Identifying regulatory mechanisms of androgen receptor variants by DNA-PKcs

Abstract

The androgen receptor (AR) is a master regulator of prostate cancer (PCa) development and progression, hence current therapies target the AR signalling pathway to inhibit tumour growth. The generation of alternatively spliced AR variants (AR-Vs) is a major resistance mechanism observed in patients who progress to the advanced castrate-resistant PCa (CRPC) stage of disease. In contrast to full-length AR (FL-AR), AR-Vs are constitutively active and drive the growth of PCa without the requirement of activating androgens. Furthermore, AR-Vs are refractory to the current repertoire of AR-targeting therapies hence there is a major drive to develop treatments that can inhibit these aberrantly functioning receptors. Targeting AR-V co-regulatory proteins, that are required for enabling their function, represents a tractable means for inactivating AR-Vs in advanced disease. DNA-PKcs, a key kinase in the DNA damage response, has been shown to regulate FL-AR transcriptional activity and is upregulated in both PCa and CRPC. Given shared co-regulator dependencies of FL-AR and AR-Vs, and the observation that AR-Vs regulate DNA damage, we hypothesised that DNA-PKcs may influence AR-V activity as a co-regulator. Using proximity biotinylation, we show that DNA- PKcs is a prominent AR-V interacting protein in the presence and absence of DNA damage and demonstrate that DNA-PKcs regulates AR-V transcriptional activity and protein abundance in CRPC cell lines. Furthermore, DNA-PKcs inhibition and depletion has anti-proliferative effects in several CRPC cell lines in the absence of DNA damage. Global transcriptomic analysis revealed a novel role for DNA-PKcs in the regulation of alternative splicing that is important in the generation of AR-Vs. Interrogation of the regulatory role of splicing associated genes by DNA-PKcs revealed that DNA-PKcs regulates expression of the RNA binding protein, RBMX, which was then validated as a key regulator of AR-V synthesis. In conclusion, targeting DNA-PKcs or RBMX are potential therapeutic options for AR-V positive PCa patients.