Project Summary African American (AA) bladder cancer (BLCA) patients have worse survival and oncologic outcomes compared to their European American (EA) counterparts. We contend that the lower survival of AA patients is driven in part by altered tumor-specific biological activities. We have identified through mass spectrometry, genome-wide transcriptomics, and functional biology in ancestry-verified cell lines and tumors from AA and EA BLCA patients a pattern of mitochondrial dysregulation and immune activation disproportionately present in AA BLCA patients. Based on this insight, we first characterized downstream metabolic changes and discovered that mitochondrial complex I dysregulation was observed more frequently in AA BLCA patients and correlated with more aggressive BLCA. In addition, targeted inhibition of complex I resulted in improved cancer outcomes in a preclinical model. Moreover, we found that AA tumors containing increased complex I activity demonstrated a higher glutamine flux into the TCA cycle by activation of GLS1, leading to accumulation of fatty acids via reductive carboxylation. Furthermore, AA tumors with increased mitochondrial complex 1 activity showed enhanced expression of the key complex I gene NDUFB8. Knockdown (KD) of NDUFB8 resulted in reduced mitochondrial complex 1 activity, glutamine metabolism, ATP production, and regression of BLCA growth. Lastly, we found that AA tumors with increased mitochondrial complex 1 activity also demonstrated concurrent activation of key immune signaling pathways, including IFN signaling pathways, suggesting a potential interaction between tumor metabolic activities and immune signaling. At the level of basic science, this project aims to identify the metabolic and immunologic basis of BLCA disparities in AA patients by metabolomic analysis of the OXPHOS-GLS1 metabolic axis and by using cutting-edge single-cell RNA sequencing and imaging mass cytometry to profile the tumor immune microenvironment and analyze tumor cell-immune cell interactions. At the translational level this work will lay the foundation to test therapeutic strategies targeting complex 1 alone or in combination with GLS1 inhibitor in AA BLCA patients. If successful, these metabolic therapeutic strategies may be further combined with immunotherapeutic interventions for AA BLCA patients.