Prostate cancer is the most common malignancy in men and the second leading cause of male cancer-related death in the United States. The main focus of targeted molecular therapies in advanced prostate cancer has been the androgen receptor signaling. While first- and second generation drugs that target androgen receptor signaling have efficacy and have increased overall survival, most patients relapse with tumors that are refractory to these treatments. More effective monotherapies targeting androgen receptor signaling are in development but are unlikely to achieve durable responses, due to the development of resistance. Thus, there is a critical need for agents targeting alternative molecular drivers of prostate cancer. The transcription factor Ets Related Gene (ERG), which is overexpressed through gene fusion with the androgen-responsive gene Transmembrane protease, serine 2 (TMPRSS2) in ~ 50% of advanced prostate tumors, is a key driver of prostate cancer development and progression. Ablation of ERG would disrupt a key oncogenic transcriptional circuit and could be a promising therapeutic strategy for prostate cancer treatment. We hypothesize that ERG stability in prostate cancer cells is modulated by the interplay of ubiquitination and deubiquitination, and that the proteins regulating the ubiquitinatin state of ERG may include enzymes that can be therapeutically targeted with small molecule inhibitors. We recently found that (1) the deubiquitinase USP9X stabilizes ERG, (2) the deubiquitinase inhibitor WP1130 causes a rapid increase of ubiquitinated ERG, which results in ERG depletion, (3) WP1130 inhibits the growth of ERG-expressing prostate tumors in vivo, and (4) a novel analog KRL-113 has better potency than WP1130 in targeting ERG. In this research proposal, we seek to build on our work to identify, characterize and optimize small molecules that cause ERG depletion, and to evaluate small molecule inhibitors affecting ERG turnover in preclinical models. To accomplish these aims, we will use rational design to further optimize the ERG-ablating compound KRL-113 in order to improve its pharmacological efficacy and specificity. In parallel, we will screen a chemical compound library to identify additional lead compounds that reduce ERG protein levels. We will characterize the effects of optimized KRL-113 analogs and new lead compounds on the proteome of prostate cancer cells. Finally, we will test the ability of KRL-113 and its analogs to reduce ERG protein levels and inhibit tumor growth in both mouse xenograft and human primary prostate tumor explant models.