PFKFB3 and PFKFB4 inhibition modulates the response of metabolically plastic resistant melanoma cells to BRAF/MEK-targeted therapy

Abstract Background Despite the development of selective inhibitors targeting oncogenic BRAF mutations, melanoma therapy remains limited by acquired resistance driven by multiple mechanisms, including metabolic reprogramming. PFKFB isoenzymes regulate cellular adaptation to therapeutic stress and may therefore represent promising therapeutic targets. Methods We investigated whether inhibition of PFKFB3 and/or PFKFB4 enhances the response of melanoma cells to the BRAF inhibitor vemurafenib using parental and BRAFi-resistant WM9 and A375 melanoma cell lines. Basal and inhibitor-induced bioenergetic changes were analyzed using Seahorse technology. PFKFB isoenzyme expression was assessed by quantitative real-time PCR and Western blotting. Cell viability was evaluated using ATP-based metabolic assays and SRB-based cell number analysis, accompanied by fluorescence-based DNA staining cytotoxicity assessment. Drug interactions were analyzed in SynergyFinder using HSA and ZIP models. PFKFB3 and PFKFB4 inhibition was additionally tested under increasing levels of combined BRAF/MEK inhibition with vemurafenib and cobimetinib. Statistical analyses included t-tests and ANOVA. Experiments were performed across multiple inhibitor concentrations to capture dose-dependent and cell-line-specific responses. Results Bioenergetic profiling revealed lineage-specific metabolic remodeling associated with resistance and context-dependent responses to PFKFB inhibition, identifying PFKFB4 as a key regulator of metabolic plasticity irrespective of baseline metabolic configuration. PFKFB3 and PFKFB4 inhibition differentially affected metabolic activity, cell number, and cytotoxicity. Consistent with bioenergetic analyses, highly glycolytic WM9-resistant cells with limited glycolytic reserve showed particular vulnerability to PFKFB4 inhibition, with a pronounced dose-dependent decrease in viability and increased cytotoxicity. Combining PFKFB inhibitors with vemurafenib improved therapeutic efficacy mainly through complementary rather than strongly synergistic effects. Importantly, PFKFB3 and PFKFB4 inhibition remained effective across different levels of vemurafenib/cobimetinib treatment, although response magnitude and pattern differed between A375 R and WM9 R cells. Combined PFKFB3 and PFKFB4 inhibition generally reduced viability more strongly than either inhibitor alone. Conclusion Constraining the ability of cells to dynamically reallocate energy metabolism, particularly through PFKFB4 inhibition, renders BRAFi-resistant melanoma cells metabolically inflexible and less capable of tolerating therapeutic stress. The maintained activity of PFKFB3 and PFKFB4 inhibition during BRAF/MEK-targeted treatment supports metabolic targeting as a complementary therapeutic strategy. Targeting metabolic flexibility rather than individual metabolic pathways may therefore offer a promising approach in BRAFi-resistant melanoma.