NAMPT and NNMT released via extracellular vesicles and as soluble mediators are distinguished traits of BRAF inhibitor resistance of melanoma cells impacting on the tumor microenvironment.

Drugs targeting mutant BRAF and MEK oncogenes are effective in melanoma, even though resistance rapidly develops. This complex picture includes acquired intrinsic tumor and tumor microenvironmental-mediated mechanisms. Here we show that melanoma cells resistant to BRAF inhibitors (BRAFi) overexpress the rate-limiting enzymes involved in nicotinamide (NAM) metabolism nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide N-methyltransferase (NNMT). Remarkably, these cells release NAMPT and NNMT both in the free-form or loaded into extracellular vesicles (EVs). NAMPT is emerging as a key mediator of resistance to BRAFi in melanoma, primarily due to its established role in NAD biosynthesis. Although previously identified as a soluble extracellular factor in this tumor, its presence within EVs released by melanoma cells has not been reported until now, highlighting a previously unrecognized mechanism through which NAMPT may influence the tumor microenvironment (TME). NNMT was revealed to increase in melanoma lesions compared to benign nevi. Here, we report for the first time its overexpression in resistant melanoma cell lines at intracellular and extracellular levels (secreted both as a soluble factor and into EVs). NNMT expression is increased in BRAF-mutated melanoma patients, suggesting a link between its upregulation and the BRAF oncogenic signaling. Moreover, NNMT levels positively correlate with gene signatures associated with pro-inflammatory signaling, immune cell migration, and chemokine-mediated pathways. NNMT pharmacological inhibition and genetic silencing significantly reduce resistant melanoma cell growth. In addition, we found that BRAFi-resistant cells are more sensitive to NNMT inhibition, highlighting a trait of vulnerability of BRAFi-resistant melanomas. Lastly, we proposed for the first time a tetrameric NNMT:TLR4 binding model offering a plausible structural and mechanistic basis for their association. Our functional results indicated that exogenous NNMT treatment is able to trigger NF-κB pathway, one of the main TLR4-dependent signaling, sharing this cytokine-like properties with NAMPT, and opening a future deeper exploration of its functional role in the extracellular space. Overall, the identification of NAMPT and, surprisingly also NNMT, included in EVs and abundantly released from resistant melanoma cells supports the impact of these moonlighting proteins involved in nicotinamide metabolism as mediators of BRAF/MEK inhibitors resistance with tumor intrinsic and potentially tumor microenvironment-mediated mechanisms. Interfering with nicotinamide metabolism could be a valid strategy to counteract drug resistance acting on the multifactorial tumor-host interactions.