Abstract 4412: Characterization and targeting of an androgen receptor pre-mRNA stem loop structure to inhibit AR-V7 splicing in prostate cancer

Abstract Alternatively spliced mRNAs can contribute to aggressiveness and resistance to androgen receptor pathway inhibitors (ARPIs) in castration-resistant prostate cancer (CRPC). Among these spliced mRNAs, the androgen receptor (AR) splice isoform AR-V7 participates in AR signaling independent of androgen levels or ARPIs, such as enzalutamide. AR-V7 detection correlates strongly with ARPI treatment resistance and poor patient outcomes in CRPC. AR-V7 incorporates cryptic exon 3 (CE3) within the AR gene that prematurely terminates the full-length transcript. While several proteins have been described that may promote splicing of CE3, the impact of the pre-mRNA structures in this region is unknown. Using selective 2′-hydroxyl acylation analyzed by primer extension with mutational profiling (SHAPE-MaP), we developed the first experimentally informed pre-mRNA secondary structure model of the CE3 3′ splice site from cellular RNA. In this structural model, we observe a novel stem loop structure, which we hypothesized could inhibit AR-V7 biogenesis. We used an AR-V7 minigene assay to evaluate structure-modulating mutations in AR-V7 biogenesis and observed concordant changes in AR-V7 abundance in response to stabilization of this repressive structure, leading us to term this structure the cryptic exon 3-splicing inhibitory stem loop (CE3-SISL). Protein binding site identification of the region through the Δ-SHAPE pipeline suggests a repressive protein interaction that may be lost in androgen independence and ARPI resistance. Together, these data suggest the importance of RNA structure in AR-V7 biogenesis and indicate a potentially targetable interface for the development of AR-V7-depleting agents, as we have exemplified using structure-guided antisense oligonucleotides (ASOs) that are able to inhibit AR-V7 splicing during enzalutamide treatment. These ASOs would represent the first RNA structure-targeted splicing modulators being developed for patients with AR-V7-driven CRPC. Citation Format: Marek D. Zorawski, James P. Falese, Riley S. Spingler, Jason A. Somarelli, Andrew J. Armstrong, Amanda E. Hargrove. Characterization and targeting of an androgen receptor pre-mRNA stem loop structure to inhibit AR-V7 splicing 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 4412.