Abstract 5659: High-throughput screening reveals novel synergistic drug combinations for metastatic castration-resistant prostate cancer

Abstract Metastatic castration-resistant prostate cancer (mCRPC) remains a significant clinical challenge, with patients often developing resistance to androgen receptor (AR)-axis-targeted therapies such as Abiraterone and Enzalutamide. A key resistance mechanism involves the AR splice variant AR-V7, which drives tumor progression and poor prognosis. To address the need for novel, effective treatment strategies, we conducted unbiased high-throughput screening (HTS) to discover highly synergistic drug combinations that improve therapeutic efficacy, overcome resistance mechanisms, and minimize toxicity. Leveraging a robotic automation platform, we quantitatively screened 2, 480 mechanistically annotated compounds from an oncology-focused library, targeting over 800 biological pathways, across 8 prostate cancer cell lines representing various stages of disease progression. Single-agent cytotoxicity was evaluated using an area under the 11-point dose-response curve (AUC) metric, identifying therapeutically vulnerable targets in mCRPC, including tubulin, MCL-1, CDK9, BRD4, and PI3K/AKT. This led to the selection of 42 compounds for pairwise study of all possible 861 dual-drug combinations in two AR-V7-expressing mCRPC cell lines, LNCaP-95 and VCaP-CR. Utilizing the Excess Highest Single Agent (HSA) model to quantify synergy/antagonism, this large-scale analysis revealed highly synergistic combinations, particularly between MCL-1 and BcL-xL inhibitors. These results guided the development of a novel triple combination therapy to simultaneously target CDK9-regulated productive RNA polymerase II phosphorylation (Enitociclib), BRD4-mediated epigenetic remodeling (Pelabresib), and pro-survival BcL-xL (Navitoclax). Notably, the triple combination demonstrated robust synergy at low doses, inducing apoptosis as early as 4 hours post-treatment through rapid Caspase 3/7 cleavage. Further mechanistic investigation through RNA-sequencing revealed significant downregulation of key oncogenes, including MYC and AR-V7, alongside disruption of downstream transcriptional pathways. Importantly, the triple combination was validated in patient-derived mCRPC organoid models LuCaP167 and LuCaP136, confirming its translational potential. These findings provide a strong rationale for clinical development of multi-drug targeted combination regimens to overcome AR-V7-driven resistance and deliver durable responses with reduced toxicity for mCRPC patients with limited treatment options. Our approach also highlights the power of HTS to unbiasedly innovate synergistic therapies while uncovering critical biological mechanisms underlying cancer drug resistance. Citation Format: Jessica D. Kindrick, Benjamin C. Brim, Kinjal Bhadresha, Xiaohu Zhang, Keith T. Schmidt, Michele Ceribelli, Douglas K. Price, Cindy Chau, Craig J. Thomas, William D. Figg. High-throughput screening reveals novel synergistic drug combinations for metastatic castration-resistant 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 5659.