Correlation of galeterone-induced degradation of the androgen receptor with inhibition of a deubiquitinating enzyme.

Abstract

345 Background: Galeterone is a highly selective oral small molecule drug candidate that disrupts androgen receptor (AR) signaling. It degrades the AR (IC50 ~1µM), is a potent CYP17 lyase inhibitor ( < 50nM), and possesses AR antagonist activity (~600nM). Galeterone-induced AR degradation was observed in models having either full-length AR or known constitutively active truncated forms of the AR receptor that lack the ligand binding domain (LBD), AR-V7 and AR567es. The LBD of the AR is not required for galeterone-dependent AR degradation. Galeterone-induced degradation activity is blocked by co-administration of the proteasome inhibitor MG132. Furthermore, galeterone-induced degradation of AR can be blocked by selective knock-down of the E3 ligases, Mdm2 and CHIP. Methods: We utilized a series of biochemical and cell-based in vitro studies to further elucidate and characterize additional signaling molecules in the proteasomal dependent mechanism of galeterone-induced AR degradation. Results: We screened a panel of 22 deubiquitinating enzymes (DUBs) in vitro and demonstrated that galeterone inhibited enzymatic activity of the DUB, USP12 with IC50 in the single digit micromolar range. In addition, we used surface plasmon resonance to demonstrate that dose-dependent inhibition of USP12 activity involves direct binding of galeterone to USP12 and USP12/UAF1 complexes with a KD of ≤ 10µM. Conclusions: Interestingly, USP12 is a co-activator of AR and selective knock-down of this DUB has been shown to increase AR degradation. USP12 has been linked to regulation of the phosphatases PHLPPs through ubiquitination. PHLPPs dephosphorylate AKT, providing an important regulatory mechanism for controlling the PI3K/AKT pathway. Since it is known that galeterone induces an increase in pAKT and pMdm2, the latter being a substrate of activated AKT, this suggests that inhibition of USP12 regulates pAKT levels through enhanced degradation of PHLPPs via increased ubiquitination. These data suggest that a differentiating mechanism of galeterone from other AR targeting agents is through inhibition of USP12, leading to enhanced AR degradation.