Defining Mechanisms of Progression and Treatment Resistance in Localized Bladder Cancer PI: Eugene Pietzak, MD SUMMARY Our overall goal is to develop therapies that selectively target molecular alterations responsible for progression of bladder cancers from non-invasive to the often-lethal muscle-invasive disease state. For patients with non- muscle invasive bladder cancer (NMIBC), the current standard is bacillus Calmette-Guérin (BCG), a nonspecific immunotherapy instilled directly into the bladder lumen. While BCG can reduce the risk of disease recurrence, a proportion of patients subsequently progress to muscle-invasive bladder cancer (MIBC). Our preliminary results indicate that this disease state, termed “secondary MIBC”, is resistant to cisplatin-based chemotherapy. The goals in the current proposal are to understand the genomic basis for treatment resistance to BCG and to identify alternative molecularly directed treatments that can achieve disease cure without the need for radical surgery. The studies proposed are based on preliminary data indicating that cytotoxic chemotherapy sensitivity in bladder cancer is influenced by somatic and germline genomic profiles, in particular mutations in DNA damage response (DDR) pathway genes, most commonly within the nucleotide excision repair gene ERCC2. As our preliminary data suggest that mutations in DDR pathway genes may also confer sensitivity to BCG, we hypothesize that prior treatment with BCG results in cross-resistance to subsequent systemic chemotherapy. To test this hypothesis, we will leverage several prospectively assembled bladder cancer cohorts, including tumor pairs collected pre-BCG and following progression to MIBC. These cohorts will be used to validate DDR mutations as predictors of BCG and cisplatin-based chemotherapy sensitivity and to identify mechanisms of progression from NMIBC to secondary MIBC. As genomic heterogeneity is common in bladder cancer, we will supplement bulk sequencing studies with multi-regional sequencing and analysis of cell-free DNA from urine to define the influence of tumor heterogeneity on cancer outcomes in early-stage bladder cancer. Our preliminary analyses of high-risk NMIBC and secondary MIBC have also identified ERBB2 mutations/amplifications as potential mediators of progression to muscle-invasive disease. Several unique patient cohorts will be used to define the frequency of ERBB2 mutation/ amplification and HER2 overexpression in high-risk NMIBC and secondary MIBC. Prior functional studies of the role of HER2 in bladder cancer pathogenesis have been impeded by a lack of patient-derived models with ERBB2 mutations and gene amplification. We will thus leverage a recently developed biobank of patient-derived organoid models containing ERBB2 mutation/amplification to study the associations between ERBB2 mutational status/HER2 expression, oncogenic dependence on HER2, and sensitivity to HER2-directed antibody drug conjugate therapy, a promising break...