PROJECT SUMMARY Acinar-to-ductal metaplasia (ADM) is a regenerative state that leads to repair of the pancreas after injury. During ADM, acinar cells transdifferentiate to a duct-like cell and become proliferative. ADM is typically reversible; however, ADM is also a risk factor for the development of pancreatic ductal adenocarcinoma. Activating mutations in KRAS lead to persistent ADM and progression to pancreatic intraepithelial neoplasia (PanIN) and cancer. Many studies describe how cell metabolism is reprogrammed in cancer, though little is known about the role of metabolism in regulating precancerous stages, like ADM and PanIN. I hypothesize that 1. cell metabolism is altered in ADM to upregulate redox homeostasis, and 2. healthy acinar cells maintain a metabolic microenvironment that is restrictive for ADM and PanIN progression. Previous work shows that genes for NADPH-producing enzymes, Glucose-6-phosphate dehydrogenase (G6pd) and Malic enzyme 1 (Me1) are upregulated during ADM to maintain redox homeostasis and glutathione recycling. Preliminary also suggest that glutathione biosynthesis pathways are necessary for controlled ADM development. Aim 1 will mechanistically focus on Me1 and determine how Me1-loss contributes to ADM and PanIN formation. The experiments proposed in Aim 1 use genetically engineered mouse models of pancreatic cancer, steady-state metabolomics, isotope tracing, and ex vivo primary acinar cell culture. Aim 2 will address if altered glutathione biosynthesis promotes ADM formation. It will interrogate if blocking cystine import (via loss of a subunit of the system xC– antiporter) and a rate-limiting enzyme in glutathione synthesis increases reactive oxygen species in the cell and promotes ADM. Aim 3 (R00 focus) will determine if healthy acinar cells contribute to a restrictive environment for the development of ADM and PanIN, even when oncogenic Kras is present. Preliminary experiments suggest that healthy acinar cells secrete metabolites to inhibit adjacent cells from undergoing ADM. Pilot spatial transcriptomic experiments have also identified potential metabolic genes important for ADM restriction. This aim uses inducible mouse models of Kras-driven pancreatic cancer, metabolomics, and spatial transcriptomics. Together, the aims presented in this proposal will provide new mechanistic insights on how metabolic pathways control the formation of precancerous states in the pancreas.