Abstract 5650: Small molecule CB001 downregulates cFLIP, potentiates the effects of androgen receptor inhibitors in prostate cancer models, and enhances NK cell activity

Abstract Background: Androgen deprivation therapy (ADT) and androgen receptor inhibitors (ARi) represent a cornerstone treatment for prostate cancer (PCa). However, resistance often leads to castration-resistant prostate cancer (CRPC). Dysregulation of the extrinsic apoptosis pathway contributes to therapeutic resistance in PCa. Overexpression of FADD-like IL-1β-converting enzyme-inhibitory protein (cFLIP), an endogenous caspase-8 inhibitor, promotes PCa progression and ADT resistance. Targeting cFLIP may represent a novel therapeutic strategy for PCa. This study investigated the potential of CB001, a cFLIP inhibitor, to enhance the effect of ARi and overcome resistance in PCa models. Methods: We investigated the prevalence of cFLIP molecular alterations in PCa and its impact on patient survival using The Cancer Genome Atlas (TCGA) dataset. We investigated cytotoxic effects of CB001 [0-10µM] in ARi-sensitive (LNCaP) and -resistant (22rv1, DU145, PC3) PCa cell lines. Combination indexing for dual treatment with CB001 [0-5µM] ± ARi (enzalutamide [0-150µM] or darolutamide [0-150µM]) was performed on SynergyFinder. cFLIP, cleaved caspase 8, and cPARP expression were investigated after treatment with CB001 (10µM) ± ARi (10-100µM) at 24 hours by western blot (WB). PCa cells were co-cultured 1:1 with natural killer cells (NKc) in the presence of CB001 (10µM), and tumor cell killing was quantified using the MetaXpress cytoplasmic/nuclear staining software. Results: Amplifications and deletions of the gene encoding cFLIP were identified in up to 2.7% and 1.34% of PCa tumors in TCGA, respectively. Solid tumors with cFLIP alterations had inferior overall survival compared to wild-type tumors. Single-agent CB001 cytotoxicity displayed a spectrum of sensitivity across different PCa cell lines, with DU145 (3.1µM) and neuroendocrine-phenotype PC3 (11.1µM) requiring higher IC50s than LNCaP (1.1µM) and 22rv1 (0.7µM). CB001 treatment decreased cFLIP expression and increased PARP and caspase 8 cleavage by WB consistent with extrinsic apoptosis pathway activation. CB001 plus ARi was synergistic; combination of CB001 with ARi induced apoptosis in ARi-resistant cell lines 22rv1, DU145, and PC3, reversing apoptosis resistance to single-agent ARi. CB001 also increased immune-mediated NKc killing of 22rv1 (p=7.3e-4), DU145 (p=2.41e-4), and PC3 (p=9.83e-4) cells in co-culture experiments. Conclusion: CB001 demonstrates potent cytotoxicity and synergizes with ARi in PCa cell lines to overcome resistance to apoptosis with single-agent ARi. In addition to direct tumor cytotoxicity, CB001 enhances NK cell-mediated killing of PCa cell lines. Targeting cFLIP may represent a novel therapeutic strategy for PCa. Citation Format: Claire Lin, Connor Purcell, Shengliang Zhang, Vida Tajiknia, Wafik S. El-Deiry, Benedito A. Carneiro. Small molecule CB001 downregulates cFLIP, potentiates the effects of androgen receptor inhibitors in prostate cancer models, and enhances NK cell activity [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 5650.