PROJECT SUMMARY Bladder cancer is responsible for 80,000 cancer diagnoses and 20,000 deaths annually in the US. Approximately one-fourth of bladder cancer patients will be diagnosed with muscle-invasive bladder cancer (MIBC), an aggressive but potentially curable disease state. In the US, the most common treatment for MIBC is bladder removal (i.e., radical cystectomy), but a bladder-sparing approach using concurrent chemoradiotherapy (CRT) is associated with similar disease outcomes and improved quality-of-life. MIBCs harbor numerous recurrent genetic alterations, including frequent alterations in DNA repair pathway genes, particularly nucleotide excision repair (NER; ~15% of tumors) and homologous recombination repair (HRR; ~10% of tumors), and we and others have shown that NER and HRR deficiency confers unique biological properties and therapeutic sensitivities. In the past two years, two antibody-drug conjugates (ADCs) have been approved for treatment of chemotherapy- refractory metastatic bladder cancer: enfortumab vedotin (EV) combines a nectin-4 targeting antibody with the microtubule poison vedotin whereas sacituzumab govitecan (SG) combines a trop-2 targeting antibody with the type I topoisomerase inhibitor govitecan. Multiple EV and SG clinical trials are now on-going in multiple bladder cancer settings; however, the interaction of ADCs with RT in bladder cancer has not been investigated. Our central hypothesis is that radiation upregulates nectin-4 and trop-2 expression on bladder cancer cells, and therefore that combining EV or SG with radiation is a compelling synthetic combination strategy for MIBC treatment. Our hypothesis is supported by preliminary data from a bladder cancer cell line demonstrating radiation-induced increase in nectin-4 and trop-2 expression as well as synergistic cell killing when EV or SG is combined with radiation. However, comprehensive analysis across preclinical models that represent the molecular diversity of MIBC is currently lacking. The overall goal of the proposed work is to investigate the impact of radiation on expression of nectin-4 and trop-2 in bladder tumors and to define the activity of EV and SG with radiation across molecularly annotated human MIBC preclinical models. In Aim 1, we will measure radiation dose- and time-dependent changes in nectin-4 and trop-2 at the gene and protein levels across bladder cancer cell lines and patient-derived xenograft models. We will also test the combined effect of EV plus RT and SG plus RT on cellular properties and tumor response in vitro and in vivo. In Aim 2, we will test the impact of NER and HRR deficiency on sensitivity to EV- and SG-based treatments using a suite of isogenic cellular systems available in our lab. Given the known mechanisms of vedotin and govitecan, we expect NER and HRR deficiency to increase sensitivity to SG- but not EV-based radiation combinations. Together, we expect these studies to reveal a novel and compelling synthetic combinati...