PROJECT SUMMARY Cisplatin (Cis)-based Neoadjuvant chemotherapy (NAC) is the standard of care prior to cystectomy, for patients with muscle-invasive bladder cancer (MIBC). Up to 30% of patients respond and show no residual tumor at cystectomy with >80% survival, but “non responders” have <30% chance of surviving 5 years. Thus, improving the effectiveness of Cis-based NAC will greatly improve outcomes in BC. Through whole-genome CRISPR-Cas9 synthetic lethal screens in Cis-resistant human BC cell lines, we discovered NPEPPS as a novel and druggable target whose expression determines sensitivity to Cis. NPEPPS was the only one of 13 M1 aminopeptidases found to be synthetic lethal with Cis. Depletion of NPEPPS enhanced Cis therapy and reduced growth in animal models. To find how NPEPPS drives these two phenotypes, we used mass spectrometry (MS) to identify the proteins that are in complex with NPEPPS in BC cells. We found NPEPPS in complex with subunits (LRRC8A- E) of the volume regulated anion channel (VRAC), a recently identified mechanism of platinum (Pt) cellular import. In BC cells, LRRC8A/D depletion increases resistance to both cisplatin and carboplatin, while NPEPPS depletion had the opposite effect. Supporting a role in human BC growth, NPEPPS expression is associated with poor patient outcome regardless of chemotherapy use. Leveraging our MS results, we developed an approach to prioritize candidate genes found in complex with NPEPPS that most likely affect growth, are associated with aggressive disease, and are prognostic markers. Thus, we propose the Guiding Hypothesis that NPEPPS drives Pt resistance and tumor growth in BC by inhibiting VRAC activity and interacting with genes regulating cell proliferation respectively. Specific Aims test this hypothesis with the Objective to lay the foundation for novel approaches to improve the outcomes for BC patients. In Aim 1 we will test the hypothesis that NPEPPS aminopeptidase activity is required for Pt resistance and growth using enzymatically dead NPEPPS mutants in vitro and in vivo. Next, we evaluate our top candidate gene CHD2, a chromatin regulator and putative tumor suppressor for its role in NPEPPS-driven tumor growth and the dependency of this role on NPEPPS enzymatic activity. In Aim 2 we will determine the role of LRRC8A/D in NPEPPS-mediated Pt resistance. We have used molecular modeling to identify residues on NPEPPS that interact with LRRC8A/D. Site-directed mutagenesis of these residues will test the hypothesis that direct interaction of NPEPPS with LRRC8A/D reduces the ability of VRACs to properly function and contributes to NPEPPS-mediated Pt resistance in vitro and in vivo. To establish the preclinical rationale for the effect of novel, anti-neoplastic agents that circumvent Pt resistance, Aim 3 will test the hypothesis that Tosedostat (Tose), a clinically well-tolerated aminopeptidase inhibitor, enhances the sensitivity of BC to Pt, and NPEPPS expression is required for this. The ...