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.