PROJECT SUMMARY The impact of mitochondrial biology on human cancers is broad because these organelles are critical regulators of metabolism, proliferation, metastasis, and cell death. Indeed, mitochondrial aberrations are common in multiple cancer types – not only do mitochondrial dysfunctions correlate with disease pathogenesis, but aberrant mitochondria also negatively impact upon chemotherapeutic success. Within a cell, mitochondrial homeostasis is maintained by a process referred to as “mitochondrial dynamics”, which is essential for efficient ATP generation, mitochondrial metabolites/substrates distribution, and mitochondrial DNA (mtDNA) integrity. Homeostatic mitochondrial dynamics result from the cumulative nature of complementary cycles of mitochondrial division and fusion. Work from my laboratory demonstrates: (1) the mitochondrial division machinery is essential for cellular transformation, (2) mitochondrial division is chronically activated in RAS-transformed murine cells and human cancer lines harboring oncogenic mutations within the MAPK pathway, (3) chronic mitochondrial division is sufficient to initiate mitochondrial dysfunction and cancer cell metabolism, and (4) FDA-approved targeted therapies that inhibit oncogenic MAPK signaling turn off the mitochondrial division machinery. While the above studies link chronic mitochondrial division to cancer biology, mechanistic explanations for how chronic mitochondrial division promotes organelle dysfunction and cancer phenotypes are scarce. In this R01 application, our goals are to (1) provide key mechanistic details into the process and contributions of mitochondrial dysfunction during oncogenic transformation, and (2) develop novel translational tools focused on the detection and inhibition of chronic mitochondrial division to enhance cancer prognosis and treatment. As our expertise and resources are in dermatology, we will focus on melanoma. The presence of mtDNA mutations and mitochondrial aberrations in cancer have been described for decades, but the molecular events that drive these changes and their impact on cancer biology remain speculative. To address this knowledge gap, we recently completed an unbiased screen using normal melanocytes and melanoma cell lines to understand how chronic mitochondrial division impacts on mitochondrial function, and identified that loss-of-function mtDNA mutations are essential for cancer cell metabolism, proliferation, and tumorigenesis. We hypothesize that oncogene- induced chronic mitochondrial division promotes mtDNA mutations and organelle heterogeneity to instigate transformation. Based on our data, chronic mitochondrial division is an early event during melanomagenesis, and provides strong prognostic value and therapeutic potential. This project emerged following years of effort to identify how chronic mitochondrial division impacts cancer mechanisms, prognosis, and treatment.