Investigating the RNA Structural Mechanism of AR-V7 Splicing in Prostate Cancer

Abstract

In castration-resistant prostate cancer (CRPC), detection of androgen receptor (AR) splice isoform AR-V7 correlates strongly with resistance to AR pathway inhibitors (ARPI) and poor patient outcomes. AR-V7 incorporates transcript-terminating cryptic exon 3 (CE3) from the AR gene, which excludes the ligand binding domain from the AR protein, thereby enabling AR to participate in transcriptional regulation independent of androgens. While several CE3 splicing regulatory proteins have been described, the impact of pre-mRNA structures in this region is unknown. We performed SHAPE-MaP to develop the first experimentally informed secondary structure model of the CE3 3' splice site from cellular RNA. In our model, we observe a previously undescribed RNA stem loop structure, which we hypothesized could inhibit AR-V7 biogenesis. Using an AR-V7 minigene assay to evaluate structure-modulating mutations, we observed decreased AR-V7 abundance in response to stabilization of this structure, leading us to term it the cryptic exon 3-splicing inhibitory stem loop (CE3-SISL). Protein binding site identification using ∆SHAPE suggests a protein interaction that may be lost in androgen independence. Using quantitative proteomics, we nominate candidate interactors, such as NOVA1, that may help explain the repressive function of CE3-SISL. Together, these data support the importance of this RNA structure in AR-V7 biogenesis and suggest a potential target for AR-V7-depleting agents. As a proof of concept, we developed several CE3-SISL-targeting antisense oligonucleotides (ASOs), which show almost complete inhibition of AR-V7 biogenesis in both the minigene assay and in endogenous AR-V7-expressing cells with or without the ARPI enzalutamide. These ASOs represent the first RNA structure-targeted splicing modulators in development against AR-V7. We further screen RNA-targeted small molecules against CE3-SISL in the hopes of developing new therapies for patients with AR-V7-driven CRPC.