Project Summary Colorectal cancer (CRC) is the second leading cause of cancer-related death in the US, with a 5-year survival rate of only 60%. The poor disease outcome associated with CRC highlights an urgent need to understand the cellular mechanisms that influence initiation and progression of CRC. Several of the well-known genetic drivers of CRC such as KRas and PIK3CA are dominant regulators of metabolic reprogramming during cancer progression. Altered tumor metabolism facilitates generation of molecules important for cell growth, signaling, and survival; yet, our knowledge of the precise mechanisms that regulate metabolism and survival in chemotherapy resistant CRC remains incomplete. One family of proteins important in coordinating metabolism with cellular survival and stress responses is the NAD+-dependent sirtuin superfamily. Our preliminary data demonstrate that the loss of a mitochondrial localized sirtuin, SIRT4, occurs in CRC and results in the reprogramming of nucleotide biosynthesis to shift metabolites away from salvage nucleotide metabolism and upregulate de novo nucleotide biosynthesis. We hypothesize this metabolic switch contributes to increased CRC cell proliferation and resistance to chemotherapy. Our proposal will test this hypothesis in two complementary, but independent Aims. First, using a biochemical approach, Aim 1 will examine the mechanism by which SIRT4-mediated metabolic reprogramming increases cell proliferation by examining SIRT4 activity and substrates. We will also examine the role of the metabolic by-products downstream of SIRT4-mediated activity. Next, Aim 2 will test the consequence of clinically relevant SIRT4 loss in physiological models of CRC using organoids, novel genetically engineered mouse models, and patient derived xenograft (PDX) models. Finally, we will examine the consequences of SIRT4 loss on CRC metabolism and chemotherapy resistance in vivo. This project will provide an unprecedented map of metabolic reprogramming in CRC at a single cell level and improve understanding of how CRC metabolism changes in the context of chemotherapy resistance, opening the door for development of novel therapeutic strategies that leverage mitochondrial metabolism to treat chemotherapy resistant cancers.