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The YAP–TEAD complex promotes senescent cell survival by lowering endoplasmic reticulum stress

Nature Aging volume 3, pages 1237–1250 (2023)Cite this article

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Abstract

Sublethal cell damage can trigger senescence, a complex adaptive program characterized by growth arrest, resistance to apoptosis and a senescence-associated secretory phenotype (SASP). Here, a whole-genome CRISPR knockout screen revealed that proteins in the YAP–TEAD pathway influenced senescent cell viability. Accordingly, treating senescent cells with a drug that inhibited this pathway, verteporfin (VPF), selectively triggered apoptotic cell death largely by derepressing DDIT4, which in turn inhibited mTOR. Reducing mTOR function in senescent cells diminished endoplasmic reticulum (ER) biogenesis, triggering ER stress and apoptosis due to high demands on ER function by the SASP. Importantly, VPF treatment decreased the numbers of senescent cells in the organs of old mice and mice exhibiting doxorubicin-induced senescence. Moreover, VPF treatment reduced immune cell infiltration and pro-fibrotic transforming growth factor-β signaling in aging mouse lungs, improving tissue homeostasis. We present an alternative senolytic strategy that eliminates senescent cells by hindering ER activity required for SASP production.

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Fig. 1: CRISPR screen identifies YAP–TEAD pathway as essential for senescent cell viability.
Fig. 2: YAP–TEAD inhibition triggers apoptosis by derepressing DDIT4, causing endoplasmic reticulum stress.
Fig. 3: Inhibition of mTOR-dependent endoplasmic reticulum biogenesis by DDIT4 induces endoplasmic reticulum stress and senolysis.
Fig. 4: VPF treatment reduces senescent cell burden in vivo.

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Data availability

RNA-seq fastq files generated for this study are available at the Gene Expression Omnibus under accession number GSE221254 to provide access to all datasets in this study. YAP–TEAD targets were identified by both existing evidence on the literature and curation with MAGIC database. Source data are available with this paper.

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Acknowledgements

This research was supported in its entirety by the NIA IRP, NIH. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Authors and Affiliations

  1. Laboratory of Genetics and Genomics, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA

    Carlos Anerillas, Krystyna Mazan-Mamczarz, Allison B. Herman, Rachel Munk, Kwan-Wood Gabriel Lam, Dimitrios Tsitsipatis, Jennifer L. Martindale, Gisela Altés, Martina Rossi, Yulan Piao, Jinshui Fan, Chang-Yi Cui, Supriyo De, Kotb Abdelmohsen & Myriam Gorospe

  2. Translational Gerontology Branch, National Institute on Aging Intramural Research Program, National Institutes of Health, Baltimore, MD, USA

    Miguel Calvo-Rubio, Amanda Garrido & Rafael de Cabo

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Contributions

C.A. and K.M.-M. conceived and designed the study. S.D., K.A., R.d.C. and M.G. supervised the study. C.A., K.M.-M. and M.G. wrote the article. C.A., A.B.H., R.M., M.C.-R., A.G., D.T., J.L.M., G.A., M.R. and C.-Y.C. performed wet-lab and mouse experiments. K.M.-M., K.-W.G.L., Y.P., J.F. and S.D. performed bulk and single-cell sequencing and bioinformatic analyses. All authors reviewed and edited the article.

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Correspondence to Carlos Anerillas or Myriam Gorospe.

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Nature Aging thanks Ming Xu, Iván Moya and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 CRISPR screen optimization, validation, and YAP-TEAD inhibition in other senescence models.

a, Cell viability assessment by direct cell counting of senescent WI-38 cells treated with puromycin (1 µg/ml, 48 h) 72 h after being transduced with the Brunello library at the indicated MOIs. The gray bars represent the expected viability if the transduction efficiency was complete while the teal bars represent the viability observed for each of the MOIs after puromycin treatment. b, Cell viability as assessed by direct cell counting of WI-38 cells transfected with the indicated siRNAs and rendered senescent after treatment with etoposide for 6 days (ETIS). c, Analysis of the levels of the indicated mRNAs in proliferating (P) or ETIS WI-38 cells transfected with the indicated siRNAs 24 h before either treatment with etoposide (50 µM) or no treatment, and culture for an additional 6 days. d, Representative western blot analysis (n = 3 independent experiments) of the levels of phosphorylated YAP (S127), YAP, phosphorylated MOB1 (T35), MOB1, and ACTB levels at the indicated conditions. e,f, Analysis of BrdU incorporation (e) and SA-β-Gal staining (f) in the indicated cell types, rendered senescent by etoposide (ETIS), ionizing radiation (IRIS), or replicative exhaustion (RS). Scale bar 100 µm. g, Caspase 3/7 activity measured in RS and IRIS WI-38 cells treated for 72 h with the indicated doses of Verteporfin (VPF). h, i, Cell viability as assessed by direct cell counting (h) and Caspase 3/7 activity measurement (i) for the indicated models of senescence along with proliferating controls, after either no treatment or treatment with VPF for 72 h at the indicated doses. Graphs in (b, c, e, g–i) represent the means and each individual value as a dot ±s.d. n = 3 independent replicates; significance (*P < 0.05, **P < 0.01, ***P < 0.001) was determined using two-tailed Student’s t-test. Unless indicated, statistical tests were performed relative to untreated or proliferating controls.

Source data

Extended Data Fig. 2 Extended analysis of YAP-TEAD inhibition.

a, b, Representative western blot analysis (a) and quantification (b) of the levels of YAP and TEAD proteins after immunoprecipitation experiments with the indicated antibodies (IgG or anti-TEAD) in ETIS WI-38 fibroblasts that were either untreated or treated with VPF for 48 h. IgG bands are indicated with arrows placed on the left side of the panel. Inputs are also included. c, Heat map displaying the differential expression of the YAP–TEAD-dependent transcripts (by row Z-Score) in the conditions described in (a). Proliferating untreated cells were included as a baseline control. d, GSEA of the association (enrichment score) with the gene set ‘YAP1_up’ of ETIS WI-38 cells treated with VPF (48 h) compared to untreated senescent cells (-). e, RT-qPCR analysis of the levels of ANKRD1 and TGFB2 mRNAs in ETIS WI-38 cells after treatment for 48 h with the indicated YAP–TEAD inhibitors. Untreated controls were also included for comparison. f, Heat map representing the differential expression (Row Z-Score) among the conditions described in (e) for the indicated transcripts. g, Heat map displaying the differential expression (Row Z-Score) of the indicated transcripts in siCtrl and siTEAD2 ETIS WI-38 cells. h, GSEA of the association with the gene set ‘Hallmark: Epithelial-Mesenchymal Transition’ for the conditions described in (d). i, RT-qPCR analysis of the indicated pro-apoptotic mRNAs for the conditions described in (e). j, Representative Western blot (n = 3 independent experiments) of the levels of ATF6, XBP1s, and loading control ACTB for the conditions described in (d). k, GSEA plot showing the association (enrichment score) of the gene set ‘GOBP: PERK-mediated UPR’ with the conditions described in (d). l, Western blot analysis of the levels of phosphorylated EIF2A (S51) and loading control ACTB in WI-38 cells transfected with siCtrl or siPERK, rendered senescent with etoposide (ETIS) and then either left untreated or treated with 1.5 µM VPF for 48 h. m, n, Cell viability assessment by direct cell counting (m) and RT-qPCR analysis of PERK mRNA levels (n) in the conditions described in (l), but here treated with VPF for 72 h. o, p, Maximal cisternae thickness (o) and disorganization score (p) as measured by TEM in the groups described in (c). Thirty cells were analyzed for each condition. q, RT–qPCR analysis of the indicated transcripts either untreated or treated with 1.5 µM VPF for 8 h. r, Relative binding to the regulatory region of the DDIT4 gene or a negative control (Neg Ctrl) DNA in YAP ChIP samples of ETIS WI-38 cells that were untreated or treated with 1.5 µM VPF (48 h). s, RT-qPCR analysis of the levels of DDIT4 and p53 mRNAs in WI-38 cells transfected with the indicated siRNAs, rendered senescent with etoposide (ETIS) and either left untreated or treated with 1.5 µM VPF for 48 h. Proliferating WI-38 cells transfected with siCtrl were included as controls. Graphs in (b, e, i, m, n, q–s) display the means and the individual values as dots ±SD n = 3 independent replicates; graphs in (o, p) show the means and the individual values as dots ±s.d. of n = 30 different cells. Significance (*P < 0.05, **P < 0.01, ***P < 0.001) was calculated using two-tailed Student’s t-test.

Source data

Extended Data Fig. 3 Analysis of mTOR inhibition and VPF treatment on ER stress in senescent cells.

a, Cell viability as assessed by direct cell counting of proliferating (P) or ETIS WI-38 fibroblasts that were either left untreated or treated with 100 nM Torin1 for 72 h. b, Representative micrographs showing the differences in viability in the conditions from (a). Scale bar, 100 µm. c, Western blot analysis of the levels of ATF6, XBP1s, and ACTB in the conditions described in (a), at 48 h instead. d, RT-qPCR analysis of the levels of PUMA, PMAIP1, and TNFRSF10B mRNAs in the conditions described in (c). Untreated P cells were included as baseline controls. e, Direct cell counting after treating as indicated for 72 h (1.5 µM VPF, 100 nM Torin1, or both) in ETIS WI-38 cells. f, RT-qPCR analysis of the indicated transcripts for the treatments described in (e), in this case for 48 h. g, Dot plot representation of the values calculated for the ER-positive relative area per cell (60 cells per condition) in ETIS WI-38 cells either untreated or treated with VPF (1.5 µM) or Torin1 (100 nM) for 48 h. h, Micrographs showing the areas corresponding to the endoplasmic reticulum (ER) in red for the indicated treatments as in (g). Phosphatidylcholine (PtdCho) was simultaneously supplemented at 50 µM where indicated. Scale bar, 100 µm. i, Heat map representation of the differences in SASP mRNA levels represented by row Z-Score for the indicated transcripts when comparing the conditions described in (g). Untreated P WI-38 cells were included as baseline controls. j, Heat map of the row Z-Score calculated for the differences in the secretion of the indicated SASP members among the groups described in (g), including proliferating (P) WI-38 cells as a control for baseline secretion. k, UMAP plot representation of the scRNA-seq data from ETIS WI-38 cells (no VPF treatment) showing the expression score specified in the legends, associated with the indicated gene sets (SASP, a custom gene set of 132 markers; ER stress, GOBP: Response to ER stress; and Oxidative Phosphorylation, Hallmark: Oxidative Phosphorylation). l, Western blot analysis of phosphorylated EIF2A (S51) and ACTB levels in WI-38 cells transfected with the indicated siRNAs, rendered senescent by treatment with etoposide for 6 days, and then either left untreated or treated with 100 nM Torin1 for 48 h. m, Cell viability measurement by direct cell counting of the conditions described in (l), here treated for 72 h. n, o, RT-qPCR analysis (n) and Bioplex analysis of the conditioned media (o) to assess SASP production and secretion in WI-38 cells transfected with siCtrl or siRELA, and rendered senescent with etoposide for 6 days. Proliferating controls transfected with siCtrl siRNA were included. Graphs in (a, d–f, m, n) represent the means and individual values (dots) of n = 3 independent replicates; plot in (g) shows the individual values of 20 different cells from each of the 3 independent replicates analyzed, making a total 60 individual values; Significance (*P < 0.05, **P < 0.01, ***P < 0.001) was calculated using two-tailed Student’s t-test.

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Extended Data Fig. 4 Analysis of senescence markers in naturally aged and doxorubicin-treated mice.

a Representative immunofluorescence images of p21 (red) and p16 (green) in mouse liver and kidney from the groups described in Fig. 4a. Scale bar (white), 200 µm. b, Quantification of the percentage of p16-positive, p21-positive, or p16/p21 double-positive cells in the liver and kidney samples represented in (a). c, RT-qPCR analysis of p16 and p21 mRNA levels (normalized to Actb mRNA) in liver and kidney for the conditions described in Fig. 4a. d, Schematic representation of the treatment regimen carried out to trigger doxorubicin-induced senescence in vivo in mice (10 mg/kg), along with 4 consecutive treatments with DMSO (Vehicle) or VPF (50 mg/kg) from day 6 onward. Samples were collected at day 10 after doxorubicin treatment. e, RT-qPCR analysis of p21 mRNA levels in lung, liver, and kidney from the groups described in (d). Untreated mice were included as baseline controls. f, g, Quantification (f) and representative images (g) of p21 immunofluorescence in the conditions described in (d). Scale bar (white), 200 µm. h, Serum measurement of GDF15 levels for the experimental groups described in (d, left, and Fig. 4a, right). Graphs in (b, c) display the means and the individual values as dots ± s.d. of the included mice (more details in Supplementary Table 6), while graphs in (e, f, h) display the means and the individual values as dots ± s.d. of n = 6 mice per group; significance (*P < 0.05, **P < 0.01, ***P < 0.001) was calculated using one-way ANOVA.

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Extended Data Fig. 5 Physiological benefits of senolytic ABT-737 and VPF treatments in naturally aged mice.

a, Pictures of the 24 m.o. mice from the different groups described in Fig. 4a at the end of the experiment. b, UMAP clustering of the scRNA-seq performed in lungs from the indicated groups. c, Feature plot displaying Cdkn2a mRNA expression in the indicated experimental groups. Each plot was obtained by merging the two samples sequenced for each experimental group, shown in (b). d, Plots displaying the signals obtained through flow cytometry analysis of single-cell lung suspensions from the indicated groups. FSC-H axis represents the signals obtained for the forward scatter, while the PE-Cy7-A axis corresponds to CD45 staining with such fluorophore. Cells considered CD45+ are colored in blue, and the percentages are specified on the top right corner of each box. e, Dot plot displaying the association of the indicated cell types and conditions with the top 15 transcripts from Hallmark: Inflammatory Response gene set. The size of the dots represents the percentage of cells expressing such transcript while the intensity of red indicates the relative expression value. f, GSEA plots displaying the association of the indicated cell types from Old DMSO condition (compared to the rest of the experimental groups) with the indicated gene sets (Reactome: Translation; Safford T Lymphocyte Anergy; Reactome: Interleukin 6 Family Signaling; Reactome: Antigen Activates B cell Receptor BCR Leading To Generation Of Second Messengers). g, h, Masson’s trichrome (MTC) staining performed in liver and kidney from the indicated groups, representative images of MTC staining (g) in blue, and quantification of the blue area present at each sample divided by the total area in red (h). Scale bar, 200 µm. i, j, Serum analysis of blood urea nitrogen (i) and AST (j) levels for the indicated experimental groups. Plots in (h–j) represent the means and the individual values as dots ± s.d. of the included mice (more details in Supplementary Table 6); significance (*P < 0.05, **P < 0.01, ***P < 0.001) was calculated using one-way ANOVA.

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Supplementary information

Reporting Summary

Supplementary Table 1

Abundance of the sgRNAs in the different conditions identified by next-generation sequencing of the CRISPR–Cas9 whole-genome screen. Data are plotted in the heat map in Fig. 1f. The data were pre-filtered here to show the significantly less abundant sgRNAs in all t = 14 replicates compared tot = 0.

Supplementary Table

Twofold changes comparing t = 14 to t = 0 in the whole-genome CRISPR–Cas9 screen included in the study for each of the detected sgRNAs, analyzed by Wald test in Deseq2. The different statistical parameters for each comparison between t = 14 and t = 0 are displayed on the rightmost columns. baseMean, average of normalized counts; log2FoldChange, log2fold change; lfcSE, standard error of log2FoldChange; stat, Wald statistic; pvalue, Wald test Pvalue; padj, Benjamini-Hochberg adjusted Pvalue.

Supplementary Table 3

List of transcripts included for the gene set ‘SASP’ used in the scRNA-seq analysis displayed in Fig. 3n.

Supplementary Table 4

Association P values and the −log10(P values) of the indicated gene sets (specified below) for each of the lung scRNA-seq clusters specified in Fig. 4g. The data included in this table are plotted in a heat map in Fig. 4h. The top 200 transcripts (obtained from the log1p average expression values for each condition per cluster) increased in old DMSO at least 2-fold relative to young DMSO and 1.5-fold relative to both old VPF and old ABT-737 were included for the analysis with Enrichr, which calculated the statistical values by itself. The P values obtained for each of the indicated gene sets are specified.

Supplementary Table 5

Percentages of each of the indicated lung cell types in the different conditions analyzed through scRNA-seq. The data included in this table are plotted in Fig. 4i.

Supplementary Table 6

Table showing the mice included in the aging study, with all the samples taken from each for all the specified subsequent analyses.

Supplementary Table 7

List of the primers used for the qPCR analysis of the specified transcripts.

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Anerillas, C., Mazan-Mamczarz, K., Herman, A.B. et al. The YAP–TEAD complex promotes senescent cell survival by lowering endoplasmic reticulum stress. Nat Aging 3, 1237–1250 (2023). https://doi.org/10.1038/s43587-023-00480-4

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