PROJECT ABSTRACT/SUMMARY During tumorigenesis, mitochondrial function is altered by fusion/fission dynamics that control organelle structure and impact overall cell metabolism. Signaling from oncogenic RAS fragments mitochondrial tubules and causes metabolic changes that support tumor growth. However, the precise role of these oncogene- driven mitochondrial changes on cancer metabolism is unclear, especially when considering the diverse metabolic environments in which tumors develop. For example, colorectal cancer (CRC) initiates in the gut where the microbiota produces high quantities of short chain fatty acids (SCFAs) that are metabolized by normal colonocytes. During CRC tumorigenesis, mutations in the KRAS oncogene occur at the transition to adenomas, suggesting that mitochondrial adaptation in the gut may be critical for progression of primary tumors. And yet, the primary site of CRC metastasis is the liver, which provides a very different set of nutrients for metabolism and growth. Recognizing the complexity of metabolic networks both inside and outside a developing tumor, we propose a systems biology approach to examine the role of RAS-induced mitochondrial fission in CRC. Specifically, our objective is to identify metabolic adaptations that permit mitochondrially fragmented CRC cells to grow in the unique metabolic environment of the gut and metastatic CRC cells to grow in the liver. While the fragmentation of the mitochondrial network can impact tumor metabolism in multiple ways, we hypothesize that RAS-induced mitochondrial fragmentation leads to hyper- compartmentalization of specific metabolic reactions within the mitochondrial matrix that depend on low- abundance mitochondrial proteins. We predict that these adaptations create unique vulnerabilities in CRC cells as they switch from normal SCFA metabolism to promote biosynthesis and energy generation. The specific aims are to 1) curate a metabolic model of human CRC cells that incorporates the system-wide impact of mitochondrial fragmentation and the availability of microbe-derived SCFAs; 2) instantiate metabolic models of CRC with data characterizing in vivo metabolic states to assess impacts of gut microbiota metabolism and mitochondrial fragmentation; and 3) evaluate the impact of metabolic adaptations to mitochondrial organelle stress on CRC colonization and growth as liver metastases. Successful completion of this project will provide a better understanding of CRC metabolism that may one day point to dietary interventions or shifts in the gut microbiota that predictably influence organelle adaptation.