结合 ULK1 缺陷和 p53 恢复的合成致死率通过 ROS/NLRP3 信号直接上调 GSDME 的转录和裂解激活,诱导热猝死。

IF 11.4 1区 医学 Q1 ONCOLOGY
Wei Chen, Kai-Bin Yang, Yuan-Zhe Zhang, Zai-Shan Lin, Jin-Wei Chen, Si-Fan Qi, Chen-Fei Wu, Gong-Kan Feng, Da-Jun Yang, Ming Chen, Xiao-Feng Zhu, Xuan Li
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引用次数: 0

摘要

背景:在许多肿瘤中,泛素连接酶 MDM2 的高表达是导致 p53 失活的主要原因,因此它是一个很有前景的治疗靶点。然而,由于 p53 诱导的反馈会增强 MDM2 的表达,MDM2 抑制剂在临床试验中失败了。这凸显了寻找有效的适应性基因型或靶点组合的迫切需要:方法:利用 TP53 野生型癌细胞进行了全基因组 CRISPR/Cas9 基因敲除筛选,以确定可调节对 MDM2 抑制剂反应的基因,结果发现 ULK1 是一个候选基因。研究人员采用 MTT 细胞活力测定法、流式细胞仪和 LDH 分析法评估了 ULK1 基因敲除与 p53 基因激活相结合对热休克的激活和合成致死效应。为了证实 p53 直接介导 GSDME 的转录,并确定 p53 在 GSDME 启动子中的结合区,研究人员进行了双荧光素酶报告实验和 ChIP-qPCR 实验。构建了ULK1敲除/过表达细胞,以研究ULK1在体外和体内的功能作用。主要通过 qPCR、western blot 和 ELISA 等方法研究了 ULK1 缺失激活 GSMDE 的机制:结果:通过高通量筛选,我们发现 ULK1 是 MDM2 抑制剂 APG115 的合成致死基因。结果:通过筛选,我们发现 ULK1 是 MDM2 抑制剂 APG115 的合成致死基因。从机理上讲,p53通过直接介导GSDME转录来诱导基础水平的热休克,从而促进热休克的启动。此外,ULK1 的耗竭会降低有丝分裂,导致受损线粒体的积累和随后活性氧(ROS)的增加。这反过来又会通过 NLRP3-Caspase 炎症信号轴裂解并激活 GSDME。这一分子级联使 ULK1 成为 p53 激活细胞介导的热凋亡启动的关键调节因子。此外,有丝分裂在铂耐药肿瘤中得到增强,ULK1耗竭/p53激活对这些肿瘤具有协同致死效应,直接通过GSDME诱导热噬:p53在激活GSDME转录中起着直接作用,而ULK1的缺乏会引发ROS-NLRP3信号通路的上调,从而导致GSDME的裂解和激活。这些发现强调了 p53 在决定化脓过程中的关键作用,为 p53 恢复疗法的临床应用提供了新的途径,并提出了潜在的组合策略。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Synthetic lethality of combined ULK1 defection and p53 restoration induce pyroptosis by directly upregulating GSDME transcription and cleavage activation through ROS/NLRP3 signaling.

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.

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来源期刊
CiteScore
18.20
自引率
1.80%
发文量
333
审稿时长
1 months
期刊介绍: The Journal of Experimental & Clinical Cancer Research is an esteemed peer-reviewed publication that focuses on cancer research, encompassing everything from fundamental discoveries to practical applications. We welcome submissions that showcase groundbreaking advancements in the field of cancer research, especially those that bridge the gap between laboratory findings and clinical implementation. Our goal is to foster a deeper understanding of cancer, improve prevention and detection strategies, facilitate accurate diagnosis, and enhance treatment options. We are particularly interested in manuscripts that shed light on the mechanisms behind the development and progression of cancer, including metastasis. Additionally, we encourage submissions that explore molecular alterations or biomarkers that can help predict the efficacy of different treatments or identify drug resistance. Translational research related to targeted therapies, personalized medicine, tumor immunotherapy, and innovative approaches applicable to clinical investigations are also of great interest to us. We provide a platform for the dissemination of large-scale molecular characterizations of human tumors and encourage researchers to share their insights, discoveries, and methodologies with the wider scientific community. By publishing high-quality research articles, reviews, and commentaries, the Journal of Experimental & Clinical Cancer Research strives to contribute to the continuous improvement of cancer care and make a meaningful impact on patients' lives.
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