PROJECT SUMMARY Metastasis is responsible for more than 90% of cancer patient mortality yet there are no therapies that specifically target metastatic disease. Many of the current in vitro models of metastasis focus on the molecular mechanisms of migration, invasion and/or surviving anoikis, but cannot recapitulate the complexity of the environment in which metastasis occurs in vivo. Conversely, in mouse models of metastasis, it has been difficult to examine the molecular mechanisms that enable cells to proceed through each distinct step of metastasis due to limited material that can be isolated and infrequency of metastatic events in these models. For these reasons little is known about the challenges facing metastasizing cells in vivo, and how they are overcome. We have previously established a clinically relevant model of melanoma metastasis, using patient-derived xenografts (PDX) in immunocompromised mice, that recapitulates the outcome of the disease of the patient in mice, to dissect the metastatic cascade into distinct steps. Using this model, we have shown that metastasizing melanoma cells undergo reversible metabolic adaptations to withstand oxidative stress in part through an increased dependence on NADPH-generating enzymes in the one-carbon pathway. Our preliminary data also show an increase in NADP+ levels in metastatic nodules compared to subcutaneous tumors, suggesting an increase in de novo NADP+ synthesis. NADP+ is generated from NAD+ by NAD+ kinase (NADK). We observe higher levels of NADK in metastatic nodules compared to subcutaneous tumors, where metastatic nodules express the isoform of NADK with the highest activity, while subcutaneous tumors do not. We will test the hypothesis that metastasizing melanoma cells upregulate a specific isoform of NADK to increase NADP+ production, increase oxidative stress resistance and survival at different steps of the metastatic cascade. Using both melanoma cell lines and PDX tumor cells, Aim 1 will determine the role of different NADK isoforms in oxidative stress resistance, Aim 2 will define the mechanism of transcriptional regulation of NADK isoforms, and Aim 3 will establish the role of different NADK isoforms as metastatic drivers in vivo. In addition, Aim 3 will test how perturbation of oxidative stress in different organelles impacts metastasis. Together this work will significantly contribute to our understanding of a novel mechanism of metabolic plasticity through upregulation of a specific NADK isoform and identify organelle-specific metabolic pathways as novel therapeutically targetable vulnerabilities in melanoma metastasis.