Abstract Acquired drug resistance prevents chemotherapy and targeted therapy from achieving stable and complete responses. Emerging evidence implicates a key role for nonmutational mechanisms during early stages of acquired drug resistance. Drug-tolerant “persister” cells are a model of nonmutational cancer drug resistance in which small fractions (<5%) of cells within cancer cell lines survive cytotoxic drug exposure despite lacking resistance-conferring mutations. These residual surviving cells are quiescent, occupy a unique chromatin state, and exhibit reversible drug resistance: removal of drug allows regrowth of cells which become resensitized to drug treatment. Importantly, this phenomenon has been observed across a wide range of cancer lineages with both chemotherapy and targeted therapy. Persister cells remain quiescent for weeks to months during constant drug exposure, however some persister cells eventually regrow and acquire irreversible drug resistance-conferring mutations that did not preexist. This model mirrors the common clinical scenario in which tumor response is followed by relapse with acquired resistance-conferring mutations. Targeting nonmutational resistance may therefore present a therapeutic opportunity to eliminate residual surviving tumor cells to prevent relapse. We have performed an unbiased functional genomics analysis of HER2+ breast cancer persister cells which survive HER2 targeted therapy. Utilizing RNAseq, pathway analysis, RNAi screens and chemical inhibitor screens, we discovered that persister cells undergo a switch to mesenchymal gene expression and are uniquely susceptible to inhibition of the phospholipid hydroperoxidase GPX4. This finding is not restricted to breast cancer, rather we have observed that all persister cells tested including those derived from melanoma, lung, ovarian and pancreatic cancer, from either targeted therapies or chemotherapies, are each specifically sensitive to loss of GPX4 function. Parental, drug naive cancer cells and normal human cells are insensitive to GPX4 inhibition. Mechanistically, we found that GPX4 dependency in persister cells is due to a disabled antioxidant program characterized by global downregulation of antioxidant genes, decreased levels of reducing cofactors glutathione and NADPH, and immediate ROS increase upon GPX4 inhibition. These findings point toward a GPX4 inhibition strategy to prevent tumor relapse by inducing ferroptosis in the residual pool of surviving persister cells. Citation Format: Matthew J. Hangauer, Vasanthi S. Viswanathan, Matthew J. Ryan, Dhruv Bole, Jake Eaton, Stuart L. Schreiber, Frank McCormick, McManus T. Michael. Drug-tolerant persister cancer cells are vulnerable to GPX4 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1006. doi:10.1158/1538-7445.AM2017-1006