Tyrosine phosphorylation and dimerization cooperatively activate NAMPT to enable NAD+ synthesis in cancer

<jats:p>Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD⁺ salvage pathway, is frequently upregulated in cancer, yet mechanisms regulating its catalytic activity remain undefined. We identify NAMPT as a direct substrate of multiple proto-oncogenic tyrosine kinases, including ALK, insulin receptor, IGF1R, and PDGFRA. Phosphoproteomics identified NAMPT Y188 as the major phosphorylation site, including the oncogenic fusion kinase NPM1::ALK. NAMPT interacted with NPM1::ALK in the cytoplasm, nucleus, and mitochondria, while Y188 phosphorylation enhanced catalytic activity, NMN/NAD⁺ biosynthesis, and downstream metabolism. Conversely, the Y188F mutant reduced enzymatic activity, proliferation, and clonogenicity, whereas disrupting dimerization similarly impaired phosphorylation and function. Interactome analyses showed phosphorylation and dimerization cooperatively remodel NAMPT-associated networks, enriching phosphorylated dimers for metabolic/redox regulators and monomeric NAMPT for ribosome biogenesis. NAMPT inhibition suppressed the growth of both ALK inhibitor-sensitive and -resistant lymphoma cells and enhanced the efficacy of ALK inhibition, revealing kinase-dependent NAMPT activation as a metabolic vulnerability in oncogene-driven cancers.</jats:p>