Abstract 251: Resistance to androgen receptor pathway inhibitors is mediated by a pro-survival stress response converging on altered mitochondrial energetics

Purpose: The current front-line therapies for men with metastatic prostate cancer are novel androgen receptor pathway inhibitors (ARPIs), such as abiraterone acetate or enzalutamide, which target the androgen receptor (AR) pathway. These therapies significantly prolong overall survival; however, acquired resistance to these drugs is common over several years. Therefore, there is a major unmet clinical need to identify mechanisms of resistance and innovative therapies to treat this therapy-resistant, metastatic disease. Experimental Procedures: To identify common molecular mechanisms of acquired ARPI resistance we developed a panel of three enzalutamide-resistant (enzaR) cell lines, LNCaP-enzaR, CS2-enzaR, and LN95-enzaR, by chronic, long-term exposure to increasing doses of enzalutamide. Analysis of the genome, transcriptome and phospho-proteome of enzaR prostate cancer cell lines were evaluated by comparative genomic hybridization (aCGH), RNA-seq and reverse phase phospho-protein arrays. Oxygen consumption rate was measured by Seahorse assays. To decipher the correlation between stress response and ATP hydrolysis, we have utilized small molecule p38 inhibitor (SB203580) to test if p38 is related to ATP hydrolysis and validated the result by western blot. The functional role ATPIF1 was deciphered by ATPIF1 siRNA transfection in LNCaP cells. Results: Our genomic and functional studies have pinpointed a pro-survival cellular stress response as convergently activated during the acquisition of AR-dependent enzalutamide resistance. This pro-survival stress response leads to induction of metabolic quiescence and activation of p38/MAPK signaling, which triggers reversal of mitochondrial ATP synthase. Instead of canonical “forward” ATP synthase activity to generate ATP, the ATP synthase runs in “reverse”, hydrolyzing ATP to maintain a proton gradient across the mitochondrial membrane and permit survival. These features create collateral sensitivity to organic cations that target non-canonical polarization of the mitochondrial membrane that is critical to cellular persistence. In short, our findings support the hypothesis that AR inhibition induces a pro-survival response characterized by mitochondrial energetic changes that can be targeted as novel treatments for AR therapy-resistant prostate cancer. Conclusion: We have discovered non-canonical ATP synthase reversal as a key targetable mechanism by which cancer cells maintain mitochondrial polarization in the face of therapy-induced resource depletion to sustain cell survival. Future studies are aimed at clinical translation of novel agents to target these persister populations. Citation Format: Shibjyoti Debnath, Kathryn Ware, Bea Thomas, Kelsey H Fisher Wellman, Andrew Armstrong, Jason Somarelli. Resistance to androgen receptor pathway inhibitors is mediated by a pro-survival stress response converging on altered mitochondrial energetics [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 251.