Department of Radiation Oncology, Sun Yat-sen University Cancer Center; State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine; Guangdong Key Laboratory of Nasopharyngeal Carcinoma Diagnosis and Therapy, Guangzhou, China.
State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, 651 Dongfeng Road East, Guangzhou, China.
J Exp Clin Cancer Res. 2024 Aug 30;43(1):248. doi: 10.1186/s13046-024-03168-8.
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.
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。这凸显了迫切需要找到一种有效的适应性基因型或靶标组合。
使用 CRISPR/Cas9 全激酶组敲除筛选，在 TP53 野生型癌细胞中鉴定出调节 MDM2 抑制剂反应的基因，发现 ULK1 是候选基因。通过 MTT 细胞活力测定、流式细胞术和 LDH 测定评估细胞焦亡的激活和联合 ULK1 耗竭与 p53 激活的合成致死效应。双荧光素酶报告基因检测和 ChIP-qPCR 用于确认 p53 直接介导 GSDME 的转录，并鉴定 GSDME 启动子中 p53 的结合区域。构建 ULK1 敲除/过表达细胞，在体外和体内研究 ULK1 的功能作用。通过 qPCR、western blot 和 ELISA 主要研究了 ULK1 耗竭激活 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。研究表明，ULK1 的缺失显著增加了细胞的敏感性，导致典型的细胞焦亡。在机制上，p53 通过直接介导 GSDME 转录来启动细胞焦亡，从而促进细胞焦亡的发生，导致基础水平的细胞焦亡。此外，ULK1 的缺失减少了线粒体自噬，导致受损线粒体的积累，并随后增加活性氧（ROS）。反过来，NLRP3-Caspase 炎症信号轴切割并激活 GSDME。该分子级联反应使 ULK1 成为 p53 激活细胞中介导细胞焦亡起始的关键调节因子。此外，在铂耐药肿瘤中增强了线粒体自噬，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 缺乏可与 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 缺乏触发 ROS-NLRP3 信号通路的上调，导致 GSDME 切割和激活。这些发现强调了 p53 在决定细胞焦亡中的关键作用，并为 p53 恢复治疗的临床应用提供了新的途径，并提示了潜在的联合策略。
