Synthetic lethality of combined ULK1 defection and p53 restoration induce pyroptosis by directly upregulating GSDME transcription and cleavage activation through ROS/NLRP3 signaling

Abstract

Background: High expression of ubiquitin ligase MDM2 is a primary cause of p53 inactivation in many tumors, making it a promising therapeutic target. However, MDM2 inhibitors have failed in clinical trials due to p53-induced feedback that enhances MDM2 expression. This underscores the urgent need to find an effective adaptive genotype or combination of targets.

Methods: Kinome-wide CRISPR/Cas9 knockout screen was performed to identify genes that modulate the response to MDM2 inhibitor using TP53 wild type cancer cells and found ULK1 as a candidate. The MTT cell viability assay, flow cytometry and LDH assay were conducted to evaluate the activation of pyroptosis and the synthetic lethality effects of combining ULK1 depletion with p53 activation. Dual-luciferase reporter assay and ChIP-qPCR were performed to confirm that p53 directly mediates the transcription of GSDME and to identify the binding region of p53 in the promoter of GSDME. ULK1 knockout / overexpression cells were constructed to investigate the functional role of ULK1 both in vitro and in vivo. The mechanism of ULK1 depletion to activate GSMDE was mainly investigated by qPCR, western blot and ELISA.

Results: By using high-throughput screening, we identified ULK1 as a synthetic lethal gene for the MDM2 inhibitor APG115. It was determined that deletion of ULK1 significantly increased the sensitivity, with cells undergoing typical pyroptosis. Mechanistically, p53 promote pyroptosis initiation by directly mediating GSDME transcription that induce basal-level pyroptosis. Moreover, ULK1 depletion reduces mitophagy, resulting in the accumulation of damaged mitochondria and subsequent increasing of reactive oxygen species (ROS). This in turn cleaves and activates GSDME via the NLRP3-Caspase inflammatory signaling axis. The molecular cascade makes ULK1 act as a crucial regulator of pyroptosis initiation mediated by p53 activation cells. Besides, mitophagy is enhanced in platinum-resistant tumors, and ULK1 depletion/p53 activation has a synergistic lethal effect on these tumors, inducing pyroptosis through GSDME directly.

Conclusion: Our research demonstrates that ULK1 deficiency can synergize with MDM2 inhibitors to induce pyroptosis. p53 plays a direct role in activating GSDME transcription, while ULK1 deficiency triggers upregulation of the ROS-NLRP3 signaling pathway, leading to GSDME cleavage and activation. These findings underscore the pivotal role of p53 in determining pyroptosis and provide new avenues for the clinical application of p53 restoration therapies, as well as suggesting potential combination strategies.

Keywords: MDM2 inhibitor; Mitophagy; Pyroptosis; Reactive oxygen species; TP53; ULK1.

Fig. 1

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. 2

ULK1 deficiency sensitizes cancer cells to APG-115 in vitro and in vivo. A, B Cell viability was measured by an MTT assay. A2780 cells and TOV21-G cells expressing sgRNAs targeting ULK1 were treated with idasanutlin for 3 days. C, D Cell viability was measured by an MTT assay. A2780 cells or TOV21-G cells were treated with idasanutlin and an ULK1 inhibitor for 3 days. E Illustration showing the workflow of the animal experiments. F The image shows tumors excised at the end of the experiment. G Average tumor weight in each group of nude mice (*** P < 0.001). H Tumor volume curves of each group of nude mice (** P < 0.01)

Fig. 3

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. 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

ULK1 deficiency induces GSDME activation through upregulation of ROS. A RNA was extracted from the indicated cells, and the expression of NLRP3, caspase 8 and IL1B was analyzed by qRT‒PCR. B Immunoblot analysis of NLRP3-Caspase pathway proteins and GAPDH from extracts of the indicated cells. C LDH release was detected using an LDH Cytotoxicity Detection Kit (Beyotime) in indicated cells. D, E The indicated A2780 cells were treated with 10 µM APG-115 for 24 h. IL-1β, IL-18 and IFN-γ concentrations in the indicated culture media were measured by ELISA. G Quantification of intracellular ROS in the indicated cells following treatment with 10 µM APG-115 for 24 h. H The indicated A2780 cells were treated with 4 mM NAC for 24 h. RNA was extracted from the indicated cells, and the expression of NLRP3, caspase 8 and IL1B was analyzed by qRT‒PCR. I The indicated A2780 cells were treated with 4 mM NAC for 24 h. Immunoblot analysis of NLRP3-Caspase pathway proteins and GAPDH from extracts of the indicated cells. J LDH release was detected using an LDH Cytotoxicity Detection Kit (Beyotime) in indicated cells. K-M The indicated A2780 cells were treated with 10 µM APG-115 and 4 mM NAC for 24 h. IL-1β, IL-18 and IFN-γ concentrations in the indicated culture media were measured by ELISA. N Survival fractions of the indicated cells following treatment with APG-115 and NAC for 72 h. O Survival fractions of control, siCaspase3 and siCaspase8 cells following treatment with APG-115

Fig. 6

The synergistic induction of pyroptosis by p53 activation and ULK1 depletion depends on mitochondria quality control. A, B Immunoblot analysis of ULK1, TOMM20, HSPD1, TIM23 and GAPDH expression in indicated cells. The indicated A2780 cells were treated with10 µM APG-115 for 24 h. C Immunofluorescence images of A2780 cells with deletion of ULK1 and treatment with 10 µM APG-115 for 24 h. The cells were costained with MitoTracker Deep Red and DAPI. The white line represents 10 µm. D, E Flow cytometric analysis of FITC- and PI-stained control, siFUNDC1, and siPARK A2780 cells treated with 10 µM APG-115 for 24 h. F, G Survival fractions of control, siFUNDC1, and siPARK A2780 cells following treatment with 10 µM APG-115 for 24 h. H, I LDH release was detected in models of mitophagy deficiency or macroautophagy deficiency following treatment with 10 µM APG-115 for 24 h. ns: P > 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. J Schematic of mechanisms underlying the synergy between MDM2i and ULK1 deficiency in TP53 wild-type cells. See main text for a detailed description

Fig. 7

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

Fig. 8

Schematic of ULK1 deficiency and p53 activation promote pyroptosis by activating GSDME directly (By Figdraw)