Abstract B038: High-throughput drug screening identifies a novel synergistic therapeutic strategy co-targeting nuclear export and translation initiation in prostate cancer

Abstract Metastatic castration-resistant prostate cancer (mCRPC) remains a significant therapeutic challenge, as patients frequently develop resistance to standard androgen receptor (AR)-directed agents. A central mechanism of resistance involves the emergence of the AR splice variant AR-V7, which promotes tumor growth and is strongly associated with poor clinical outcomes. To address the need for novel treatment strategies capable of overcoming AR-driven resistance, we performed an unbiased high-throughput drug screen to identify highly synergistic combinations with the potential to improve efficacy at low doses, minimizing toxicity. Leveraging a robotic automation platform, we evaluated 2,480 mechanistically annotated compounds, targeting over 800 biological pathways, across 8 prostate cancer cell lines. Single-agent cytotoxicity assays revealed key vulnerabilities, including the nuclear export receptor XPO1. Based on these findings, 42 compounds were advanced for testing in two AR-V7-expressing mCRPC models, LNCaP-95 and VCaP-CR, using 10×10 dose matrices to evaluate all 861 possible dual-drug combinations. Application of the ExcessHSA synergy model identified highly synergistic interactions, especially between XPO1 and EIF4A1 inhibitors. Validation through targeted screening across 7 cell lines and 3 patient-derived prostate cancer organoid models demonstrated robust synergy at low, clinically achievable doses. These results guided the development of a novel combination therapy simultaneously targeting XPO1-mediated nuclear export (Eltanexor) and EIF4A1-regulated translation initiation (Zotatifin). Mechanistic analyses displayed that a single administration of this combined treatment, assessed by live-cell imaging, significantly reduced proliferation and induced apoptosis, with effects sustained over one week. Global proteomic profiling of the combination treatment demonstrated downregulation of key oncogenic drivers, including AR, CCND1, SLC2A1, and CDK4, along with suppression of AR-V7-specific target genes. Gene set enrichment analyses also revealed concordant reductions in cell cycle and metabolic pathways, notably G2M checkpoint, E2F, MTORC1, glycolysis, and MYC targets. Furthermore, subcellular fractionation, immunoblotting, and immunofluorescence confirmed marked decreases in AR-FL, AR-V7, and MYC, with striking nuclear accumulation of the apoptotic mediator p53, implicating it as a critical initiator of combination-induced cell death. Importantly, in vivo the combination achieved significant tumor regression in LNCaP-95 and LuCaP167-CR xenografts at doses more than tenfold below established tolerability limits, with no measurable weight loss. Collectively, these findings position XPO1-EIF4A1 co-inhibition as a potent and mechanistically defined therapeutic strategy to synergistically disrupt oncogenic signaling, overcome AR-V7-driven resistance, and achieve durable responses with reduced toxicity. Building upon these substantial preclinical findings, we now prepare to advance this combination into early-phase clinical evaluation for mCRPC. Citation Format: Jessica D. Kindrick, Kinjal Bhadresha, Xiaohu Zhang, Erica L. Beatson, Spencer S. Gaut, Patrick J. Signorelli, Benjamin C. Brim, Jessica L. Horner, Michele Ceribelli, Cindy C. Chau, Craig J. Thomas, William D. Figg. High-throughput drug screening identifies a novel synergistic therapeutic strategy co-targeting nuclear export and translation initiation in prostate cancer [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 B038.