The deadliest complication of late-stage melanoma is leptomeningeal metastasis (LMM), where the cells spread to the cerebrospinal fluid (CSF) space and the membrane coverings of the brain and spinal cord. Very little is known about the biology of this disease and there are no FDA-approved therapies for LMM. This highlights a major gap in knowledge and an unmet need in the clinical care of late-stage melanoma patients. Our single-cell RNAseq studies of melanoma patient specimens demonstrate that the LMM microenvironment is uniquely immune-suppressed and is dominated by inactive, dysfunctional T cells and alternatively activated macrophages. The tumor cells in the LMM upregulate mediators of lipid metabolism, including enzymes involved in fatty acid, lipid and carnitine synthesis. This is coupled with an increase in exogenous carnitines and lipids found in the cerebrospinal fluid of patients with LMM compared to those without. Although the expression of PD1 on CD8+ T cells in the LMM are comparable to those found at other sites of metastasis, tumors at LMM do not respond to PD1 inhibition despite responding at other sites of disease. We will test the hypothesis that increased lipid metabolism in the tumor and upregulation of exogenous fatty acids, lipids and carnitines fuel a shift in the metabolism of infiltrating macrophages and CD8+ T cells to favor fatty acid oxidation and increased oxidative phosphorylation, supporting immune dysfunction at the leptomeninges. We will utilize our extensive single-cell RNAseq data from LMM patient and mouse samples to create a comprehensive map of metabolic processes in the immune microenvironment surrounding the upregulated lipid metabolism in the tumor. Stable isotope tracing, targeted metabolic assays and media supplementation and depletion will be performed on patient macrophage and CD8+ T cells in the context of autologous LMM-derived CSF and primary tumor cultures to measure the effects of exogenous carnitines, fatty acids and lipids on promoting fatty acid oxidation and oxidative phosphorylation. Previous studies suggest lipid metabolism plays a critical role in mediating immune responses in cancer and support tumor survival under conditions of metabolic stress. We will determine if targeting the lipid metabolism using inhibitors of fatty acid synthase or CPT1a would promote tumor cell death in the nutrient poor LMM microenvironment and restore anti-tumor function of macrophages and CD8+ T cells in LMM. We will utilize primary cultures of human LMM in co-culture with autologous macrophages and T cells to examine if targeting fatty acid synthase or CPT1a would promote anti-tumor polarization of macrophages and activation of T cells. We will use in vivo models of LMM to test if inhibition of fatty acid synthase or CPT1a will attenuate the immune- suppressive environment and sensitize the tumors to anti-PD1 therapy. Overall, this proposal will identify the metabolic drivers promoting the pro-tumorigenic inte...