CRL3KEAP1 complex licenses genotoxic stress-induced tumor cell death

项目总结/摘要 PA-17-449：癌细胞中细胞死亡途径的相互作用 存活率和对治疗的抗性（R21）。一般来说，如果肿瘤细胞发生凋亡， 细胞表达功能性p53。遗传毒性应激也可诱导p53非依赖性凋亡， XIAP和cIAP 1/2的自身消耗，这导致“ripoptosome”复合物的形成。 核糖体可以刺激caspase-8介导的细胞凋亡。突变的p53和半胱天冬酶-8 在人类癌症中经常被检测到。Caspase-8突变或失活通常导致 抗凋亡。然而，在少数情况下，它使细胞对另一种细胞高度敏感。 程序性细胞死亡的一种形式，称为“坏死性凋亡”。激酶RIPK 3及其底物MLKL是 坏死性凋亡的关键因素尽管一些研究表明，DNA损伤可以 诱导坏死性凋亡（或不确定的坏死），分子机制在很大程度上是未知的。在此， 我们使用了一种无偏见的全基因组CRISPR敲除方法来识别新的参与者， DNA损伤诱导的坏死性凋亡。我们鉴定了Cullin 3（Cul 3）-RING E3泛素连接酶 （CRL 3 KEAP 1）复合物作为一个有希望的候选者，介导DNA损伤诱导的坏死性凋亡。 我们的工作模型是，CRL 3 KEAP 1复合物需要降解一种未鉴定的底物， 让DNA损伤诱导的坏死性凋亡进展，从而许可基因毒性诱导的细胞 死亡我们将在本提案中努力检验这一假设。我们将决定 CRL 3 KEAP 1底物是什么以及它如何阻止细胞死亡。拟议的研究将 为DNA损伤剂诱导的细胞死亡提供了新的机制见解。我们不断努力 有可能改变我们触发肿瘤细胞死亡的方式，特别是在那些有p53的肿瘤中， caspase-8和/或KEAP 1突变。

PROJECT SUMMARY/ABSTRACT This application is responsive to PA-17-449: The Interplay of Cell Death Pathways in Cancer Cell Survival and Resistance to Therapy (R21). In general, genotoxic stress triggers apoptosis if tumor cells express functional p53. Genotoxic stress can also induce p53 independent apoptosis due to the auto-depletion of XIAP and cIAP1/2, which leads to the formation of “ripoptosome” complex. Ripoptosome can stimulate caspase-8-mediated apoptosis. Mutated p53 and caspase-8 have been frequently detected in human cancers. Caspase-8 mutation or inactivation often leads to resistance to apoptosis. However, in a few cases, it renders cells highly susceptible to another form of programmed cell death, termed “necroptosis.” Kinase RIPK3 and its substrate MLKL are the critical players for necroptosis. Although several studies have indicated that DNA damage can induce necroptosis (or undefined necrosis), the molecular mechanism is largely unknown. Herein, we have used an unbiased genome-wide CRISPR knockout approach to identify new players in DNA damage-induced necroptosis. We identified Cullin3 (Cul3)-RING E3 ubiquitin Ligase (CRL3KEAP1) complex as a promising candidate that mediated DNA damage-induced necroptosis. Our working model is that CRL3KEAP1 complex needs to degrade an unidentified substrate(s) to let the progression of DNA damage-induced necroptosis, thus licensing genotoxicity-induced cell death. We will make efforts to test this hypothesis in this proposal. We will determine what the CRL3KEAP1 substrate(s) is(are) and how it(they) can halt cell death. The proposed studies will provide new mechanistic insights on DNA damage agents induced cell death. Our ongoing efforts have the potential to change how we trigger cell death in tumors, especially in those with p53, caspase-8, and/or KEAP1 mutations.