Histone-modifying enzymes in androgen-deprived prostate cancer: EZH1 as a synergistic therapeutic target

Background: Androgen deprivation therapy (ADT) is the cornerstone of treatment for advanced prostate cancer (PC). While ADT induces apoptosis and senescence, residual tumor cells frequently persist and drive castration resistance. The initiation of ADT may create vulnerabilities that can be therapeutically exploited. We investigated the role of histone-modifying enzymes (HMEs) in supporting tumor cell survival after ADT, hypothesizing that their upregulation contributes to persistence and represents a target for synergistic therapy. Methods: High-risk prostatectomy samples treated with 3 months of ADT were compared to untreated controls using bulk RNA sequencing (n=10 vs. 26). Publicly available datasets (Wyatt GSE550162; Sowalsky GSE1831003) were interrogated for validation. The expression of EZH1/2 and H3K27me3 was assessed in hormone-sensitive PC (HSPC) cell lines LNCaP, LAPC4 and VCaP treated with ADT (CSS: charcoal-stripped serum) at different timepoints by Western blotting. A tissue microarray was constructed to evaluate the protein expression of EZH1 in 30 ADT-treated and 29 untreated post-prostatectomy HSPC tumors using immunohistochemistry and automated VECTRA imaging and quantification. HSPC cell lines and patient-derived organoids were treated with bicalutamide or darolutamide in combination with dox-inducible EZH1 shRNAs or FDA-approved EZH1/2 inhibitor valemetostat to examine synergistic inhibition of cell growth. Results: High-risk prostatectomy samples identified four consistently upregulated HMEs: EZH1, MECOM, SIRT1, and GCN5, KDM6B, PRDM5/6/16 after ADT. Among these, EZH1 and MECOM showed increased protein expression with ADT in a validation tissue microarray of 59 patients with EZH1 correlated with adverse clinical features including and PSA recurrence in the ADT-treated cohort. In LNCaP and VCaP cell lines EZH1 expression rose or persisted after early ADT, while EZH2 declined with increased H3K27 methylation levels. Total H3K27me3 levels increased. EZH1 expression positively associates with a subset of EMT and stem cell markers. EZH1 knockdown reduced survival of ADT-treated cells, and patient-derived organoid models showed synergistic cell death with combined either bicalutamide or darolutamide and EZH1/2 inhibition (valemetostat). Conclusions: ADT induces EZH1 upregulation, which promotes prostate cancer cell persistence potentially through regulation of cancer stemness and epithelial-to-mesenchymal transition. These findings establish EZH1 as a mediator of survival after ADT and a promising target for synergistic therapeutic inhibition. Funding Acknowledgements: This study was supported in part by DoD PC150211, UW Prostate SPORE P50CA269011, and NIH/NCIR01CA76184, P30 CA014520. COI: none