NAMPT in drug-resistant melanoma: linking NAMPT-dependent metabolic reprogramming and immune regulation

Introduction Targeted-therapy and immune checkpoint inhibitors (ICIs) have notably improved the treatment of BRAF-mutated metastatic melanoma (MM) patients; however resistance to treatment dramatically impacts on the survival of patients. BRAF(i)nhibitors-resistant MM cells showed increased amounts of NAD, an essential redox cofactor, supporting their metabolic adaptations underlying the acquisition of drug resistance. This was obtained selectively overexpressing the rate-limiting NAD-biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT). NAMPT-NAD axis becomes a driver of melanoma progression and resistance to BRAFi, that it may be therapeutically targeted. Material and Method NAMPT-NAD axis was studied using biochemical, enzymatic, immunohistochemical assays. Immunoprecipitation of NAMPT following mass spectrometry (MS) analysis was used to define NAMPT interactome. In silico analysis using TCGA database, western blot analysis and RT-PCR were used to detect and confirm protein expression and signaling pathways activation. Results and discussion The BRAF oncogenic signaling and NAMPT expression are molecularly linked. We found that mutations in the BRAF oncogene positively correlate with NAMPT expression in TCGA melanoma cohort and in tissue from MM patients. The over-expression of NAMPT correlates with its gene amplification. Analyzing the nature of this genetic amplification we found a co-amplification of others genes including PIK3CG that we started to evaluate functionally. A second set of preliminary data revealed a significant increase in the nuclear localization of NAMPT in resistant cells, also as NAMPT nuclear fraction chromatin-bound. We performed NAMPT immunoprecipitation following MS in cellular extract to identify NAMPT-interacting proteins. Data showed enrichment of NAMPT-interacting nuclear proteins and proteins involved in RNA processing, translation, metabolic processes, cellular response to stress and immune response among others. Starting to analyze NAMPT-immune gene signature relationship we found in TCGA melanoma cohort a positive correlation between NAMPT expression and interferon signaling, including CD274, IRF1, STAT1 genes that will be further investigated. Conclusion The multiple roles of NAMPT as intracellular and soluble protein are known; here we speculate that NAMPT could have an essential and unknown function in the nucleus and in regulating immune responses, with a possible impact on ICIs activities.