Abstract B075: Transcriptional reprogramming mediates ARSi resistance in Rb-deficient CRPC

Abstract Background: Prostate Cancer (PCa) is initially treated with Androgen Deprivation Therapy (ADT), but it eventually progresses to Castration-Resistance Prostate Cancer (CRPC) due to acquired treatment resistance. Enzalutamide, a second-generation androgen receptor signaling inhibitor (ARSi), is a current standard treatment for CRPC. However, its long-term efficacy is limited by resistance development. A subset of CRPCs harbor biallelic RB1 deletions, yet the mechanisms through which RB1-loss tumors progress under ARSi treatment remain poorly defined. In this study, we established an RB1-loss CRPC model that acquires resistance through long-term enzalutamide exposure and aimed to elucidate the associated molecular alterations. Hypothesis: We hypothesize that RB1-loss prostate cancer cells undergo distinct transcriptional and metabolic reprogramming during long-term ARSi exposure. Our goal is to characterize these molecular changes and identify key pathways driving enzalutamide resistance. Methods: RB1 was knocked out in C4-2 cells (an AR-positive/PTEN-negative CRPC line) using CRISPR/Cas9-mediated silencing, followed by prolonged high-dose enzalutamide treatment for approximately six months to generate a fully resistant line (called ER6 cells). Intermediate lines were collected at 1.5 (ER1.5) and 3 (ER3) months. Functional assays were performed to validate therapeutic resistance, and integrated RNA-seq and ChIP-seq analyses were conducted to define transcriptional and epigenetic alterations associated with resistance. Results: The ER6 cells exhibited cross-resistance to multiple ARSi agents, including apalutamide. Protein analyses revealed further loss of expression of the Rb-like protein, RBL2, resulting in complete depletion of the Rb repressive program. Notably, ER6 cells retained AR expression but lacked expression of neuroendocrine markers, instead displaying reduced expression of luminal drivers (FOXA1, HOXB13) and increased expression of AP-1 components (JUN) and EMT markers, suggesting a transition toward an EMT-like phenotype. JUN silencing restored FOXA1 expression and reduced EMT markers, suggesting an important role for AP-1 in this reprogramming process. Furthermore, ER6 cells displayed enhanced MAPK pathway activation, shown by increased phospho-ERK levels. RNA-seq analysis was consistent with these findings, revealing enrichment of EMT, Ras-MAPK, and inflammatory pathways in resistant cells. Conclusion: This study demonstrates that RB1-loss CRPC tumors can acquire ARSi resistance through alternative mechanisms independent of neuroendocrine differentiation. Our findings suggest that AP-1–mediated transcriptional reprogramming and MAPK pathway activation may be key drivers of this adaptive resistance. These data uncover potential therapeutic vulnerabilities and highlight the AP-1 and MAPK pathways as potential targets for treating ARSi-resistant, Rb-deficient prostate cancer. Citation Format: HyeonYeong Sun, Yaozong Su, Changmeng Cai, Jill Macoska, Songqi Zhang, Jaeweon Jeong, Mingyu Liu. Transcriptional reprogramming mediates ARSi resistance in Rb-deficient CRPC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Prostate Cancer Research and Treatment; 2026 Jan 20-22; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(2_Suppl):Abstract nr B075.