Abstract
Senolytics, drugs that kill senescent cells, have been proposed to improve the response to pro-senescence cancer therapies; however, this remains challenging due to a lack of broadly acting senolytic drugs. Using CRISPR/Cas9-based genetic screens in different senescent cancer cell models, we identify loss of the death receptor inhibitor cFLIP as a common vulnerability of senescent cancer cells. Senescent cells are primed for apoptotic death by NF-κB-mediated upregulation of death receptor 5 (DR5) and its ligand TRAIL, but are protected from death by increased cFLIP expression. Activation of DR5 signaling by agonistic antibody, which can be enhanced further by suppression of cFLIP by BRD2 inhibition, leads to efficient killing of a variety of senescent cancer cells. Moreover, senescent cells sensitize adjacent non-senescent cells to killing by DR5 agonist through a bystander effect mediated by secretion of cytokines. We validate this ‘one-two punch’ cancer therapy by combining pro-senescence therapy with DR5 activation in different animal models.
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Data availability
RNA-seq data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) under accession codes GSE102639 and GSE191255 and the ArrayExpress database under accession code E-MTAB-9970. NF-κB signature analysis (Fig. 2a) is in GEO under accession code GSE102639. RNA sequencing of the alisertib and etoposide-treated cell line panel is in ArrayExpress under accession code E-MTAB-9970 (Fig. 2d–e and Extended Data Fig. 3a–f). RNA-sequencing data of NEO2734-treated PC9 and Hep1, SASP-medium-treated A549, CFI-400945-treated A549 cells are in GEO under accession code GSE191255 (Fig. 5e and Extended Data Fig. 10d). Source data for Figs. 1–7 and Extended Data Figs. 1–10 have been provided as Source Data files. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.
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Acknowledgements
We thank members of the Bernards laboratory and J. Borst and I. Verbrugge for helpful discussion and thoughtful feedback. This work was supported by the European Research Council as ERC-787925 (R.B.), 19-051-ASP from the Mark Foundation (R.B.), KWF-12539 from the Dutch Cancer Society (RL.B.), 81920108025, 82011530441 and 82073039 from the National Natural Science Foundation of China (H.J., W.Q. and R.B.) and 22XD1423100 from the program of Shanghai Academic/Technology Research Leader (H.J.).
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Contributions
All authors contributed to the experimental design and analyzed experiments. L.W. and R.B. conceived the study. L.W., R.B. and R.L.B designed the screen platform. L.W. and H.J. designed in vivo studies. C.L. analyzed screens. L.W., H.J., W.Z., R.L.B., W.Q. and R.B supervised the study. L.W., H.J. and R.B. wrote the paper. All authors discussed the results and commented on the manuscript.
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L.W. and R.B. are named inventors on a patent application based on the study (owned by the Netherlands Cancer Institute). L.W. and R.B. are current part-time employees of Oncosence. R.B. is shareholder of Oncosence. R.B. is the board member of LIXTE Biotechnology. The remaining authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Senolytic screen optimization.
a, Senescence-associated beta-galactosidase staining in one week of senescence inducers (0.5 µM Alisertib, 1 µM Barasertib, 50 nM CFI-400951 or 2 µM Etoposide) treated A549 and SK-Hep1 cells. The black scale bars present 100 µm. b, SENCAN classifier analysis18 on different senescence inducers treated A549 cells. c, Western blot analysis of P21 and phosp-RB in A549 treated with 50 nM CFI-400945 for 1 week. The result is representative of at least 3 repeats with similar results. d, Western blot analysis of CAS9 expression in A549-iCas9 clone on 24 hours treatment of 1 µg/ml doxycycline. The result is representative of at least 3 repeats with similar results. e, Incucyte growth curve on A549-iCas9 and the parental cells. f, A549 cells were infected with pXRP-011 and treated with or without 1 µg/ml doxycycline for a week. Afterwards, the GFP signals were measured by flow cytometry to determine gene editing efficiencies. (GFP depletion were checked at least 3 times with similar results). Error bars in this figure panel a, e represent as mean ± standard deviations, N = 3 independent experiments, statistical significance was calculated by two-tailed t-test.
Extended Data Fig. 2 Senolytic screens and validation in additional models.
a, Schematic of CRISPR-based senolytic screen workflow in SK-Hep1 and PC3. Genome-wide Brunello gRNA collection lentivirus was introduced to the SK-Hep1-iCas9 or PC3-iCas9 cells. After 7 days of 0.5 µM alisertib treatment, cells were switched to 1 µg/ml doxycycline (DOX) for 10 days. Illumina deep sequencing was used to determine changes in library representation. b, Top hits from SK-Hep1 and PC3 were selected based on the fold depletion of S2 divided by S1 (N = 3 independently viral infected cell cultures). c, Real-time PCR analysis of cFLIP expression on SK-Hep1 cFLIPKO clone. d, Senescence inducers treated SK-Hep1 parental and cFLIPKO cells were incubated with caspase-3/7 green apoptosis assay reagent. The black scale bars present 100 µm. e, CellTiter-Blue measurement in the SK-Hep1 parental and cFLIPKO cells treated with senescence inducers for one week. f, Quantification of colony-formation result in 0.5 µM alisertib and 10 µM Z-VAD-FMK treated A549 parental and cFLIP-KO cells. g, Senescence-associated beta-galactosidase staining on PC3 cells with one week of 0.5 µM Alisertib treatment. The black scale bars present 100 µm. h, Real-time PCR analysis of cFLIP expression on PC3 cells infected with shRNAs against cFLIP. i, Quantification of colony-formation result in 0.5 µM alisertib treated PC3 cells infected with shRNAs against cFLIP. Error bars in this figure panel c-i represent as mean ± standard deviations, N = 3 independent experiments, statistical significance was calculated by two-tailed t-test.
Extended Data Fig. 3 Enrichment of NF-kB signatures, death receptor related gene expression upon senescence induction and extrinsic apoptosis priming in senescent cells.
a-f, Transcriptomic analysis of death receptor genes TNFSF10 (TRAIL), TNFRSF10C (DCR1), TNFRSF10D (DCR2), TNFRSF10A (DR4), TNF and TNFRSF1A (TNFR1) from 13 cell lines made senescent by etoposide and alisertib (ArrayExpress E-MTAB-9970). g, Real-time PCR analysis of DR4, DR5, cFLIP, TRAIL in SK-Hep1 cells treated with 0.5 µM alisertib. h, Real-time PCR analysis of TRAIL and DR5 in cFLIP knockout SK-Hep1 cells with shRNAs against these genes. i, Cell viability assessed by colony formation assay in the cFLIPKO cells with shRNAs against TRAIL or DR5 and treated with 0.5 µM alisertib for 7 days. Error bars in this figure panel g-i represent as mean ± standard deviations, N = 3 independent experiments, statistical significance was calculated by two-tailed t-test.
Extended Data Fig. 4 Lz-TRAIL-mediated senolysis in additional alisertib induced senescent cell models.
a, Incucyte cell proliferation assay showing 0.5 µM alisertib-induced senescent A549 cells were treated with 100 ng/ml Lz-TRAIL. b, Senescence-associated with beta-galactosidase staining in A549 parental and alisertib-induced senescent cells treated with 200 ng/ml Lz-TRAIL. c, Senescence-associated with beta-galactosidase staining in a panel of cell lines treated with alisertib. The black scale bars present 100 µm. d, Dose-response curves of alisertib-induced senescent and proliferating CDKN2A mutant cell models upon Lz-TRAIL treatment (the result represents at least 3 repeats with similar results). e, Cell viability-based colony formation assay with alisertib-induced senescent and proliferating CDKN2A wild-type cell models upon 100 ng/ml Lz-TRAIL treatment. Error bars in this figure panel a-c, e represent as mean ± standard deviations, N = 3 independent experiments, statistical significance was calculated by two-tailed t-test.
Extended Data Fig. 5 Death receptor agonist-mediated senolysis in additional models induced by different senescence inducers.
a, Caspase-3/7 green apoptosis assay on alisertib-induced senescent SK-Hep1 cells treated with 100 ng/ml Lz-TRAIL. b, Cell viability accessed by Colony formation in the alisertib-induced senescent and proliferating SK-Hep1 cells treated with 100 ng/ml Lz-TRAIL, 10 µM Z-VAD-FMK. c, Dose-response curve of 0.5 µM alisertib-induced senescent and proliferating SK-Hep1 cells treated with conatumumab (The result represents at least 3 repeats with similar results). d, Colony formation assay in drug-induced (one week of 1 µM Barasertib, 100 nM CFI-400945, 2 µM Etoposide treatment) senescent SK-Hep1 cells treated with conatumumab. e, Senescence-associated with beta-galactosidase staining in 7 days post-IR 10 Gy on SK-Hep1 cells. The black scale bars present 100 µm. f-g, Colony formation (f) and Western blot of cl-PARP (g) in the IR-induced senescent and proliferating SK-Hep1 cells treated with 1 µg/ml conatumumab and 10 µM Z-VAD-FMK. h, Cell viability accessed by colony formation in the IR-induced senescent and proliferating SK-Hep1 cells treated 100 ng/ml Lz-TRAIL. i, Senescence associated with beta-galactosidase staining in 7 days 10 Gy post-ionizing irradiated (IR) A549 cells. The black scale bars present 100 µm. j-k, Colony formation assay (j) and western blot of cl-PARP (k) in the 10 Gy IR-induced senescent and proliferating A549 cells treated with 100 ng/ml Lz-TRAIL and 10 µM Z-VAD-FMK. l, Cell viability accessed by colony formation of 0.5 µM alisertib-induced senescent and 10 µg/ml LPS cultured cells treated with the 4 µg/ml conatumumab or 200 ng/ml Lz-TRAIL. m, Real-time PCR of DR5, cFLIP, TRAIL in in LPS treated and alisertib-induced senescent A549 cells. The results in panel g, k are representative of at least 3 repeats with similar results. Error bars in this figure panel a, b, d-f, h-j, l, m represent as mean ± standard deviations, N = 3 independent experiments, statistical significance was calculated by two-tailed t-test.
Extended Data Fig. 6 in vivo study of conatumumab-mediated senolysis.
a-b. Tumor growth of A549 (a) and SK-Hep1 (b) cells in flanks of Balb/c nude mice subcutaneously injected with 5 million cells. When tumors reached approximately 150mm3, assigned to either vehicle control, 25 mg/kg alisertib, conatumumab (5 µg/dose for SK-Hep1, 10 µg/dose for A549) and combination (6 mice per group for A549, 8 mice per group for SK-Hep1). The data are presented as mean ± s.e.m. Two-way ANOVA test was applied for xenograft experiments (a, b) for statistical analysis c, Fixed tissues were dehydrated and embedded in paraffin. Sections of 2-4 µm were prepared and immunostained with P21 to indicate senescent cells in the tumors of A549 and Sk-Hep1. The black scale bars present 50 µm. d-e, Immunohistochemistry analysis of P21 expression. Error bars in this figure panel d,e represent as mean ± standard deviations, N = 12 tumor tissue slides, statistical significance was calculated by two-tailed t-test.
Extended Data Fig. 7 Potential resistant mechanisms of death receptor activation mediated senolysis.
a, CRISPR-based resistance screen was performed on cFLIP null SK-Hep1 cells to identify genes whose inhibition can result in resistance to alisertib mediated senolysis. The cFLIP null SK-Hep1 cells were infected with genome-wide Brunello gRNA collection lentivirus and cultured with 0.5 µM alisertib and 1 µg/ml doxycycline for 14 days. The control arm is without alisertib. Deep-sequencing was used to determine changes in library representation. b, Top hits were selected by the fold enrichment upon senolysis (N = 3 independently viral infected cell cultures). c, Real-time PCR of Bid in cells A549 and SK-Hep1 with shRNA against Bid. d, Cell viability accessed by colony formation assay on Bid knockdown senescent A549 and SK-Hep1 cells treated with conatumumab. e, Real-time PCR of Caspase 8 in cells A549 with shRNA against Caspase 8. f, Cell viability on Caspase 8 knockdown senescent A549 cells treated with conatumumab. g, Cell viability on FADD suppressed senescent A549 cells treated with Lz-TRAIL. h, Real-time PCR of FADD in cells A549 with shRNA against FADD. i, Dose-response curve in 1 µM alisertib-induced senescent PLC/PRF/5 cells treated with conatumumab, the result represents at least 3 times with similar results. j, Real-time PCR of FADD in cells A549 infected with FADD overexpression vector. k, Cell viability on FADD overexpressed senescent A549 cells treated with Lz-TRAIL. Error bars in this figure panel c-h, j, k represent as mean ± standard deviations, N = 3 independent experiments, statistical significance was calculated by two-tailed t-test.
Extended Data Fig. 8 Examination of senolytic potentials with BRD2 inhibition and/or death receptor 5 activation on additional senescent models.
a, Western blot analysis on tamoxifen (4-OHT)-inducible mutant HRASV12 vector infected BJ cells. b, Senescence associated with beta-galactosidase staining in proliferating and HRASV12 induced senescent BJ cells infected with an shRNA viral vector against TP53. The black scale bars present 100 µm. c, Cell viability accessed by colony formation assay on TP53 knockdown senescent BJ cells treated with 10 µg/ml as an extremely high dose of conatumumab. d, Senescence associated beta-galactosidase staining in one week of senescence inducers (0.5 µM PF-06873600, 100 nM doxorubicin and the combination of 5 nM trametinib plus 0.5 µM palbociclib) treated A549 and SK-Hep1 cells. The black scale bars present 100 µm. e, Western blot analysis of senescence and death receptor signaling markers on A549 and SK-Hep1 treated with different senescence inducers. f, Colony formation using low dose 0.25 µM of NEO2734 and 0.125 µg/ml conatumumab on SK-Hep1, 0.25 µM of NEO2734 and 1 µg/ml conatumumab on A549 cells made senescent by one-week treatment of different senescence inducers. g, Western blot analysis on FADD on the knockout cells. h, TIDE analysis of Caspase 8 knockout cells. i, Colony formation using low dose 0.25 µM of NEO2734 and 1 µg/ml conatumumab on FADD, CASP8 or DR5 null A549 senescent cells. The data in panel a, e-h represent at least 3 repeats with similar results. Error bars in this figure panel b-d, i represent as mean ± standard deviations, N = 3 independent experiments, statistical significance was calculated by two-tailed t-test.
Extended Data Fig. 9 Death receptor activation mediated immune surveillance and the evidence of | SASP-mediated bystander effect.
a, Fixed tissues were dehydrated and embedded in paraffin. Sections of 2-4 µm were prepared and immunostained with P21 to indicate senescent cells in the tumors of A549. The black scale bars present 50 µm. b, the quantification of the P21 immunostaining (N = 18 tumor tissue slides). c, Transcriptomic analysis of CD95 (FAS) in A549 cells treated with 1 week of 0.5 µM alisertib. d, Dose-response curves in 0.5 µM alisertib-induced senescent A549 and SK-Hep1 cells treated with CD95 ligand (FASLG). e, Senescence associated beta-galactosidase staining on one week of different SASP media cultured A549 cells (the media were collected from the senescent models induced by different treatments). 0.5 µM Alisertib-induced senescent cells were used as the control. The black scale bars present 100 µm. f-g, SASP medium was collected from the alisertib-induced senescent cells and mixed with an equal volume fresh medium. Ctrl medium was collected from proliferating cells and mixed with an equal volume fresh medium. Afterwards, The SASP medium or Ctrl medium were added to new proliferating A549 (f) and SK-Hep (g) cells and treated with NEO2734 and Conatumumab. h, Cell viability accessed by colony formation of SASP medium cultured A549 cells treated with the combination of 0.25 µM NEO2734 plus 4 µg/ml conatumumab and 10 µM Z-VAD-FMK. The results in panel d, e represent at least 3 times with similar results. Error bars in this figure panel b represent for N = 18 tumor tissue slides. Results in panel f-h represent by N = 3 independent experiments. These results represent as mean ± standard deviations, statistical significance was calculated by two-tailed t-test.
Extended Data Fig. 10 SASP-mediated bystander effect with BRD inhibitor and DR5 agonist combination is regulated by NF-kB signaling.
a, The proliferating A549 cells were cultured with SASP medium from different treatment-induced senescent A549 cells and treated with combination of 0.25 µM NEO2734 plus 4 µg/ml conatumumab or 1 µM navitoclax. b, A549 cells were cultured with different concentrations of SASP media and treated with the combination of 0.25 µM NEO2734 plus 4 µg/ml conatumumab. c, in vitro competition assay, to study the bystander effect of conatumumab plus NEO2734 in a heterogeneous population containing proliferating and senescent cells. GFP and RFP cells were generated through infection with a lentiviral vector coding for H2B-GFP and H2B-RFP. RFP labeled cells were induced to senescent by one week of 0.5 µM alisertib treatment. GFP labeled cells were used as proliferating cells. Different ratios of GFP and RFP cells were initially mixed and treated with the drug combination. The gray scale bars present 100 µm. d, RNA sequencing was performed on A549 cells cultured with SASP medium for 1 week and followed by gene set enrichment analysis (GSEA) of SASP medium cultured cells versus ctrl medium cultured cells for multiple independent NF-KB signaling gene sets (N = 1 RNA-sequencing data). e, P65 knockdown A549 cells were cultured with SASP medium and treated with 0.25 µM NEO2734 plus 4 µg/ml conatumumab. Error bars in this figure panel a-c, e represent as mean ± standard deviations, N = 3 independent experiments, statistical significance was calculated by two-tailed t-test.
Supplementary information
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Wang, L., Jin, H., Jochems, F. et al. cFLIP suppression and DR5 activation sensitize senescent cancer cells to senolysis. Nat Cancer 3, 1284–1299 (2022). https://doi.org/10.1038/s43018-022-00462-2
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DOI: https://doi.org/10.1038/s43018-022-00462-2
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