泛素连接酶 MDM2 的高表达是许多肿瘤中 p53 失活的主要原因，使其成为一个有前途的治疗靶点。然而，由于 p53 诱导的反馈增强了 MDM2 的表达，MDM2 抑制剂在临床试验中失败了。这强调了寻找有效的适应性基因型或靶标组合的迫切需要。使用TP53野生型癌细胞进行了全激酶组CRISPR/Cas9敲除筛选，以鉴定调节对MDM2抑制剂反应的基因，并发现ULK1作为候选基因。通过 MTT 细胞活力测定、流式细胞术和 LDH 测定来评估细胞焦亡的激活以及 ULK1 耗竭与 p53 激活相结合的综合致死效果。进行双荧光素酶报告基因测定和 ChIP-qPCR 来证实 p53 直接介导 GSDME 的转录，并鉴定 p53 在 GSDME 启动子中的结合区域。构建 ULK1 敲除/过表达细胞以研究 ULK1 的体外和体内功能作用。主要通过 qPCR、蛋白质印迹和 ELISA 研究 ULK1 缺失激活 GSMDE 的机制。通过高通量筛选，我们确定 ULK1 是 MDM2 抑制剂 APG115 的合成致死基因。结果表明，ULK1 的缺失显着提高了敏感性，细胞会发生典型的细胞焦亡。从机制上讲，p53 通过直接介导诱导基础水平焦亡的 GSDME 转录来促进焦亡启动。此外，ULK1 耗竭会减少线粒体自噬，导致受损线粒体的积累以及随后活性氧 (ROS) 的增加。这反过来又通过 NLRP3-Caspase 炎症信号轴裂解并激活 GSDME。分子级联使 ULK1 成为 p53 激活细胞介导的细胞焦亡起始的关键调节因子。此外，铂类耐药肿瘤中线粒体自噬增强，ULK1缺失/p53激活对这些肿瘤具有协同致死作用，通过GSDME直接诱导细胞焦亡。我们的研究表明，ULK1缺陷可以与MDM2抑制剂协同诱导细胞焦亡。 p53 在激活 GSDME 转录中发挥直接作用，而 ULK1 缺陷会触发 ROS-NLRP3 信号通路的上调，导致 GSDME 裂解和激活。这些发现强调了 p53 在确定焦亡中的关键作用，并为 p53 恢复疗法的临床应用提供了新途径，并提出了潜在的组合策略。© 2024。作者。

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.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.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.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.© 2024. The Author(s).