Prostate cancer (PC) that recurs following standard of care androgen-deprivation therapy is termed castration-resistant prostate cancer (CRPC) and is currently incurable. CRPC is marked by reactivation of androgen receptor (AR) signaling, eventual metastasis and, in some cases, further evolution to AR null/AR indifferent neuroendocrine PC (NEPC). BUB1B (BUBR1) is a master regulator of a seven gene network that is upregulated in and drives CRPC. BUB1B is overexpressed in PC compared to benign prostate tissue and is further elevated in metastatic CRPC (mCRPC) and NEPC. BUB1B is an essential component of the spindle assembly checkpoint (SAC) and aberrant BUB1B expression has been associated with development of several cancers; however, its role in PC remains understudied and there are no drug-like inhibitors available. Depletion of BUB1B halts AR+ and AR- CRPC cell proliferation. Furthermore, ectopic BUB1B expression in the less aggressive predecessor of CRPC, androgen dependent (AD) PC, is sufficient to confer castration resistance. Expression of constitutively active AR variants (AR-Vs) is one mechanism underlying progression to CRPC and clinical resistance to 2nd generation AR antagonists such as enzalutamide. In CRPC cells, BUB1B knockdown decreased AR and constitutively active AR-V protein stability leading to decreased ligand- independent AR transcriptional activity, which is a driver of most CRPC and metastatic CRPC. Thus, BUB1B is a promising therapeutic target for CRPC including, but not restricted to, currently intractable constitutively active AR-V expressing tumors. This proposal will test the hypotheses that: (1) BUB1B kinase activity is essential for progression to lethal CRPC by increasing AR / AR-V levels, which can drive CRPC; (2) in AR null/AR indifferent NEPC, BUB1B promotes growth through yet to be defined AR-independent mechanisms; and (3) BUB1B represents a promising but untapped therapeutic vulnerability that can be exploited for treating deadly CRPC and NEPC. Aim 1 will evaluate the mechanisms underlying BUB1B kinase growth-promoting effects in CRPC and NEPC and will determine the requirement for BUB1B kinase activity in stabilizing AR/AR-V7 as well as explore mechanisms of BUB1B promotion of CRPC and NEPC. BUB1B protein will be evaluated in TMAs containing bone and visceral metastasis, which will be useful for future patient stratification. Aim 2 will establish BUB1B as a tractable druggable target by evaluating and prioritizing recently identified small molecule inhibitors that target the BUB1B kinase domain at the ATP binding site. A NanoBRET assay will be developed for BUB1B drug optimization and advancement. Aim 3 will develop, characterize, and evaluate drug-like potent and selective small molecule BUB1B kinase inhibitors. Preliminary data support the proposed use of a highly scalable and innovative computational screening pipeline that integrates AI-based kinase activity predictions, protein homology modeling, and ph...