Abstract 2756: Epigenetic drivers of enzalutamide resistance in prostate cancer: targeting of DNMTs and EZH2 as therapeutic strategies to overcome enzalutamide resistance in prostate cancer

Background: Prostate cancer results in over 35,000 deaths in USA, annually. Enzalutamide (ENZ) treatment enhances overall patient survival. The development of ENZ-R is marked by adaptive cellular mechanisms, including cellular plasticity and the emergence of CRPC-NEPC and CRPC-DNPC phenotypes. The role of epigenetic mechanisms in CRPC remains less understood. Recent studies, including those from our laboratory, have pointed to increased expression and activity of DNA methyltransferases (DNMTs) and the PRC2 component EZH2 during prostate cancer progression. However, the direct role of DNMTs in ENZ-R has not been thoroughly evaluated. In this study, we explored the potential of DNMT inhibitors and EZH2 inhibitors to sensitize prostate cancer cells to enzalutamide. Methods: Enzalutamide-resistant (LNCaP-ENZ-R) cells were developed by maintaining the cells in a medium containing 5 μM ENZ for a minimum of six months, while Short-term treatment with ENZ was conducted for 72h. Where indicated DNMTi (5-AZA-dC) and EZH2i (GSK-126 ) and Enz were used at 5uM concentrations. Proliferation was assessed using Incucyte. Colony formation and cell migration were assessed. Protein and mRNA level were determined by western blot and RT-PCR techniques. Results: We observed that treatment of PCa cells with ENZ increased levels of DNMTs in both short-term (treatment of the LNCaP cells for 72 h) and established ENZ resistant (LNCaP-ENZ-R) cells. We also observed a gradual transformation from epithelial morphology to neuroendocrine phenotype in ENZ-R cells, characterized by spindle-shaped and elongated structures in LNCaP-ENZ-R cells. Moreover, in ENZ-R cells AR levels did not change but AR target (PDEF, PSA, and KRT18) were significantly downregulated, while markers associated with EMT (Snail) and NEPC (CHGA and enolase) were upregulated. Treatment of the cells with a DNMT inhibitor (5-AZA-dC) resulted in a decrease in DNMT levels in both ENZ-sensitive and -resistant cells. EZH2 expression showed no notable changes in either ENZ-sensitive or -resistant cells, however, H3K27me3 levels were increased with ENZ treatment. Additionally, LNCaP cells were sensitive to DNMTs and EZH2 inhibitors, and inhibiting both DNMTs and EZH2 substantially inhibited the growth and proliferation ENZ-sensitive as well as of ENZ-R-PCa cells. We also observed that treatment with inhibitors of DNMT and EZH2 decreased the scratch wound closure and clonogenic activity of PCa cells. Conclusion: Overall, our findings reveal that the DNA and histone methylation pathways play an important role in prostate cancer and development of ENZ-Resistance, and that targeting these epigenetic regulators by small molecule inhibitors, may help overcome therapy resistance in PCa. Supported in part by: Funds from VA Merit Award: I01BX005351, NIH/NCI RO1: CA242839, and LSUHSC, NOLA. Citation Format: Santosh Lamichhane, Brandon E. Burow, Sweaty Koul, Hari K. Koul. Epigenetic drivers of enzalutamide resistance in prostate cancer: targeting of DNMTs and EZH2 as therapeutic strategies to overcome enzalutamide resistance in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2756.