Subcellular Characterization of Nicotinamide Adenine Dinucleotide Biosynthesis in Metastatic Melanoma by Using Organelle-Specific Biosensors

Aim: Nicotinamide adenine dinucleotide (NAD⁺) plays central roles in a wide array of normal and pathological conditions. Inhibition of NAD⁺ biosynthesis can be exploited therapeutically in cancer, including melanoma. To obtain quantitation of NAD⁺ levels in live cells and to address the issue of the compartmentalization of NAD⁺ biosynthesis, we exploited a recently described genetically encoded NAD⁺ biosensor (LigA-circularly permutated Venus), which was targeted to the cytosol, mitochondria, and nuclei of BRAF-V600E A375 melanoma cells, a model of metastatic melanoma (MM). Results: FK866, a specific inhibitor of nicotinamide phosphoribosyltransferase (NAMPT), the main NAD⁺-producing enzyme in MM cells, was used to monitor NAD⁺ depletion kinetics at the subcellular level in biosensor-transduced A375 cells. In addition, we treated FK866-blocked A375 cells with NAD⁺ precursors, including nicotinamide, nicotinic acid, nicotinamide riboside, and quinolinic acid, highlighting an organelle-specific capacity of each substrate to rescue from NAMPT block. Expression of NAD⁺ biosynthetic enzymes was then biochemically studied in isolated organelles, revealing the presence of NAMPT in all three cellular compartments, whereas nicotinate phosphoribosyltransferase was predominantly cytosolic and mitochondrial, and nicotinamide riboside kinase mitochondrial and nuclear. In keeping with biosensor data, quinolinate phosphoribosyltransferase was expressed at extremely low levels. Innovation and Conclusions: Throughout this work, we validated the use of genetically encoded NAD⁺ biosensors to characterize subcellular distribution of NAD⁺ production routes in MM. The chance of real-time monitoring of NAD⁺ fluctuations after chemical perturbations, together with a deeper comprehension of the cofactor biosynthesis compartmentalization, strengthens the foundation for a targeted strategy of NAD⁺ pool manipulation in cancer and metabolic diseases.