PROJECT SUMMARY ABSTRACT Herein, a group of collaborative merit review applications (CMA) aim to advance the precision management of bladder cancer (BCa), especially focused on the early stage initiation of urothelium as a model of dynamic epithelial changes in response to smoking and deployment-related carcinogens. Malignancies are the second most common cause of death among Veterans and BCa is the fourth most common cancer in the VA. Among tumor types, 70% of BCa is confined to the superficial part of the bladder (Stages T1, Ta, and CIS), with the remainder invasive of the muscle or metastatic. If BCas are identified at an earlier stage, nearly all of these tumors are treatable with a combination of surgery and intracavitary therapy. Yet, there are currently no validated or recommended screening procedures to identify asymptomatic BCas and there are no methods to identify at-risk patients at an earlier and more curable stage. The proposed CMAs aim to address these limitations and to significantly disrupt BCa prevention, detection, risk stratification and precision treatment by dissecting the genetic and molecular foundations of early stage BCa. The projects include the following: CMA1 aims to determine the genetic and immune-suppressive landscape of CIS to identify new therapeutics and immunotherapies. CMA2 investigates the plasticity of the urothelium to determine how PPARg can direct epithelial differentiation as a possible modulator of CIS. CMA3 will examine the epigenetic basis of urothelial differentiation and the role of LSD1-inhibitor, Methysticin, as a chemopreventative agent to restore the epigenetic imbalance of the urothelium. Finally, CMA4 will develop artificial intelligence algorithms for enhanced cystoscopy imaging technologies or BCa detection and risk stratification. These CMAs are linked both intrinsically among each other and extrinsically with all contributors already supported by VA R&D with Merit Awards focused on BCa to maximize synergy and ensure success. Rationale: More than 80% of Veterans report a history of tobacco smoking with 90% of Veterans with BCa self-reported smokers. Unlike lung, prostate or colorectal cancer, there are no screening protocols recommended for Veterans at risk for BCa. There is no primary care recommendation for uniform evaluation of blood in the urine, and no urinary tests have a high negative predictive value that can replace cystoscopy. Almost all patients with BCa develop blood in the urine at some time, but there is often delays in pursuing an evaluation by months to years that lead to tumor progression due to lack of referrals to urologic surgery for evaluation. Once diagnosed, the urothelium is often challenging to follow and up 20% of invasive tumors will progress to higher stage cancer. Treatment for early stage invasive bladder cancer is dependent on BCG immunotherapy, but BCG is frequently unavailable and underutilized for maintenance and 30% of BCas become BCG unresponsive. Therefore, the three major challenges for improving survival for patients with BCa are 1) early detection of high-risk tumors 2) identification of progression to higher stage cancer and 3) treatment resistance to BCG immunotherapy. Our preliminary data suggest that the urothelium has plasticity in early stage BCa that, if understood at the genetic, epigenetic and molecular level, could be treated and driven to a more indolent cancer. Based on our preliminary studies and the gaps in diagnosis and treatment we hypothesize that the urothelium can be influenced by the state of epithelial differentiation and driven towards a more stable state if detected at early time point.

Project Narrative This Collaborative Merit Award proposal (CMA) is an integrated, synergistic approach focused on the pre- invasive stage of bladder cancer (BCa), carcinoma in situ (CIS). CIS is the spreading edge of BCa, identified by abnormal, proliferating cells. The overall strategy of the CMA is to pursue mechanistic strategies to improve diagnostic and molecular therapies targeted at CIS. In CMA1, we focus on the genetic, epigenetic and expression-based pathways of proliferation, the hallmark of CIS. Our central hypothesis is that alterations of the RB1 pathway cause activation of E2F and the Enhancer of Zeste-2 (EZH2) causing cellular proliferation and immune suppression. In this proposal we will define the genetic and immunologic landscape of CIS in Veterans with smoking, and deployment related occupational chemical and biologic warfare exposure to identify new therapies for Veterans with BCa. This proposal is highly integrated with the other three proposals and will serve as a translational resource to investigate molecular mechanisms from CMAs 2-4 in Veterans with BCa.