PROJECT SUMMARY Many common oncogenes and tumor suppressors directly regulate metabolic pathways that support cancer cell survival, growth and proliferation. Metabolites also contribute to the regulation of the chromatin landscape: multiple cellular metabolites serve as critical co-substrates of enzymes that deposit or remove chemical modifications on histones and DNA. Oncogenic mutations in several metabolic enzymes result in the pathological accumulation of metabolites that interfere with normal maintenance of histone and DNA modifications. However, absent these specific metabolic mutations, whether the more general cancer- associated metabolic alterations driven by common oncogenes and tumor suppressors likewise affect the regulation of the chromatin landscape remains poorly understood. Using mouse models of pancreatic cancer harboring reversible expression of the tumor suppressor p53, we discovered that p53 controls levels of intracellular alpha-ketoglutarate (αKG), an obligate co-substrate of a family of αKG-dependent dioxygenases that includes the ten-eleven (TET) family of DNA methylcytosine oxidases. Restoring p53 function in malignant pancreatic cancer cells triggered intracellular αKG accumulation, which was both necessary and sufficient to increase markers of TET activity, induce tumor cell differentiation and blunt tumor progression. Our findings raise the possibility that p53-mediated accumulation of αKG and concomitant changes in the chromatin landscape and gene expression profiles contribute to the tumor suppressive function of wild-type p53. The goal of this work is to determine how wild-type p53 functions to regulate cellular αKG levels in response to oncogenic stress and how αKG contributes to p53-mediated tumor suppression. We hypothesize that regulation of metabolic pathways by p53 promotes accumulation of αKG, thereby activating gene expression programs that safeguard against malignant progression. To address this hypothesis, we will determine the mechanisms by which p53 regulates αKG (Aim 1); elucidate the pathways through which αKG induces tumor differentiation (Aim 2), and test whether αKG is a barrier to malignant progression (Aim 3). The proposed experiments will reveal how metabolic alterations that commonly occur in human tumors contribute to the maintenance of the malignant state and identify pathways that can be targeted to enforce tumor suppressive outputs even in malignant cells.