 53BP1 nuclear foci in treated versus control (nontreated) cells. DNA damage positive cells percentage was calculated relative to the DAPI staining (total nuclei count). γH2AX and 53BP1 foci were quantified in more than 50 nuclei from three different fields of each chamber. For DNA synthesis evaluation EdU positive cells were similarly counted and reported to total nuclei (DAPI).

Cells were seeded in 6-well plates and treated 24 h after seeding. At each indicated time, cells were trypsinized, washed with PBS and fixed in cold ethanol (70%) for 24 h. Cells were then washed with PBS and stained for 30 min at room temperature with a 25 μg/mL PI solution containing 100 μg/mL RNAse A. The PI fluorescence signal was detected using the Fortessa flow cytometer (BD Biosciences, Mississauga, ON) and analyzed with FlowJo™ v10.8 Software (BD Life Sciences).

We adapted the SA-β-Gal protocol used by Fleury et al. (Debacq-Chainiaux et al., 2009; Fleury et al., 2019). Briefly, cells were seeded in 6-well plates and treated 24 h after seeding. At the endpoint (day 9 post-treatment), cells were washed once with PBS, fixed with 10% formalin for 5 min, washed again with PBS, then incubated at 37 °C for 12–16 h (depending on the cell line) in a staining solution composed of 1 mg mL⁻¹ 5-bromo-4-chloro-3-inolyl-β-galactosidase in DMSO (20 mg mL⁻¹ stock), 5 mM potassium ferricyanide, 150 mM NaCl, 40 mM citric acid/sodium phosphate, and 2 mM MgCl₂, at pH 6.0. Finally, cells were washed twice with PBS and at least four representative pictures per well were taken for quantification using EVOS™ FL Digital Inverted Fluorescence Microscope from Thermo Fisher Scientific (Carlsbad, CA, United States). Each experiment was performed in duplicate (6 well plate) and repeated 3 times.

Nuclear size measurements were conducted using the ImageJ software (https://imagej.nih.gov/ij/) to quantify nuclear area based on DAPI nuclear staining. This was applied to high-resolution images acquired from the EVOS™ FL Digital Inverted Fluorescence Microscope (Thermo Fisher Scientific).

Cells were seeded in Petri dishes (100 mm) and allowed to adhere for 24 h before each treatment. At the indicated times, cells were lysed in mammalian protein extraction reagent (MPER, Thermo Fisher Scientific, Waltham, MA) containing a protease and phosphatase inhibitor cocktail (Sigma-Aldrich Inc., St. Louis, MO). After protein quantification (Pierce BCA Protein Assay Kit, Thermo Fisher Scientific), 15 μg of total protein were separated using stain-free 4%–15% gradient Tris-glycine SDS-polyacrylamide gels (Mini PROTEAN^® TGX Stain-Free^™ Gels, Bio-Rad Laboratories, CA) and transferred onto PVDF membranes (Amersham Hybond, GE Healthcare Life Sciences, Mississauga, ON, Canada). Immunodetections were performed using enhanced chemiluminescence (Thermo Fisher Scientific) to detect peroxidase-conjugated secondary antibodies bound to primary antibodies. A ChemiDoc MP Imaging System (Bio-Rad Laboratories) was used to detect chemiluminescence. The stain-free technology (Bio-Rad Laboratories) was used to quantify protein loading in the gel/membrane. Immunoreactive band intensities were quantified using ImageJ software (https://imagej.nih.gov/ij/).

Conditioned media - CM were prepared by incubating cells in 200 µL RPMI complete medium (FBS 8%) per well using 96-well treated culture microplate (Falcon ^®; #353072) for 48 h and stored at −80 °C until probed. Levels of IL-8 were assessed using ELISA (R&D Systems (IL-8 #DY208). The data were normalized to cell number and reported as concentration of secreted protein per mL per cell. We also analyzed the CM using multiplex ELISA. 40 secreted factors were screened utilizing the V-Plex human kit supplied by Meso Scale Discovery (MSD; #K15209D), in accordance with the protocol provided by the manufacturer. The data were normalized to cell number and reported as concentration of secreted protein per mL per cell or as log2 fold changes of secreted protein to appropriate control.

The following antibodies were used: phospho-histone γ-H2AX (clone JBW301, EMD Millipore, Temecula, CA) (dilution for immunofluorescence 1/2500); 53BP1 (clone 305, Novus Biologicals, Littleton) (dilution for immunofluorescence 1/2500). p53 (clone DO-1, Santa Cruz, 1/5000) ERK-1/2 and phospho-ERK-1/2, 1/2500, Cell Signaling, #4370S; phospho-Rb (Ser 807/811), Cell Signaling, 1/2500; phospho-p90-RSK, Cell Signaling, 1/2000; p21, BD, 1/2000; GAPDH, Cell Signaling, 1/2500; Tubulin-a, 1/5000, Cell Signaling, 3873P); Bcl-2, Santa Cruz, #A0807, 1/1000; Bcl-XL, Cell Signaling, #5446, 1/1000.

To assess the impact of diverse cancer therapies on melanoma cells, we used four human melanoma cell lines representing distinct clinical features (Figure 1A, left), including wild-type BRAF (Mel-SK23), NRASQ61 K mutation (Mel-1102), and BRAFV600E and TP53 mutations (Mel-624.38 and Mel-526). Lentiviral labeling with H2B-GFP enabled cell nucleus visualization, and cells were subjected to various therapies (Figure 1A, right), including Dabrafenib and Trametinib (BRAF and MEK inhibitors respectively). We monitored cell proliferation through live cell imaging (Supplementary Figures S1C–E).

Dabrafenib alone inhibited the proliferation of Braf V600E mutant melanoma cells, whereas Mel-SK23 and NRASQ61K Mel-1102 cells maintained their proliferative potential (Supplementary Figure S1C). Trametinib alone hindered the proliferation of all cell lines, with higher efficacy in Mel-624.38 and Mel-526 cells (Supplementary Figure S1D). The combination of BRAF and MEK inhibitors (BMi) halted the proliferation of Braf mutant cells (Supplementary Figures S1B, E). Alternatively, the NRASQ61 K mutated Mel-1102 cells exhibited a limited response to BRAF inhibition due to paradoxical ERK activation, a phenomenon where RAF inhibitors can induce the activation of ERK signaling in cells with upstream activating mutations such as NRAS (Supplementary Figure S1B) (Lai et al., 2022). Overall these findings confirm the genetic profile of MAP-kinase signaling in our melanoma cell line panel.

We then also exposed the panel of melanoma cells to conventional chemotherapies and radiotherapies, including a combination of carboplatin and paclitaxel (10 µM of Carboplatin +30 nM of Paclitaxel, referred to as CP) and ionizing radiation (10 Gy of X-ray, referred to as XRA). Remarkably, all melanoma cells, irrespective of their mutation status, exhibited substantial proliferation arrest after CP or XRA treatment (Figure 1B).

Cancer cells can respond to anti-cancer therapies in various ways, such as cell death, senescence, mitotic catastrophe, autophagy, or altered proliferation (Fu et al., 2021). We focused on melanoma cell fate decisions following exposure to CP, XRA, or BMi. Live cell imaging revealed that CP and XRA treatments led to nuclei enlargement within 2 days, suggesting increased ploidy and genome instability. Subsequent flow cytometry analysis identified G2/M accumulation and increased 8N polyploid cells after CP and XRA (Figure 1E). Conversely, BMi-treated cells demonstrated G1 phase accumulation and lacked these features (Supplementary Figures S1C, S2A, S1F). These results emphasize the distinct cellular responses triggered by different treatment modalities.

We then examined cell death in treated melanoma cells using real-time imaging with dynamic propidium iodide (PI) incorporation. CP and XRA induced some cell death, whereas BMi did not (Figure 1D). Notably, cell death occurred earlier in wild type p53 cells with CP and XRA treatment, while in mutated p53 cells it occurred later, indicating distinct responses based on p53 status.

Following CP and XRA treatments, a proportion of melanoma cells underwent cell death, while surviving cells exhibited a senescent phenotype. The potential role of cellular senescence in mediating tumor microenvironment responses to cancer therapies led us to investigate senescence hallmarks in these cells.

We assessed the proliferative capacity of surviving cells through a 24-h Edu pulse assay, measuring DNA synthesis (Figure 2A top). After 9 days of CP and XRA treatments, Mel-SK23 and Mel-1102 cells displayed a significant reduction in Edu-positive cells (about 20% residual positivity), while Mel-624.38 and Mel-526 cells had around 40% of cells remaining Edu-positive (Figure 2A, Supplementary Figure S2B). Furthermore, cell cycle distribution analysis revealed a G2/M phase arrest following S phase, with increased aneuploidy (8N) and genome instability in p53-mutated melanoma cells compared to wild-type p53 cells.

Additionally, SA-β-Gal staining indicated a significant increase in beta-galactosidase activity in melanoma cells 9 days after CP and XRA treatment (Figures 2B, C). Immunofluorescence assays demonstrated the presence of persistent DNA double-strand break damage foci marked by 53BP1 and phosphorylated histone H2AX in these senescent cells (Figures 2D, E).

Three out of four cell lines also exhibited elevated levels of p21 proteins in p53 dependent (Supplementary Figures S4A–D) and p53 independent manner (Supplementary Figures S4G, H). Moreover, Mel-SK23 and Mel-1102 cells exhibited increase expression of both Bcl-2 and Bcl-XL at day 9 following CP treatment, while Mel-624.38 and Mel-526 did not (Supplementary Figures S4A, B). Together these observations highlight the presence of senescent cells following CP and XRA treatments.

Accumulated DNA damage response (DDR) foci and genome instability contribute to cellular senescence induction (Ghadaouia et al., 2021). The activation of NF-kappaB, crucial for upregulation of SASP cytokines (Elliott et al., 2001; Wang et al., 2017), is another consequence of DDR foci and genome instability. Considering the importance of the SASP factor IL8 in melanoma pathogenesis (Filimon et al., 2021), we analysed IL8 levels via ELISA and observed a time-dependent increase in secretion, except in the Mel-1102 cell line (NRASQ61 K). In general CP and XRA-treated cells exhibited four to over 10 times higher IL8 secretion compared to untreated cells (Figure 2F).

To take a broader look at the SASP secretome, we confirmed IL8 using a different multiplex ELISA analysis, which also included 38 additional secreted factors. Notably, this analysis revealed a significant elevation in the levels of several key cytokines and chemokines, including, but not limited to, IL-10, IFN-γ, IL-6, IP-10, MCP-1, VEGF and sICAM-1 in CP-treated melanoma cells (Supplementary Figures S2C–G). Together these results indicate that melanoma cells display a classical senescence phenotype in response to DNA damage-inducing treatments.

To assess senescence hallmarks following combined Braf and Mek inhibition, we continuously exposed melanoma cells to BMi for 9 days. In a 24-h EdU pulse experiment (Figure 3A top), we observed that wild-type Braf melanoma cells (Mel-SK23 and Mel-1102) had a relatively high frequency of EdU-positive cells (19% and 50% respectively), while Braf V600E mutated melanoma cells showed almost no EdU incorporation (Figure 3A bottom). This suggests the inhibitors induced a more robust proliferation arrest in Braf-mutated cell lines. Importantly, we observed an increase in SA-β-Gal activity in Braf V600E mutated cells 9 days after BMi treatment, but not in wild-type Braf cells (Figures 3B, C).

Unlike cells treated with CP and XRA, those subjected to BMi did not show an increase in DDR foci (Figures 3D, E) or IL8 secretion (Figure 3F). In fact, when probing the SASP from a broader point of view using multiplex ELISA, BMi-treated cells exhibited a reduction in different SASP factors compare to untreated cells (Figure 3F; S2.C - G). Alternatively, in general, BMi-treated cells displayed increased levels of Bcl-2 or Bcl-XL anti-apoptotic proteins, consistent with senescence-associated apoptotic resistance (Supplementary Figures S4A, B). In summary these observations suggest that long-term Braf and Mek inhibition induces a senescent-like state in Braf V600E mutated melanoma cells, which is different from that induced by genotoxic stresses.

While senescence in normal cells is defined by a stable proliferation arrest, recent research in cancer cells, including Olaparib-induced senescence in high-grade serous ovarian cancer or androgen depletion-induced senescence in prostate cancer, has demonstrated the potential reversibility of a cancer senescence-like state (Fleury et al., 2019; Malaquin et al., 2020). Thus, we explored whether the senescence-like state induced by BMi is autonomously stable or require sustained inhibition of proliferation signalling. Through real-time imaging, we performed BMi treatment and release assays and observed that at least a fraction of wild-type Braf cells recovered from a BMi treatment that lasted nine or 15 days (Figure 4A). In contrast, while Braf V600E mutated cells also recovered, they experienced a significant delay compared to wild-type cells, suggesting that perhaps less cells recovered (Figure 4B).

To ascertain if recovery was widespread or limited to a subset of cells, we conducted a colony formation assay with 15 days of BMi treatment followed by a 15-day drug-free period (Figure 4C). Most wild-type Braf melanoma cells regained their proliferative capacity post-drug release (Figures 4D, E). In contrast, only approximately 30% of Braf-mutated cells regained their proliferative capacity, forming small colonies mainly consisting of 16 cells or less (Figure 4F). The cells that retain the ability to recover and proliferate may represent previously described persister cells, which can adapt to treatment (Marine et al., 2020; Chauvistre et al., 2022). Alternatively, the cell population that remain incapable of proliferation after drug release appears senescent-like, possibly via epigenetic modifications induced by prolonged Braf and Mek inhibition, which converted the majority of Braf V600E melanoma cells into a stable proliferation arrest state resembling senescence (Crouch et al., 2022). Overall, these observations support a mixed cell fate decision model in melanoma cells harboring the Braf V600E mutation in response to long-term Braf and Mek inhibition, characterized by both senescent-like cells that cannot recover after drug withdrawal (cannot form colonies), and cells that exhibit a persister phenotype (cells that maintain the ability to recover proliferation after treatment withdrawal).

Senescent cells often display senescence-associated apoptosis resistance targetable using senolytics, which encompass molecules/drugs that selectively kill senescent cells without harming non-senescent cells (Chang et al., 2016; Zhu et al., 2016; Hickson et al., 2019). Sensitivity to senolytics, particularly Bcl2-family inhibitors, varies based on cancer type and senescence inducer (Fleury et al., 2019; Malaquin et al., 2020; Lafontaine et al., 2021). Considering the distinct melanoma cell fate phenotypes induced by genotoxic stresses or BMi, we evaluated the levels of the anti-apoptotic proteins Bcl-2 or Bcl-XL. In general, both proteins were detected even in untreated cells, and at least one of these proteins was upregulated in senescent-ike BMi-induced cells consistent with the potential induction of Bcl2-family apoptotic resistance in this context. However, this was not the case during CP-induced senescence, where only p53 wild-type mel-SK23 and mel-1102 showed increased levels (Supplementary Figures S4A, B).

To directly test senolytic sensitivity, we first determined the IC50 of the tested senolytic drugs for each cell line (Supplementary Figure S1A) and employed a real-time death assay to test cell sensitivity to senolytic drugs (at a lower dose than the IC50), including: ABT-263, Bcl2 and Bcl-XL inhibitor (Wendt, 2008), A-115 (Bcl-XL inhibitor) (Tao et al., 2014), ABT-199 (Bcl2 inhibitor) (Chang et al., 2016), and Piperlongumine (PPL, a natural antioxidant product) (Wang et al., 2016). CP and XRA-induced senescent melanoma cells demonstrated significant sensitivity to these senolytics, particularly ABT-263, A-115, and ABT-199, which were more effective than PPL in inducing cell death (Figure 5. B left, C, D left, E). This suggests that both Bcl-XL and Bcl2 play critical roles in the survival of senescent melanoma cells induced by CP or XRA, regardless of whether they are induced or not during senescence (Supplementary Figures S4A, B).

To confirm the specificity of the senolytic effect, we repeated the assay 1 day after CP or XRA treatment (Figures 5B, D, right). Early after treatment, three out of four cell lines were no longer or less sensitive to these drugs, indicating that the initial sensitivity of CP or XRA-treated cells primarily arises from their senescence state, not a direct synergy with DNA damage or CP. As an exception, Mel-SK23 cells were strongly sensitive to Bcl2/Bcl-XL inhibitors immediately after CP exposure, suggesting that these cells are sensitive to Bcl2 inhibitors-induced apoptosis when exposed to DNA damage, even before senescence. Overall, these findings suggest that Bcl2/Bcl-XL inhibitors can induce cell death in DNA damage-induced senescent melanoma cells.

We then investigated the sensitivity of BMi-induced phenotypes to senolytics. Given the reported direct synthetic lethality between Mek and Bcl2/Bcl-XL inhibitors (Cragg et al., 2008; Corcoran et al., 2013; Iavarone et al., 2019) and the mixed nature of BMi-induced phenotypes (Figure 4B), we used a sequential treatment approach with or without BMi (Figure 6A).

Unlike CP and XRA-induced senescent cells, BMi-induced senescent-like and persister cells showed no or less sensitivity to Bcl2/Bcl-XL inhibitors or PPL when treated after induction of senescence and removal of BMi (Figure 6B left). However, simultaneous treatment with senolytics and BMi led to significant inscrease in cell death, especially with Bcl2/Bcl-XL inhibitors (Figure 6B right). This indicates that a direct synergy between Bcl2/Bcl-XL inhibitors and BMi, rather than with BMi-induced senescence phenotypes, triggers melanoma cell death (Airiau et al., 2016).

To confirm direct BMi-Bcl2 synergy, we conducted another cell death assay 1 day after BMi treatment (Figure 6C). We added senolytics with or without BMi. The simultaneous combination of Bcl2/Bcl-XL inhibitors and BMi induced melanoma cell death (Figure 6C right), unlike Bcl2/Bcl-XL inhibitors alone (Figure 6C left). These results show that the BMi-induced senescence-like or persister state does not sensitize melanoma cells to Bcl2/Bcl-XL inhibitors and PPL. However, as previously reported, a direct cooperation between Bcl2/Bcl-XL inhibitors and BMi used simultaneously provoke both non-senescent and senescent melanoma cell death.