PROJECT SUMMARY Dysregulated cellular metabolism is a common characteristic of cancer, but a growing body of research highlights significant differences in metabolic phenotypes both within primary tumors, and in tumor cells that metastasize to distant organs. While highly proliferative primary tumor cells often exhibit the Warburg effect, marked by a preference for aerobic glycolysis to generate ATP, genes for mitochondrial biogenesis and oxidative phosphorylation are reported to be upregulated in metastatic cancer cells. Therefore, detailed characterization of reprogrammed metabolic pathways in lung cancer may reveal useful targets to combat tumor progression and improve patient survival. In order to metastasize, cancer cells must first resist anoikis: an apoptotic cell death mechanism triggered by loss of proper contact with the extracellular matrix. Our lab has previously demonstrated that the mitochondrial enzyme glutamate dehydrogenase 1 (GDH1) contributes to anoikis resistance and metastasis by regulating the bioenergetic response through reactivation of AMPK in LKB1-deficient lung cancer. However, the role of other mitochondrial enzymes in anoikis resistance remains poorly understood. To identify other factors important for cancer cell anoikis resistance, we performed an unbiased RNAi screen targeting 120 mitochondrial enzymes in lung cancer cells and identified the ATP- specific Succinyl-CoA Synthetase beta subunit (SUCLA2) as a factor that may be important for cancer cell survival after ECM detachment. Stable knockdown of SUCLA2 sensitized lung cancer cell lines to anoikis when cultured under non-adherent conditions in vitro. Bioinformatic analysis of publicly available data indicates that higher tumor SUCLA2 mRNA expression is associated with poor patient survival, and our immunohistochemistry staining suggests that SUCLA2 protein levels are higher in metastatic lung cancer compared to matched primary tumors. Additionally, our data suggests that SUCLA2 knockdown significantly increases reactive oxygen species (ROS) production and anoikis sensitivity in ECM detached cancer cells, which can be reversed with antioxidant NAC supplementation. In the first half of this research proposal, we will employ multiple approaches to decipher how SUCLA2 modulates cellular redox status to promote anoikis resistance and tumor metastasis. The second half of the proposal lays out a plan for a productive postdoctoral fellowship (K00) that will build on applicant's predoctoral F99 training and prepare him well to start a career as an independent cancer researcher.