Project Summary: Cancers require metabolic adaptations to support the unbridled proliferation that drives tumor growth. Mutations in the mitochondrial genome (mtDNA) are observed in many cancers, but the role of these mutations in shaping cellular metabolism and tumor growth is incompletely understood. mtDNA mutations that impair components of the electron transport chain (ETC) appear to be selected against in most forms of cancer. Hürthle cell carcinoma of the thyroid (HTC) is clinically aggressive cancer uniquely enriched for loss-of-function mtDNA mutations in components of complex I of the ETC. We propose that HTC represents an ideal disease outlier in which to interrogate the role of mtDNA alterations and ETC function in cancer. In this proposal, we employ unique and highly complementary approaches to characterize the metabolic impact of mtDNA mutations in HTC and other forms of thyroid and kidney cancer. First, we have developed a clinical protocol to monitor central carbon directly in surgical patients using stable isotope tracing (Aim 1). Second, we have identified a synthetic lethal interaction encoded by complex I mutation and identified a promising small molecule therapeutic using patient-derived models (Aim 2). Finally, we have developed novel GEMMs from which to interrogate the role of complex I function in thyroid tumorigenesis (Aim 3). These approaches are highly complementary and synergistic, yet each is independently poised to bridge key knowledge gaps and lead to new insights into metabolic regulation in cancer. The overall goals of this proposal are to characterize the metabolic adaptations necessitated by complex I loss in HTC directly in patients undergoing thyroid surgery, to identify and target metabolic liabilities as a result of metabolic re-wiring downstream of complex I loss, and to determine whether complex I loss acts to promote or alter thyroid tumor formation in mice. These findings will be of immediate and direct relevance to thyroid cancer patients, provide new insights relevant to other tumors harboring mtDNA mutations and have broad implications across cancer types by providing new insights into ETC function in cancer.