Abstract 1592: Targeting cFLIP to overcome resistance of BRAF V600E melanoma cells with the small molecule CB001

Background: Over 50% of metastatic melanoma harbor activating BRAF V600E mutations. Vemurafenib selectively inhibits mutant BRAF. However, resistance to vemurafenib frequently develops within a few months of initiating treatment. Vemurafenib-induced apoptosis is inhibited by overexpression of cellular FADD-like IL-1β-converting enzyme-inhibitory protein (cFLIP), an endogenous inhibitor of caspase 8 in the extrinsic apoptosis pathway. Studies have also shown that suppression of cFLIP expression restores the extrinsic pro-apoptotic pathway in melanoma cells. Thus, targeting cFLIP with CB001, a novel cFLIP inhibitor, may represent a novel therapeutic strategy to overcome resistance to BRAF inhibitor in melanoma. Methods: We investigated the prevalence of cFLIP and BRAF molecular alterations in melanoma and its impact on patient survival using The Cancer Genome Atlas (TCGA) dataset. We investigated cytotoxic effects of CB001 (0-10µM) or the BRAF inhibitor vemurafenib (0-500nM) in BRAF V600E melanoma cell lines (RPMI-7951, SH4, SK-MEL28). Combination indexing for dual treatment with CB001 [0-10µM] and vemurafenib [0-500nM] was performed on SynergyFinder. cFLIP and cPARP expression was investigated after treatment with CB001 (10µM) +/- vemurafenib (400nM) at 24 hours by western blot (WB). Melanoma cells were co-cultured at a ratio of 1:5 with natural killer cells (NKc) in the presence of CB001 (10µM), and fluorescent co-culture images were quantified using the MetaXpress cytoplasmic/nuclear staining software. Results: cFLIP alterations are frequently enriched in patients with BRAF-mutated tumors (p= 4.049e-3), and amplification of the gene encoding cFLIP is correlated with inferior overall survival compared to wild type tumors. Single-agent CB001 showed variable cytotoxicity across melanoma cell lines, with RPMI-7951 (13.4µM) requiring a higher IC50 than SH4 (3µM) and SK-MEL28 (1.29µM). While interaction between CB001 and vemurafenib was only additive, the combination treatment did overcome apoptosis resistance in vemurafenib-resistant RPMI-7951 (vemurafenib IC50 > 500nM). CB001 with vemurafenib decreased cFLIP expression and increased PARP and caspase 8 cleavage on WB, consistent with increased extrinsic apoptosis pathway activation, in RPMI-7951. CB001 also increased immune-mediated NKc killing of RPMI-7951 (p=6.23e-4), SH4 (p=5.5e-4), and SK-MEL28 (p=5.55e-4) cells in co-culture experiments at a 5:1 NKc:melanoma ratio. Conclusion: CB001 demonstrates potent cytotoxicity in metastatic melanoma cell lines and may overcome resistance to vemurafenib via downregulation of cFLIP and activation of the extrinsic apoptosis pathway. CB001 also enhances immune-mediated NKc killing of melanoma cell lines, suggesting that targeting cFLIP has an immunomodulatory effect in addition to direct tumor cytotoxicity. Citation Format: Claire Lin, Matthew J. Hadfield, Connor Purcell, Shengliang Zhang, Vida Tajiknia, Wafik S. El-Deiry, Benedito A. Carneiro. Targeting cFLIP to overcome resistance of BRAF V600E melanoma cells with the small molecule CB001 [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 1592.