Melanoma is the deadliest form of skin cancer and expected to cause 8000 deaths in the United States in 2019. Although treatment with anti-CTLA-4 and anti-PD-1 immune checkpoint inhibitors has dramatically improved survival in advanced melanoma, many patients do not benefit from these therapies or relapse after an initial period of response. There is a clear need to identify pathways and targets that enhance therapeutic responses to immune checkpoint inhibitors in melanoma and other cancers. Ferroptosis is an iron-dependent form of regulated cell death that is morphologically, biochemically, and genetically distinct from apoptosis, autophagy, pyroptosis, and necroptosis. Dysregulation of ferroptosis has been linked to the development of multiple cancers including melanoma, and it was recently demonstrated that immune checkpoint inhibition enhances ferroptosis, and that increased ferroptosis contributes to the anti-tumor efficacy of immunotherapy. Our laboratory completed an in vivo, whole genome CRISPR screen in our YUMMER melanoma model. Data from this screen identified several ferroptosis genes as regulating anti-cancer immunity. Based on this preliminary data, I hypothesize that specific ferroptosis regulating genes are critical to an effective antitumor immune response and that inducers of ferroptosis will synergistically enhance the efficacy of immunotherapies. Aim 1 will further characterize ferroptosis gene hits from our CRISPR screen and determine whether in vivo knockout of these hits can alter tumor growth and response to immune checkpoint inhibitors. Aim 2 will examine the ability of ferroptosis inhibitors to impair the effects of immune checkpoint blockade, test the combined effects of ferroptosis inducers and immune checkpoint inhibitors, and determine the immune cells mediating any synergistic effect. Completion of this proposal will provide a preclinical framework to assess the therapeutic potential of ferroptosis inducers in combination with existing immunotherapy regimens and a genetic framework to understand these interactions.