Cytosolic NADK is conditionally essential for folate-dependent nucleotide synthesis.

Nicotinamide adenine dinucleotide kinase (NADK) catalyses the phosphorylation of NAD to produce NAD phosphate, the oxidized form of NADPH, a cofactor that serves a critical role in driving reductive metabolism. Cancer cells co-express two distinct NAD kinases that differ by localization (NADK, cytosol; NADK2, mitochondria). CRISPR screens performed across hundreds of cancer cell lines indicate that both are dispensable for growth in conventional culture media. By contrast, NADK deletion impaired cell growth in human plasma-like medium. Here we trace this conditional NADK dependence to the availability of folic acid. NADPH is the preferred cofactor of dihydrofolate reductase (DHFR), the enzyme that mediates metabolic activation of folic acid. We find that NADK is required for enabling cytosolic NADPH-driven DHFR activity sufficient to maintain folate-dependent nucleotide synthesis under low folic acid conditions. Our results reveal a basis for conditional NADK essentiality and suggest that folate availability determines whether DHFR activity can be sustained by alternative electron donors such as NADH.