PROJECT SUMMARY/ ABSTRACT Every five minutes, a new patient is diagnosed with urothelial carcinoma (UC) in the United States, resulting in the death of 18,000 patients annually. Nearly all patients with advanced UC will develop resistance to systemic treatment. Intratumoral heterogeneity (ITH) is a major contributor to treatment resistance by increasing the chance for resistant subclones to emerge. However, genetic ITH is currently not druggable and not considered in therapeutic decision-making, thus worsening drug-resistant patient phenotypes. The fundamental knowledge gap regarding genetic drivers of ITH impedes the development of effective therapeutic strategies to prevent and eliminate drug resistance. Our long-term goal is to define targetable mechanisms of ITH and treatment resistance to develop an effective precision strategy to achieve cures in patients with advanced UC. The overall objective is to define targetable mechanisms by which APOBEC3A-mediated ITH drives drug resistance and identify strategies to eliminate UC cells with APOBEC3A activity. Our central hypothesis is that APOBEC3A-induced cytidine deamination drives genetic ITH leading to the emergence of therapy-resistant UC clones and, in so doing, simultaneously creating unique targetable vulnerabilities. This hypothesis was formulated based on our published work and strong preliminary data showing that APOBEC3A expression in isogenic UC cell lines and patient-derived organoids drives genetic ITH. We found that APOBEC3A-induced, de novo mutations in the PIKC3A-AKT-MTOR signaling hub drive the resistance to erdafitinib, an FGFR3-inhibitor (FGFR3i) approved for UC treatment. Our preliminary data also revealed that APOBEC3A-induced double-stranded DNA breaks are preferentially repaired by the microhomology-mediated end-joining (MMEJ) pathway and that targeting the critical MMEJ mediator, polymerase theta (Polθ), is synthetically lethal in APOBEC3A-expressing clones. The rationale is that identifying targetable mechanisms by which APOBEC3A-induced ITH drives drug resistance and developing strategies to eliminate APOBEC3A-expressing UC cells will improve cure rates for patients. We will test our hypothesis by pursuing two specific Aims. Aim 1: Identify targetable mechanisms by which APOBEC3A- induced mutational ITH drives treatment resistance in UC. Aim 2: Identify synthetic lethal strategies to target UC tumors with APOBEC3A-induced DNA double-strand breaks. Aim 1 will use longitudinal clonal barcoding and in vitro and in vivo laboratory evolution to identify targetable APOBEC3A-driven kinase hubs that mediate FGFR3i resistance and validate them in patient samples from FGFR3i clinical trials. Aim 2 will use genetic and pharmacologic inhibition of Polθ in APOBEC3A-expressing UC models and a co-clinical trial of patient-derived UC organoids and xenografts to identify clinical biomarkers of response to APOBEC3A-MMEJ synthetic lethality. The approach is conceptually and technically inno...