Metabolism is extensively rewired in cancer cells to support biosynthesis, growth, and proliferation. It has recently become apparent that many histone and DNA modifications are sensitive to cellular metabolic state. Histone acetylation is a dynamic chromatin modification, and global levels of histone acetylation are responsive to availability of the metabolite acetyl-CoA. However, the functional significance of metabolic control of histone acetylation remains poorly understood. The Wellen lab has recently shown that oncogenic activation of the PI3K-Akt pathway in cancer cells promotes acetyl-CoA production and elevated histone acetylation levels, indicating that normal metabolic controls on histone acetylation are disrupted in cancer cells. Extensive chromatin modifications, including histone acetylation, play crucial roles in the regulation of DNA repair. The Greenberg lab has recently shown that acetylation of histone H4 lysine 16 (H4K16ac) at sites of DNA double strand breaks (DSBs) plays a key role in recruitment of DNA repair proteins such as BRCA1 and utilization of DNA repair mechanisms. DSBs are repaired either by homologous recombination (HR) or non-homologous end joining (NHEJ). HR deficiencies, such as occur with BRCA1/2 mutations, result in genome instability and are a major cause of cancer predisposition and response to emerging chemotherapies. Since histone acetylation is responsive to acetyl-CoA availability and acetyl-CoA metabolism is frequently deregulated in cancer cells, we postulated that acetyl-CoA might impact H4K16ac levels at DSB sites to modulate the DNA repair efficiency. Our preliminary data shows that manipulation of nuclear-cytoplasmic acetyl-CoA levels by silencing the metabolic enzyme ATP-citrate lyase (ACLY) indeed impacts the recruitment of repair proteins to DSB sites, with recruitment of pro-NHEJ factor 53BP1 favored over pro-HR BRCA1 upon ACLY silencing. In this R21 application, we propose to test the hypothesis that oncogene-driven glucose uptake and acetyl- CoA production impact the acetylation state at DSBs and modulate the response to DNA damaging agents. We will pursue this hypothesis by first testing whether acetyl-CoA availability is indeed a critical determinant o DNA repair mechanism by affecting the balance of HR and NHEJ factors at DSBs. We will then examine the influence of altered acetyl-CoA metabolism mediated by the PI3K-Akt and LKB1-AMPK-ACC1 pathways on the DNA repair mechanism. This study has potential to identify previously unrecognized links between cellular metabolism and the DNA damage response and to identify new approaches to sensitize cells to chemotherapeutics.