Abstract This proposal describes a 5-year research career development program focused on a non-canonical role of RB1 loss in bladder cancer. Dr. Qiang Li is an Assistant Professor of Oncology at Roswell Park Comprehensive Cancer Center in the Department of Urology. The proposal builds on the candidate’s previous experience and current research projects using genetically engineered mouse models (GEMMs) and organoids. The proposed experiments and training will enable his transition to independence as a physician scientist in bladder cancer translational research. He will be mentored primarily by Dr. David Goodrich. Dr. Goodrich is an expert in RB1 cancer biology, genetically engineered mouse models, acquired drug resistance and cancer cell plasticity. The training plan includes the following goals: (1) Enhance expertise in preclinical cancer modeling (GEMM and organoids); (2) Probe the molecular mechanisms of bladder cancer cellular plasticity and drug resistance; and (3) Gain expertise in bioinformatic analysis. RB1 mutations are predictive of pathologic response after neoadjuvant chemotherapy in bladder cancer. Other clinical observations suggest that the basal type of bladder cancer is more likely to respond to chemotherapy than the luminal type. However, the biological impact of RB1 loss on molecular subtypes of bladder cancer pathogenesis and chemotherapy response has not been investigated. Newly discovered features of the RB1 pathway in other cancer types suggest that RB1 loss promotes lineage plasticity and acquired therapy resistance. Thus, we hypothesize that RB1 loss promotes bladder cancer progression, metastasis, and cellular plasticity (luminal to basal, and therapeutic resistance). We use two transgenic mouse systems (Uroplakin II driven reverse tetracycline trans-activator, TRE-Cre) to investigate the role of RB1 loss in bladder urothelium by facilitating deletion of tumor suppressor genes (Trp53, Pten, Rb1) under control of doxycycline administration. We engineered doxycycline inducible triple knockout mice Trp53-/-: Pten-/-: Rb1-/- (referred as TKO) and double knockout mice Trp53-/-: Pten-/- (referred as DKO). We propose the following Specific Aims: (1) Define the function of RB1 loss in accelerating tumor progression, metastasis, and cellular plasticity in bladder cancer GEMMs; (2) Dissect the impact of cell-of-origin on bladder tumorigenesis, metastasis and response to chemotherapy in TKO tumors derived from basal cells versus luminal cells. Successful completion of this proposal will allow the candidate to gain valuable technical knowledge and expertise in preclinical modeling of advanced bladder cancer and further his development as an independent physician scientist. The work will also establish experimental models and analysis pipelines that will provide the foundation for the candidate’s independent research program.