Fig 2: P53 activation combined with ULK1 deficiency can initiate pyroptosis. A Evaluation of APG-115-induced pyroptosis in A2780 sgAAV1 and A2780 sgULK1 cells by phase contrast imaging. B Flow cytometric analysis of Annexin V-FITC and PI staining in A2780 ULK1 knockout cells following treatment with 10 µM APG-115 for 24 h. C LDH release was detected using an LDH Cytotoxicity Detection Kit (Beyotime) in A2780 ULK1 knockout cells following treatment with 10 µM APG-115 for 24 h. **** P < 0.0001. D, F Flow cytometric analysis of FITC staining, PI staining, and LDH release in A549 p53-overexpressing cells following treatment with TNFα + CHX for 24 h. **** P < 0.0001. E, G Flow cytometric analysis of FITC staining, PI staining, and LDH release in A2780 ULK1 knockout cells following treatment with TNFα + CHX for 24 h. H RNA was extracted from the indicated cells, and the expression of GSDMA-E was analyzed by qRT‒PCR. * P < 0.05, *** P < 0.001, **** P < 0.0001. I Western blot showing GSDME protein levels in indicated A2780 cells

Fig 3: In vitro CRISPR screening identified ULK1 as a combinatorial target with an MDM2 inhibitor. A MDM2 mRNA expression (log2(FPKM + 1)) among all paired samples from the TCGA grouped by cancer. Each point represents one sample. The P values are based on two-tailed Student’s t test. B Summary of the correlation between MDM2 expression and overall survival (OS) based on univariate Cox regression and Kaplan‒Meier analyses. Red indicates that MDM2 is a risk factor affecting the prognosis of cancer patients, and green represents protective factors. Only P values < 0.05 are shown. C Schematic diagram of the in vitro screening process used to identify novel drug combinations. D Dot plots showing gene-specific CRISPR viability scores (log fold change and RRA scores). The points ranked in the top ten are highlighted in blue. E Venn diagram showing the intersection of the top 15 genes ranked by the log fold change score and the top 15 genes ranked by the RRA score. F Heatmap of RNA-seq analysis of nine TP53 wild-type cancer cell lines. G Survival curves of APG-115 in nine TP53 wild-type cancer cell lines. H Correlation analysis between gene expression and the IC50 of APG-115. I Western blot showing ULK1 protein levels in A2780 cells and TOV21-G cells expressing sgRNAs targeting ULK1. J Cell viability was measured by an MTT assay. A2780 cells and TOV21-G cells expressing sgRNAs targeting ULK1 were treated with APG-115 for 72 h

Fig 4: P53 directly activates the transcription of GSDME. A Dot plot showing the differences in the mRNA expression of GSDME between patients with different TP53 mutation statuses. Each point represents one sample. B Bar plot showing the correlation between the mRNA expression of GSDME and that of TP53 in 1210 cell lines from the CCLE database grouped by organ system. C, D p53 affects the expression of GSDME. A2780 and A549 cells were transfected with the indicated plasmids, and the expression of GSDME was determined by immunoblotting. E, F RNA was extracted from the indicated cells, and the expression of GSDME was analyzed by qRT‒PCR. * P < 0.05, *** P < 0.001, **** P < 0.0001. G Illustration of the truncation fragments of the GSDME promoter. H, I Firefly luciferase activity was measured and normalized to that of Renilla luciferase as the internal control. ** P < 0.01, *** P < 0.001, **** P < 0.0001. J, K The published ChIP-seq dataset was reanalyzed via the UCSC Genome Browser. After p53 was activated with MDM2 inhibitors, a peak appeared in the GSDME promoter region. L The indicated HEK-293 T p53-overexpressing cells were subjected to a ChIP assay using an antibody against p53. Isotype-matched IgG was used as a negative control. ** P < 0.01. The data are representative of three independent experiments

Fig 5: Combined targeting of mitophagy and activation of p53 could be used to reverse platinum resistance. A GSVA scores of mitophagy-related genes in cisplatin-sensitive and cisplatin-resistant samples of four tumors from TCGA. The sensitivity of the tumor samples was calculated using the "pRRophetic" R package, and the optimal cutoff value of the ROC curve was selected as the cutoff point between sensitivity and resistance based on the Youden index. The P values were calculated using the Wilcoxon rank–sum test. B GSVA scores of pyroptosis-related genes in cisplatin-sensitive and cisplatin-resistant samples of four tumors from TCGA. C Illustration showing the process for generating cisplatin-resistant cell lines. D Survival fractions of the indicated cisplatin-resistant cells following treatment with cisplatin for 72 h. E Immunoblotting of ULK1, TOMM20, HSPD1, TIM23 and GAPDH in extracts of cisplatin-resistant cells. F, G GSDME expression in cisplatin-resistant cells. *** P < 0.001. H, I The indicated cisplatin-resistant cells were treated with TNFα + CHX for 24 h. ** P < 0.01, *** P < 0.001, **** P < 0.0001. J Survival fractions of the indicated cisplatin-resistant cells following treatment with cisplatin for 72 h