PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDA) is an aggressive malignancy that remains largely incurable. The dismal prognosis of PDA reflects its advanced disease stage at diagnosis and its profound resistance to existing therapies. The KRAS oncogene is mutated in 95% of PDAs and acts as a potent driver of PDA growth and maintenance. Oncogenic KRAS induces transcriptional upregulation of NFE2L2, which encodes the NRF2 transcription factor, a master regulator of redox homeostasis that protects cells from the cytotoxic/cytostatic effects of reactive oxygen species (ROS). Importantly, we found that genetic ablation of NRF2 suppresses the growth of pancreatic cancer cells both in vitro and in vivo (2). Although elevated levels of ROS are generally believed to induce cytotoxicity through irreversible damage to macromolecules, particularly DNA and lipids, we discovered that genetic ablation of NRF2 did not lead to DNA or lipid damage (2). Instead, both protein synthesis and tumor cell fitness were compromised as a consequence of reversible and selective oxidation of cysteine residues on key regulators of protein synthesis (2). Based on these observations, we hypothesize that NRF2 promotes pancreatic tumorigenesis through its ability to regulate oxidative post-translational modifications (oxPTM). Herein, we propose to elucidate the mechanisms underlying this process, determine the functions of redox control in PDA, and identify potential therapeutic targets. To this end, we will use ChIP- seq and RNAseq to identify direct Nrf2 targeting genes that govern cysteine oxidative modification (Aim 1). In addition, we will use a variety of biochemical approaches to delineate the mechanisms through which cysteine oxidation contributes to protein synthesis in PDA. We will further define transcript-specific effects of redox- dependent translation regulation through ribosome profiling (Aim 2). In addition to cysteine, we recently discovered that perturbation of NRF2 activity also leads to the reversible oxidation of the other sulfur-containing amino acid, methionine. Various biochemical and genetic approaches will be taken to assess the functional role of methionine oxidation in pancreatic tumorigenesis (Aim 3). We anticipate that our results will explain fundamental aspects of redox homeostasis in PDA and will inform the development of more effective therapies for pancreatic cancer and potentially other KRAS-driven malignancies.