Pancreatic cancer is characterized by poor overall survival and few efficacious therapeutic interventions. One area that has offered significant scope for further exploration are the numerous metabolic disturbances in preclinical models of pancreatic cancer. Given the relative hypoxia of the tumor microenvironment and the limited nutrient availability, pancreatic tumor cells undergo substantial metabolic rewiring. Several of these derangements lead to imbalances in metabolites, including S-adenosylmethionine (SAM), α-ketoglutarate (αKG), and acetyl-CoA that are crucial to the regulation of epigenetic landscapes. Previously, we have demonstrated that pancreatic cancer initiation features abundant epigenetic reprogramming in the progression to neoplasia, and that the cytoplasmic enzyme ATP citrate lyase, which produces acetyl-CoA from citrate, is essential to the neoplastic process. In mouse models, Acly deletion diminishes the enhancement of histone acetylation that occurs in response to mutant Kras. The role of mitochondrial generation of the ACLY substrate citrate in acetyl- CoA availability, and the downstream effect of mutant Kras-induced acetyl-CoA on chromatin landscapes both remain unknown. Our long-term goals are to identify how metabolic rewiring impacts the epigenome to facilitate alterations to cell fate in cancer. In this proposal, we intend to utilize acetyl-CoA to define the mitochondrial-to-nuclear connections that control epigenetic reprogramming in pancreatic cancer initiation. Our aims are to elucidate (1) the mechanisms by which mitochondrial function controls the availability of exported citrate and nucleo-cytoplasmic acetyl-CoA, and (2) the global effect of alterations in acetyl-CoA abundance on the epigenome and preneoplastic cell fate. We will utilize several perturbations to citrate synthesis and export as well as to mitochondrial structure and function to delineate cross-talk between the mitochondria and the cytosol in controlling acetyl-CoA. In addition, we will utilize acinar explants in vitro and lineage-traced mouse models of acinar-derived neoplasia to interrogate the chromatin accessibility and histone acetylation dynamics induced by alterations to acetyl-CoA. These aims therefore discriminate mitochondrial-to-cytosol and cytosol-to-nucleus relationships, respectively, in the control of the epigenetic metabolite acetyl-CoA. In turn, we intend for this demonstration of inter-organelle communication in pancreatic cancer initiation to reveal mitochondrial control of acetyl-CoA as a therapeutic vulnerability in this otherwise deadly disease.