Chemical, physical and biological factors triggering Bcl-xL-mediated apoptosis

We demonstrated recently that Bcl-xL-specific inhibitors prematurely killed virus-infected or RNA/DNA-transfected cells. Here we showed that Bcl-xL-specific agent A-1155463 prematurely killed human non-malignant as well as malignant cells and the small roundworm C. elegans when combined with DNA-damaging agent 4-nitroquinoline-1-oxide (4NQO). The synergistic effect of 4NQO-A-1155463 combination was p53-dependent, was associated with the release of Bad and Bax from Bcl-xL and with mitochondrial outer membrane permeabilization (MOMP), indicating that Bcl-xL linked DNA damage response, p53 signalling and apoptotic pathways. In addition, combinations of Bcl-xL specific inhibitors with several anticancer agents, immunosuppressant drug, antiviral drug, DNA binding probes or UV radiation also killed cells. Thus, we identified biological, chemical and physical factors triggering Bcl-xL-mediated apoptosis.


INTRODUCTION
Apoptosis is a tightly regulated process that kills cells with damaged or pathogenic DNA, RNA, and proteins (Elmore, 2007; Shim, Kim, Tenson, Min & Kainov, 2017). When apoptosis is inhibited, cells that should be eliminated may persist and become malignant (Wong, 2011). B cell lymphoma 2 (Bcl-2) family of proteins are key players in apoptosis (Kale, Osterlund & Andrews, 2018). For example, Bcl-xL, Bcl-2 and Mcl-1 are anti-apoptotic, whereas Bax, Bak and Bad are pro-apoptotic members of the family (Denisova et al., 2012;Fernandez et al., 2018;. Interaction between pro-and anti-apoptotic proteins determine the fate of a cell. In particular, alteration of the interactions could lead to release of Bax and Bak which could form a pore in the mitochondrial outer membrane that allowed cytochrome C to escape into the cytoplasm and activate the caspase cascade (Shamas-Din, Kale, Leber & Andrews, 2013).
Several chemical inhibitors were developed to bind anti-apoptotic of Bcl-2 proteins and induce cancer cells death including recently developed Bcl-PROTACs (Adams, Clark-Garvey, Porcu & Eischen, 2018) (Khan, Zhang et al. 2019, Wang, He et al. 2019, He, Zhang et al. 2020). These small molecules belong to several structurally distinct classes (Fig. 1a). First class of Bcl2i includes ABT-737 and its derivatives  and ; second class includes WEHI-539 and its derivatives, A-1331852 and A-1155463. There are other classes of Bcl-2 inhibitors containing structurally similar molecules, such as S63845 and S64315, S55746 and A1210477.
To obtain additive or synergistic effects, enhance efficacy of treatment and combat genetically heterogeneous cancers, Bcl-2 inhibitors were combined with other anticancer drugs ( Fig. 1c; bcl2icombi.info) (Chen et al., 2011;Haikala et al., 2019;Jeong, Oh, Jeong, Lee, Lee & Ahn, 2019;Shen et al., 2018). The drug combinations were also used to lower the dose of Bcl-2 inhibitors to overcome resistance and toxicity issues for non-malignant cells (Adams, Clark-Garvey, Porcu & Eischen, 2018). Dozens of the drug combinations have been reported to be active in vitro (cell culture, patient-derived cells or organoids) and in vivo (patient-derived xenograft mouse models). In addition, 109 combinations (excluding combinations with biological agents) were in clinical trials. However, only  in combination with azacytidine, decitabine or cytarabine was approved for the treatment of acute myeloid leukaemia (AML). Clinical trials with 31 combinations have been terminated, withdrawn or suspended due to adverse effects or other issues. These include trials with ABT-263 plus bendamustine and rituximab in patients with relapsed diffuse large B cell lymphoma, ABT-263 plus abiraterone acetate with or without hydroxychloroquine in patients with progressive metastatic castrate refractory prostate cancer, as well as ifosfamide,carboplatin,etoposide), or plus rifampin, or plus bortezomib and dexamethasone, ixazomib and carfilzomib (NCT02471391, NCT01423539, NCT03064867, NCT01969682, NCT02755597, NCT03314181, NCT03701321). The identification of the adverse effects prior to clinical trials is challenging, but could save lives for many cancer patients.
We noticed that many Bcl-2 inhibitors have been combined with compounds, which damaged cellular DNA, RNA or proteins by targeting DNA replication, RNA transcription and decay, as well as protein signalling and degradation pathways. Here, we shed new light on the mechanisms of actions of such combinations and identified chemical, physical and biological factors which trigger Bcl-xLmediated apoptosis. First, we showed that combination of DNA-damaging agent 4-nitroquinoline-1oxide (4NQO) together with Bcl-xL-specific inhibitor A-1155463, but not drugs alone, killed human malignant and non-malignant cells, as well as for C. elegans. The synergistic effect of the drug combination was p53 dependent. Moreover, it was associated with the release of Bad and Bax from Bcl-xL. Second, we demonstrated that several anticancer drugs (i.e. amsacrine, SN38, cisplatin, mitoxantrone, dactinomycin. dinaciclib, UCN-01, bortezamib, and S63845), as well as birth-control 17α-ethynylestradiol, immunosuppressant cyclosporin, antiviral brincidofovir, DNA binding MB2Py(Ac), DB2Py(4) and DBPy(5), as well as UV radiation triggered Bcl-xl-mediated apoptosis in non-malignant cells. Finally, we discussed the application of our findings for the development of novel options for treatment of cancer and other diseases.

Bcl2iCombi.info database
We reviewed developmental status of Bcl-2 inhibitors and their combinations with other anticancer therapeutics. We summarized the information in freely accessible database (bcl2icombi.info). The drug annotations were obtained from PubChem, DrugBank, DrugCentral, PubMed and clinicaltrials.gov databases (Kim et al., 2019;Ursu et al., 2019;Wishart et al., 2018). The database summarizes activities and developmental stages of the drug combinations and allows their interactive exploration. A feedback form is available on the website. The database will be updated upon request or as soon as a new drug combination is reported.  (7), monomeric MB2Py(Ac), dimeric DB2Py(4) and DB2Py(5) benzimidazole-pyrroles were synthesized as described previously (Koval et al., 2018) (Ivanov, Susova, Salyanov, Kirsanov & Zhuze, 2013). A library of 527 approved and emerging investigational oncology drugs were from the collection of the Institute of Molecular Medicine Finland, FIMM (www.fimm.fi/en/services/technology-centre/htb/equipment-and-libraries/chemical-libraries). A library of 48 drugs commonly dispensed in Norway was assembled based on Norwegian Prescription Database (www.norpd.no). Table S1 lists these compounds, their suppliers and catalogue numbers. A library of 50 safe-in-man broad-spectrum antivirals was published previously (Ianevski et al., 2018).
To obtain 10 mM stock solutions, compounds were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, Steinheim, Germany) or milli-Q water. The solutions were stored at −80 °C until use.

C. elegans maintenance, lifespan and toxicity assays
Standard C. elegans strain maintenance procedures were followed in all experiments (Brenner, 1974;Fang et al., 2019;Fang et al., 2016;Fang et al., 2014). Nematode rearing temperature was kept at 20 °C, unless noted otherwise. N2: wild type Bristol isolate was from Caenorhabditis Genetics Center (CGC).
For lifespan experiments, gravid adult worms were placed on NGM plates containing either A-1155463 (10 µM), 4NQO (10 µM), combination A-1155463 (10 µM) + 4NQO (10 µM) or vehicle control and seeded with OP50 to lay eggs. Progeny were grown at 20°C through the L4 larval stage and then transferred to fresh plates in groups of 30-35 worms per plate for a total of 100 individuals per experimental condition. Animals were transferred to fresh plates every 2-4 days thereafter and examined every other day for touch-provoked movement and pharyngeal pumping, until death.
Worms that died owing to internally hatched eggs, an extruded gonad or desiccation due to crawling on the edge of the plates were censored and incorporated as such into the data set. Survival curves were created using the product-limit method of Kaplan and Meier. The log-rank (Mantel-Cox) test was used for statistical analysis.
A series of toxicity experiments, including fecundity, egg hatching, larval development, were conducted using N2 Bristol isolate C. elegans, which were cultivated as previously described (Brenner, 1974) and maintained at 20°C. Briefly, for the toxicity assay animals were initially synchronised by bleaching gravid adults (adult day 1-4) to extract the eggs. Eggs were placed on nematode growth medium (NGM) plates seeded with Escherichia coli (OP50). L4 larvae were subsequently transferred onto fresh OP50-seeded NGM plates and allowed to grow to adulthood. Ten adult day 1 worms (n = 30-50/experimental condition) were transferred onto assay NGM plates with OP50 containing either A-1155463 (1, 10, 100 µM), 4NQO (1, 10, 100 µM), combination A-1155463 (10 µM) plus 4NQO (2, 10, 20 µM) or vehicle control. Animals laid eggs for three hours. Subsequently, adults were removed from the plate and the frequency of eggs laid was quantified as a measure of reproduction and egglaying capacity of worms. The following day the frequency of unhatched eggs and L1 larvae were counted in order to evaluate the efficiency of egg hatching. Subsequently, 36 h later, development to L4 larvae was assessed, as a measure of larval growth. Finally, growth of L4 larvae to adulthood was quantified after 16 h.
The toxicity assay was conducted at 20°C on 10 ml NGM plates seeded with 100 µl OP50 from an overnight culture. Drug compounds and vehicle solvents were dissolved in a total volume of 200 µl, sufficient to cover the entire surface of the plate, and were dried at room temperature for 1-2 h prior to the transfer of worms. Each chemical concentration was tested three-five times. Statistical analysis was conducted using One-Way ANOVA followed by Tukey's multiple comparison test.

Real-time impedance assay
RPE cells were grown at 37°C in 16-well E-Plates to 90% confluency. The plates were supplied with golden electrodes at the bottom of the wells and a weak electrical current was constantly applied to the cell medium. The changes in the cell adherence indexes (CI) were monitored by the xCELLigence real-time drug cytotoxicity system (ACEA Biosciences, San Diego, USA) as described previously (Solly, Wang, Xu, Strulovici & Zheng, 2004). When cells reached 90% confluency, 1 µM 4NQO, 1 µM A-1155463 or their combination were added. Control cells were treated with 0.01% of DMSO. CI were normalized and monitored for another 24 hours.

Cell viability assay
Approximately, 4x10 4 RPE or HCT116 cells per well of 96-well plate were treated with A-1155463, 4NQO or both compounds. Control cells were treated with 0.01% of DMSO. After 24 or 48 h, respectively, the viability of cells was measured using the Cell Titer Glo assay (CTG, Promega).
Luminescence was measured using PerkinElmer Victor X3 or Synergy Mx plate readers.

Cell toxicity assay
Approximately, 4x10 4 HCT116 cells per well of 96-well plate were treated with 1 µM 4NQO, 1 µM A-1155463 or their combination. Control cells were treated with 0.01% of DMSO. After 24 h, the death of cells was detected using the Cell Toxicity Assay (CTxG, Promega). Fluorescence was measured using PerkinElmer Victor X3 Reader.
Activation of apoptosis was assessed using RealTime-Glo Annexin V Apoptosis and Necrosis Assay (Promega). Luminescence was measured using PerkinElmer Victor X3 Reader.

Apoptosis and phospho-kinase arrays
Approximately, 1x10 6 RPE cells per well of 6-well plate were treated with 1 µM 4NQO, 1 µM A-1155463 or their combination. Control cells were treated with 0.01% of DMSO. After 2 h, relative levels of apoptosis-related proteins and protein kinase phosphorylation were determined using proteome profiler human apoptosis and human phospho-kinase array kits, respectively, as described in the manuals (R&D Systems, Minneapolis, MN, USA). Membranes were scanned using Odyssey Li-Cor system.

Metabolic labelling of cellular RNA and proteins
Approximately, 1x10 5 RPE cells per well of 12-well plate were treated with 1 µM 4NQO, 1 µM A-1155463 or their combination dissolved in 500 l cell growth medium. Control cells were treated with 0.01% of DMSO. The medium was supplemented with 3 l of [alpha-P32]UTP (9,25 MBq, 250 Ci in 25 l). Cells were incubated for 2 h at 37 °C. Cells were washed twice with PBS. Total RNA was isolated using RNeasy Plus extraction kit (Qiagen, Hilden, Germany). RNA was separated on 1% agarose gel. Gel was dried. Total RNA was detected using ethidium bromide. 32 P-labeled RNA was monitored using autoradiography and visualised using Typhoon 9400 scanner (GE Healthcare).
In a parallel experiment, the compounds or DMSO were added to 500 μl cysteineand methioninefree DMEM medium (Sigma-Aldrich, Germany) containing 10% BSA and 3 µl [ 35 S] EasyTag Express protein labelling mix (7 mCi, 259 MBq, 1175 Ci/mmol in 632 ml; Perkin Elmer, Espoo, Finland). After 2 h of incubation at 37 °C cells were washed twice with PBS, lysed in 2 × SDS-loading buffer and sonicated. Lysates were loaded and proteins were separated on a 10% SDS-polyacrylamide gel. 35 Slabelled proteins were monitored using autoradiography and visualised using a Typhoon 9400 scanner (GE Healthcare).

Synergy calculations
RPE cells were treated with increasing concentrations of a Bcl-2 inhibitor and other drug. After 24 h cell viability was measured using CTG assay. To test whether the drug combinations act synergistically, the observed responses were compared with expected combination responses. The expected responses were calculated based on Bliss reference model using SynergyFinder webapplication (Ianevski, He, Aittokallio & Tang, 2017). For in vitro combinatorial experiments where the whole dose-response matrix was measured, a normalized Bliss reference model, i.e. Zero Interaction Potency (ZIP) model was utilized. Unpaired t-test p-value was calculated using stats R package (v.3.6.3), to test the null hypothesis that no difference exists between synergy score mean values of two groups.

Fractionation of RPE cells
RPE cells were remained untreated or treated with 1 μM A-1155463, 1 μM 4NQO or their combination for 2 h. The cells were washed with PBS and collected in the cell lysis buffer consisting of 10 mM HEPES, pH 7.5, 10 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 0.5% Nonidet-40 and protease inhibitor cocktail. The cells were placed on ice for 20 min with vortexed mix every 5 min, and then centrifuged at 12,000 g at 4°C for 10 min. The supernatant was collected as cytoplasmic fraction.
Remaining cell pellets were washed three times with the cell lysis buffer and resuspended in the nuclear buffer consisting of 20 mM HEPES, pH 7.5, 400 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, protease inhibitor cocktail. The resuspended nuclear fraction was placed in ice for 30 min and centrifuged at 12,000 g at 4°C for 15 min. The supernatant was collected as nuclear extracts. Protein concentration of all cell fractions was determined using Bradford's reagent (BioRad, USA).

Analysis of mitochondrial membrane potential
RPE cells were remained untreated or treated with 1 μM A-1155463, 1 μM 4NQO or their combination for 2 h. The mitochondrial potential was determined using JC-1 dye staining according to the manufacturer's instruction (ThermoFisher Scientific). The cells were washed once with PBS and stained with 10 ug/ml of JC-1 dye in culture medium for 10 min. The images of JC-1 staining were captured using EVOS fluroscence microscope and the mitochondrial potential was indicated by the shift of the fluorescence intensity ratio of J-aggregate (535/590 nm) and monomers (485/530 nm).
Normal rabbit IgG (Santa Cruz Biotechnology, sc-2025) was used to control immunoprecipitation with an equal volume of cell lysate. The immobilized protein complexes together with the appropriate whole-cell extract inputs were subjected for SDS-PAGE followed by immunoblotting.
HCT116 TP53 +/+ and TP53 −/− cells remained untreated or were treated with 1 μM A-1155463, 1 μM 4NQO or both compounds. After 16 h, cells were lysed, and proteins were separated using 4-20% gradient SDS-PAGE. The proteins were transferred to a Hybond-LFP PVDF membrane. The membranes were blocked using 5% milk in Tris-buffered saline (TBS). p53 and GAPDH were detected using mouse anti-p53 and mouse anti-GAPDH antibodies as described above.

Toxicity of A-1155463-4NQO combination in C. elegans
We demonstrated recently that A-1155463 prematurely killed virus-infected or RNA/DNA-transfected cells via Bcl-xL-mediated apoptosis (Bulanova et al., 2017;Kakkola et al., 2013;Shim, Kim, Tenson, Min & Kainov, 2017). We hypothesised that not only invasive RNA or DNA (biological factors) but also DNA/RNA-damaging chemical or physical factors could trigger apoptosis in A-1155463-sencitized cells. We tested DNA-damaging agent 4NQO (Walker & Sridhar, 1975) plus Bcl-xL-specific inhibitor A-1155463 in C. elegans. The worms treated with A-1155463-4NQO combination died faster than those treated with either A-1155463 or 4NQO (Fig. 2a, b). Moreover, the worms treated with the drug combination exhibited defects in reproduction and development (Fig. 2c). The Bliss synergy scores for adult development, L4 development and egg hatching were 19, 21 and 24 respectively. Treatment with A-1155463 or 4NQO alone did not affect these stages. Thus, combination of 4NQO with A-1155463 had a severe impact on the C. elegans lifespan, reproductive system and development.

To recapitulate the in vivo toxicity of A-1155463-4NQO combination in in vitro experiments, we used human non-malignant retinal pigment epithelium (RPE) cells. PRE cells died 3 h after treatment with A-1155463-4NQO combination, whereas majority of cells treated with either A-1155463 or 4NQO
remained viable for 24 h as measured by real-time impedance assay (Fig. 3a). Another cell viability assay, which quantified intracellular ATP, demonstrated that the effect of A-1155463-4NQO combination was synergistic (ZIP synergy score, 14±3; Fig. 3b). Similar results were obtained with 4NQO in combination with another Bcl-xL-specific inhibitor, A-133852 (ZIP synergy score, 17±0; Fig.   3c). ABT-263, a pan-Bcl-2 inhibitor, also showed synergy with 4NQO (ZIP synergy score, 8±1; Fig.   3c). In contrast, combinations of 4NQO with the Bcl-2-or Mcl-1-specific inhibitors did not show a synergy (ZIP synergy scores, 3±1 and 2±1, respectively; Fig. 2c). These results suggested that the DNA-damaging agent combined with Bcl-xL-, but not Bcl-2-or Mcl1-specific inhibitors facilitated the death of human non-malignant cells.
We also tested 4NQO-A-1155463 and 4NQO-A-1331852 combinations in 3 cancer cell lines (A549, H460 and Caco-2; Fig. 3d). In addition, we tested 4NQO-A-1155463 combination in a panel of patient-derived primary cell cultures (Fig. 3e). The combinations showed synergy in all tested cells, indicating that the DNA-damaging agent combined with Bcl-xL-specific inhibitor facilitated the death of human non-malignant as well as malignant cells.

4NQO triggered overexpression of p53, A-1155463 mediated release of Bad and Bax from Bcl-xL, and concerted action of 4NQO and A-1155463 led to mitochondrial outer membrane permeabilization
Immunoblot analysis of whole-cell extracts, nuclear and cytoplasm fractions showed that p53, a key regulator of DNA-damage response and Bcl-xL-dependent apoptosis, was over-expressed and accumulated in the nucleus of RPE cells after 2 h in response to 4NQO treatment (both as single agent and in combination; Fig. 4a, b). Confocal microscopy confirmed this observation (Fig. 4c).

Immuno-precipitation of Bcl-xL-interacting partners showed that A-1155463 displaced Bad and Bax
from Bcl-xL in RPE cells after 2 h of treatment (Fig. 4a). This is in agreement with previous observations that upon treatment with a Bcl-xL inhibitor, BAX localized to the outer mitochondrial membrane and dimerize with BAK to form the pores to induce mitochondrial outer membrane depolarization (MOMP) (Kakkola et al., 2013;Lai, Chi & Chen, 2007).
We monitored MOMP with the membrane-permeant JC-1 dye. JC-1 dye exhibits potentialdependent accumulation in apoptotic mitochondria, indicated by a fluorescence emission shift from green to red. Fig. 4d shows that treatment of RPE cells with 4NQO-A-1155463 combination for 4 h decreased the red/green fluorescence intensity ratio compared to non-, 4NQO-or A-1155463-treated cells. This result indicated that concerted action of DNA-damaging agent and Bcl-xL inhibitor led to MOMP.
Of note, treatment of RPE cells for 2 h with 4NQO, A-1155463 or their combination did not substantially affect general translation as well as expression levels of several apoptotic proteins (Fig.   S1b,c). By contrast, RNA synthesis and protein phosphorylation (EGFR, ERK1/2, PYK2 and c-Jun) were affected by the combination (Fig. S1a,d). These effects could represent the immediate early responses to apoptosis.

Synergistic toxicity of Bcl-xL-specific inhibitors combined with other anticancer agents
We next tested combinations of A-1155463, A-133852, ABT-199, and S63845 with 39 DNA, RNA and protein damaging agents in RPE cells. The experiment revealed that A-133852 and A-1155463 in combination with these agents were highly synergistic, indicating that Bcl-xL was essential for induction of apoptosis in non-malignant cells under chemical insults (Fig. 6a, b). Importantly, combinations with high or low synergy scores ( from 7.5 to 47.7) induced expression of p53 after 2 h of treatment (Fig. 6c). Interestingly, combinations of S63845 with Bcl-xL, but not Bcl-2 inhibitors were highly synergistic (cytotoxic) indicating that S63845 could damage Mcl-1 or another potential cellular target (Fig. 6d).
Given that DB2Py(4) and DB2Py(5) differed from DBA(n) molecules by the pyrrole fragment and the N-methylpiperazine end group (instead of dimethylaminopropylamide; Fig. S2), these differences appeared to be essential for initiation of apoptosis in A-1155463-sensitized RPE cells. Moreover, dimerization of B2Py(n) was not essential for Bcl-xL-mediated apoptosis, since both MB2Py(Ac) and DB2Py(n) had similar synergy scores. Most probably, MB2Py(Ac) bound minor groove of cellular DNA and altered pol II transcription, which led to accumulation of aberrant RNA transcripts, that triggered apoptosis in A-1155463-sensitized RPE cells. Thus, the MB2Py(Ac), DB2Py(4) and DB2Py (5) possessed DNA-binding activity that could interfere with general transcription.
We further exploited Bcl-xL-mediated apoptosis for identification of chemical agents that damage cellular DNA, RNA or proteins. We tested library consisting of 48 drugs commonly dispensed in Norway as well as random library consisting of 50 safe-in-man broad-spectrum antiviral agents. We found that 17α-ethynylestradiol, bortezomib, cyclosporin and brincidofovir primed A-1155463sensitized RPE cells for apoptosis (Bliss synergy score >6). These results indicated that 17αethynylestradiol, cyclosporin and brincidofovir could damage cellular DNA, RNA or proteins and subsequently could induce apoptosis (Fig. 7b,c). Moreover, we confirmed that bortezomib impaired protein degradation, which could be associated with accumulation of damaged proteins, which triggered Bcl-xL-mediated apoptosis (Kim, Song, Zhang, Bartlett & Lee, 2014).

Physical factors that trigger Bcl-xL-mediated apoptosis
We next exposed A-1155463-sensitized and non-sensitized RPE cells to UVB and UVC radiation, which similarly to the 4NQO treatment, induced lesions in DNA (Walker & Sridhar, 1975). After 24 h the cell viability was measured using the CTG assay. We observed that already 8 sec of UVB or UVC exposure killed A-1155463-sensitized but not non-sensitized RPE cells (Bliss synergy scores: 31.2 and 23.8, respectively; Fig. 8a,b). Similar effect was observed in UVC-exposed A-1155463-sensitized cancer A549, H460 and Caco-2 cell lines (Fig. 8c). This indicated that UV triggered Bcl-xL-mediated apoptosis in non-malignant and malignant human cells.

DISCUSSION AND CONCLUSIONS
We showed recently that non-malignant cells infected with viruses or transfected with viral RNA or plasmid DNA were sensitive to Bcl-xL-specific inhibitor A-1155463 (Bulanova et al., 2017;Kakkola et al., 2013;Shim, Kim, Tenson, Min & Kainov, 2017). This indicated that invasive RNA or DNA, i.e. biological factors, could trigger apoptosis, and that Bcl-xL could serve as 'safety fuse' controlling the process (Fig. 9).
In addition, we demonstrated that DNA-damaging agent 4NQO killed human non-malignant and malignant cells, as well as small roundworm C. elegans, which were sensitized with A-1155463. The cell death was dependent on the concentration of both agents. The synergistic effect of 4NQO-A-1155463 combination was p53 dependent and was associated with the release of Bad and Bax from Bcl-xL. Our results suggest that intracellular receptors sensed damaged DNA, transmitted this information via p53 to anti-apoptotic Bcl-xL, which released pro-apoptotic Bax and Bad to trigger MOMP which led to cell death. Thus, when concentration of damages in the cellular factors reached critical levels, Bcl-xL triggered apoptosis (Fig. 9). The p53-dependent Bcl-xL-mediated apoptotic pathway is evolutionary conserved (from C.elegans to H. sapiens).
Importantly, systemic Bcl-xL-mediated apoptosis could be lethal for patients, whereas locally induced Bcl-xL-mediated apoptosis could have a clinical potential. For example, skin cancers could be treated with local Bcl-xL-specific inhibitor plus topical UV radiation, oncolytic virus infection or anticancer medication. Recent studies support this idea by showing, for example, that combination of radiotherapy with pan-Bcl-2 inhibitors ABT-737 and ABT-263 killed breast and small cell lung cancer cells in vitro (Table S3) (Li, Wang, Deng, Han, Hong & Lin, 2019;Wu et al., 2014). Moreover, ABT-737 sensitized chronic lymphocytic leukaemia cells to reovirus and vesicular stomatitis oncolysis (Samuel et al., 2013). In this respect, antibody-Bcl-xL inhibitor conjugates or Bcl-xL PROTACs can be used to achieve tissue selectivity and targeted delivery of the compounds (Khan, Zhang et al. 2019, Wang, He et al. 2019, He, Zhang et al. 2020).
In conclusion, we identified chemical, physical and biological factors that induced Bcl-xL-mediated apoptosis. This could provide novel opportunities for discovery of therapeutic and adverse effects of anticancer medications as well as to identify conditions to prevent aging, and treat non-and communicable diseases, given that cells with high concentration of damaged DNA, RNA or proteins or with invasive DNA and RNA molecules would be eliminated from an organism receiving a Bcl-xLspecific inhibitor.

DATA AVAILABILITY
Supplementary Data are available online.   antibodies. Nuclei were stained with DAPI. Cells were imaged using a confocal microscopy.
The mitochondrial potential was determined using JC-1 dye staining. The images of JC-1 staining were captured using fluorescence microscope and the mitochondrial potential was indicated by the shift of the fluorescence intensity ratio of J-aggregate (535/590 nm) and monomers (485/530 nm).

Figure 8.
Identification of physical factors, which trigger Bcl-xL-mediated apoptosis. (a,b) RPE cells were exposed to UVB or UVC radiation for the indicated times and covered with medium containing 1 μM A-1155463 or 0.1% DMSO. After 24 h, viability of cells was measured using the CTG assay.
Mean ± SD, n = 3. (c) Synergy scores of A-1155463-UVC combinations in 4 cell lines. Cells were treated with increasing A-1155463 concentrations and exposed UVC radiation for 0, 20, 40, 80, 160, and 240 sec. After 24 h cell viability was measured using the CTG assay. Synergy scores were quantified using Bliss model. Alternatively, DR proteins and PRRs could mediate repair or degradation of damaged or invasive molecules, respectively, which would leave cells alive. Bcl-xL-specific inhibitors facilitate death of cells and, thus, impair repair or degradation of damaged or invasive factors.