Abstract: Pancreatic ductal adenocarcinoma (PDA) is one of the most difficult challenges in oncology. Activating mutations in the K-ras oncogene are found in 95% of PDA cases, but agents are not yet available that can effectively target this (or any other) high prevalence alteration in PDA. An alternative strategy is to target critical biological processes that PDA cells depend on but normal cells can forego. An example of this is the import of exogenous cysteine (in the oxidized form of cystine) via the cystine/glutamate antiporter called System xc?. The inhibition of System xc? in many cancer cell lines has been shown to induce a peculiar form of non- apoptotic cell death, called ferroptosis, which is mechanistically distinct from necroptosis, autophagy, parthanatos, and other forms of non-apoptotic cell death. It is characterized by the rapid, iron-dependent accumulation of lipid ROS leading to loss of membrane integrity in the absence of DNA cleavage. Despite the dramatic effects of System xc? inhibition in tumor cells, germline System xc? knockout mice are viable and healthy as adults, proving that normal cells do not usually require cystine import. Cysteine is the rate-limiting precursor for the synthesis of glutathione (GSH), a non-protein tripeptide that is critical for the detoxification of reactive oxygen species (ROS). Ferroptosis can also be induced by inhibitors of glutathione peroxidase 4 (GPX4) which detoxifies lipid peroxides using GSH as a co-factor. Yet depletion of GSH itself has not been shown to induce ferroptosis, for reasons that are unclear. We hypothesize that depletion of cysteine is qualitatively distinct from the depletion of glutathione and that additional cysteine?derived metabolites play a critical role in the detoxification of lipid ROS and control of ferroptosis. Several inhibitors of System xc? have been identified that effectively induce ferroptosis in vitro, including erastin, sulfasalazine, and sorafenib, and new inhibitors are rapidly being developed. The overarching goal of this proposal is to determine the underlying mechanisms of ferroptosis induction through System xc? inhibition, including the identification of determinants of ferroptosis sensitivity. We bring to bear a range of innovative tools, including cysteine and methionine carbon labeling, mass spectrometry, chemical biology approaches, inducible lentiviral shRNA knockdown of key metabolic enzymes, systems biology techniques, small animal imaging, and translational therapeutics using genetically engineered mouse models. In addition to 2D cell line and organoid culture models, we also present pilot data from a sophisticated genetically engineered mouse model that enables the acute deletion of System xc? in established K-ras/p53 mutant pancreatic tumors. This six-allele dual recombinase mouse strategy provides an ideal genetic strategy for the evaluation of System xc? function in PDA and serves as a source for genetically?defined primary cells to facilitate our proposed mechanism studies. Finally, we will evaluate a candidate mechanistic pathway using a novel combination of two repurposed, clinically?developed agents, each of which is individually well-tolerated.