Combinational Inhibition of the eIF4F Complex, AKT1, and EZH2 Enhances Anticancer Effects in BRAF(V600E) Mutant A375 Melanoma Cells.

OBJECTIVES: The eukaryotic initiation factor 4F (eIF4F) translation initiation complex inhibitors (eIF4Fi) were recently found to hyperactivate extracellular signal-regulated kinases 1/2 (ERK1/2) signals, which contribute to acquired resistance to BRAF (B-Raf proto-oncogene, serine/threonine kinase) inhibitors in melanoma. This present study aims to elucidate how to overcome the resistance of the eIF4Fi in BRAF(V600E) mutant melanoma cells and explore the underlying mechanisms. METHODS: Melanoma A375 (vemurafenib [VEM]-sensitive) and A375R (VEM-resistant) cells were exposed to eIF4Fi RocA at varying doses and durations in vitro. We investigated the impact of RocA on the activity of ERK1/2, AKT serine/threonine kinase 1 (AKT1), eIF4E, and enhancer of zeste homolog 2 (EZH2). We then examined the impact of RocA on pro-apoptotic BH3-only proteins and proliferative proteins. We subsequently determined the effect of combined eIF4Fi, AKT1 inhibitor, EZH2 inhibitor or VEM on tumor growth in vitro and in vivo. RESULTS: RocA inhibited proliferation and induced apoptosis in A375 cells, but inhibited proliferation in A375R cells. RocA rapidly reactivated ERK1/2 at 3 h and returned to baseline levels at 48 h. However, eIF4E and AKT1 activation began at 12 h and peaked at 48 h. ERK1/2 positively regulated EZH2 and EZH2-dependent expression of c-Fos and EGR1, while AKT1 negatively regulated c-Myc, c-Jun, and BMF, but positively regulated eIF4E. RocA downregulated ERK1/2 (or EZH2, AKT1, and eIF4E) independent bcl-2 and Mcl-1 expression. AKT1i enhanced RocA-induced cell apoptosis, while EZH2i reduced RocA-induced cell proliferation. Combined CR-1-31-B, EZH2i, and AKT1i effectively overcame resistance to RocA and VEM resistance both in vitro and in vivo. CONCLUSION: The eIF4F complex inhibitor reactivates ERK1/2-EZH2 and AKT1 signaling pathways, resulting in resistance to both eIF4Fi and VEM. Combined administration of an eIF4Fi with EZH2 and AKT1 inhibitors effectively enhances sensitivity to both eIF4F complex and BRAF inhibitors.