Translational reprogramming in BRAF inhibitors resistant melanoma: the impact of NAMPT/NAD metabolism in mTOR- dependent regulation of protein synthesis

Drug resistance remains a major obstacle to effective cancer therapy. In melanoma, nicotinamide phosphoribosyltransferase (NAMPT)-dependent NAD metabolism has been shown to drive the acquisition of resistance to BRAF inhibitors (BRAFi). The mammalian target of rapamycin (mTOR) pathway represents another critical regulator in this context, controlling anabolic processes, energy metabolism, and protein translation, all of which contribute to adaptive resistance mechanisms. However, the functional interplay between NAMPT/NAD metabolism and mTOR signaling in the regulation of mRNA translation during resistance acquisition remains poorly defined, as does the impact of NAMPT activity modulation on protein synthesis efficiency in this context. In this thesis, we demonstrate that NAMPT inhibition, using two distinct inhibitors (OT 82 and FK866), inducing metabolic stress characterized by reduced intracellular NAD and ATP levels, triggers activation of AMP-activated protein kinase (AMPK), leading to decreased phosphorylation of mTOR and its downstream effector 4EBP1, a key regulator of cap-dependent translation. In parallel, we observed increased phosphorylation of eIF2α, a central translation initiation factor activated in response to cellular stress, which suppresses global protein synthesis and promotes stress granule (SG) assembly. Collectively, these molecular events resulted in robust translational arrest upon NAMPT inhibition, as confirmed by Click-it and puromycin incorporation assays, as well as polysome profiling. Notably, NAMPT interactome analysis, performed by immunoprecipitation followed by mass spectrometry, revealed enrichment of proteins involved in translational pathways, particularly in BRAFi-resistant cells. Consistently, western blot analysis detected NAMPT in 80S monosome and both light and heavy polysome fractions, supporting its physical association with the translational machinery, including ribosomal proteins and/or initiation and elongation factors. Preliminary co-immunoprecipitation experiments further demonstrated an interaction between NAMPT and eIF2α. Lastly, comparative proteomic profiling of paired BRAFi-sensitive and -resistant melanoma cell lines revealed a global reduction in protein synthesis in resistant cells, consistent with translational remodeling as a mechanism of drug adaptation and potentially linked to NAMPT function. Overall, although the precise impact of NAMPT modulation on translatome remodeling in targeted therapy (TT)-resistant melanoma cells remains to be fully elucidated, our findings support the existence of a NAMPT/NAD-dependent metabolic-translational crosstalk that may contribute to the adaptive vulnerabilities of resistant melanoma cells.