PROJECT SUMMARY Mitochondria created an evolutionary advantage for eukaryote and metazoan organization, and their impact on cell biology extends from anabolic and catabolic metabolism to determining the final moments of cell survival by engaging apoptosis. Throughout the last decade, interest in studying how mitochondria influence cancer cell biology led our laboratories to identify mechanisms linking oncogenic signaling (i.e., BRAFV600E / NRASG12V ) to multiple mitochondria-centric processes within malignant cells including altered mitochondrial dynamics, oxidative phosphorylation, and chemosensitivity. More recently, we focused on exploring how oncogenes intersect upon mitochondrial biology prior to transformation – which will likely provide molecular details into pre- malignant cell biology and early stages of disease. We commonly position our studies in the context of melanoma as we have extensive experience with primary human melanocytes, integrated cohorts of patient RNA-seq datasets and tissues, and multiple in vitro and in vivo models of early and late disease. For instance, the introduction of oncogenic signaling (BRAFV600E / NRASG12/Q60) in primary human melanocytes causes rapid oncogene-induced senescence (OIS), and this tumor-suppressive mechanism is reflected in patients who present with pre-malignant skin lesions in the clinic. Therefore, the scientific premise for this application is based on three novel observations: (i) primary human melanocytes expressing oncogenes rapidly expand their mitochondrial networks during OIS; (ii) this expansion is dictated by the undescribed activation of the ATF5- dependent mitochondrial unfolded protein response (mtUPR); and (iii) the mtUPR controls the rate and extent of OIS. While the mtUPR is a fundamental organelle-specific quality control signaling pathway that is essential to mitigate mitochondrial stress, no literature mechanistically connects oncogenic signaling to mtUPR activation, melanocyte biology, nor melanoma progression. In our preliminary experiments, we explored biochemical signaling, mitochondrial responses, cellular gain-of-function / loss-of-function approaches, and hundreds of patient samples to establish the hypothesis that the oncogene-activated ATF5-dependent mtUPR is a key signaling pathway that instructs melanocytes during OIS and its escape. In this R01 application, we propose three complimentary, but distinct, specific aims to examine this hypothesis using melanocytes, numerous models of early disease, and patient samples. Specific Aim #1: Interrogate the mechanistic relationship between oncogenic signaling, mtUPR, and OIS. Specific Aim #2: Identify the gene expression programs mediated and maintained by the mtUPR during OIS and early disease primary melanoma. Specific Aim #3: Define the impact of mtUPR activation in models of nevi and primary disease.