Abstract A019: Enzalutamide resistance is driven by adaptations that enhance AR splice variant and FOXA1 activities

Abstract

Abstract Prostate cancer (PCa) that progresses on therapy with an androgen receptor (AR) inhibitor such as enzalutamide (Enz) generally continues to express high levels of transcriptionally active AR, but mechanisms driving this persistent AR activity remain to be established. These tumors also generally express increased levels of AR splice variants that lack the ligand binding domain and hence have androgen-independent activity (with ARv7 being the most common), but the extent to which these variants contribute to AR activity, and their dependence on the full length AR (ARfl), also remain unclear. We found Enz treatment initially suppressed AR activity in VCaP cells, despite an increase in ARv7 splice variant, while Enz-resistant VCaP cells (VCaPEnzR) generated by prolonged culture in Enz had restoration of AR transcriptional activity. Notably, this restored AR activity was not associated with further increases in ARv7 expression. However, by ChIP-seq we found it was associated with increased ARv7 chromatin binding, with ARv7 binding at a subset of the sites occupied by ARfl, but not at any unique sites. Moreover, approaches using RNAi and an AR degrader targeting ARfl showed that ARv7 was the major driver of AR transcriptional activity in the VCaPEnzR cells, despite much higher levels of ARfl expression, and was not dependent on ARfl. ATAC-seq showed there was a global increase in chromatin accessibility in the VCaPEnzR cells, with the greatest increase being at ARv7 sites. This suggested these sites had decreased nucleosome occupancy, which was confirmed by MNase-seq. As FOXA1 can act as a pioneer factor to open chromatin, we next carried out FOXA1 ChIP-seq. This showed increased FOXA1 binding across all FOXA1 sites in the VCaPEnzR cells versus parental VCaP, with the greatest increases being at ARv7 binding sites and at sites that showed the greatest increases in accessibility by ATAC-seq. Notably, frameshift mutations in the C-terminus of FOXA1 are found at increased frequency in castration-resistant prostate cancer (CRPC) and may enhance FOXA1 activity, but FOXA1 was not mutated in the VCaPEnzR versus parental VCaP cells. Mass spectrometry then identified posttranslational modifications (PTMs) in FOXA1 that have been previously reported in PCa, including K270 methylation that can impair FOXA1 chromatin binding, but this was not consistently decreased in VCaPEnzR cells. In contrast, we found consistent increases in phosphorylation at C-terminal sites including T334 and Y429. Notably, the Y429 phosphorylation was reported previously to enhance FOXA1 chromatin binding and estrogen receptor activity. Together these findings indicate that progression to Enz-resistance is dependent on adaptations that enhance ARv7 activity. These adaptations include increasing chromatin accessibility at ARv7 binding sites, which may be driven by PTMs in FOXA1. Citation Format: Betul Ersoy-Fazlioglu, Larysa Poluben, Mannan Nouri, Henry W. Long, Joshua W. Russo, Steven P. Balk. Enzalutamide resistance is driven by adaptations that enhance AR splice variant and FOXA1 activities [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A019.