Targeting androgen receptor splicing in lethal prostate cancer

Abstract

Over the past decade, androgen receptor (AR) directed therapies such as abiraterone and enzalutamide have become the standard of care for treating advanced prostate cancer, improving both progression-free and overall survival. Some patients, however, never respond to these agents, while all eventually acquire resistance, leading to invariably fatal disease progression. This resistance is in part due to the development of constitutively active alternatively spliced variants of the AR (AR-SVs) that are truncated and lack the regulatory AR ligand-binding domain; the target of current AR directed therapies. Of the many AR-SVs that have been reported, AR splice variant 7 (AR-V7) is the most prevalent and the best studied, and has been associated with resistance to AR targeting therapies and poorer overall survival. In this thesis, I describe my work focused on identifying proteins that are key to the production of AR-V7, validate my findings using clinical samples and study splicing regulatory mechanisms in in vitro models of lethal prostate cancer. Through orthogonal studies I identify the 2-oxoglutarate-dependent dioxygenase JMJD6 as a key regulator of AR-V7, as evidenced by its: 1) upregulation with in vitro androgen-deprivation-resistance; 2) downregulation alongside AR-V7 by BET inhibition; 3) being the top hit of a targeted siRNA screen of spliceosome related genes. Furthermore, I demonstrate that JMJD6 protein levels increase significantly with castration-resistance (p<0.001) and are associated with both higher levels of AR-V7 (p=0.036), and shorter median survival from castration-resistant prostate cancer (p=0.048). In vitro, I show that JMJD6 knockdown reduces prostate cancer cell growth, AR-V7 levels, and recruitment of the splicing regulatory factor U2AF65 to AR pre-mRNA. Importantly, my mutagenesis studies indicate that JMJD6 enzymatic activity is key to JMJD6-mediated AR-V7 generation, with the JMJD6 catalytic machinery residing within a druggable pocket. Taken together, I conclude that JMJD6 is a druggable target for treating advanced prostate cancer.