In the intricate web of cellular metabolism, certain enzymes quietly govern critical biochemical currencies that sustain life. Among these, nicotinamide adenine dinucleotide kinase (NADK) plays a particularly intriguing role by catalyzing the phosphorylation of nicotinamide adenine dinucleotide (NAD⁺) to generate nicotinamide adenine dinucleotide phosphate (NADP⁺). This seemingly simple reaction is, in fact, a pivotal step that produces NADP⁺, the oxidized form of NADPH, which serves as a vital reducing cofactor across various anabolic pathways. Recent research highlights the conditional necessity of NADK, revealing that while it may be dispensable under routine culture conditions, its role becomes indispensable when cells are subjected to nutrient environments that closely mimic human physiological plasma, particularly when folate levels are limited.
NADK exists in two isoforms within human cells: the cytosolic NADK and mitochondrial NAD kinase 2 (NADK2). These isoforms are spatially segregated, with NADK operating in the cytosol and NADK2 residing within mitochondria. Both enzymes contribute to maintaining intracellular pools of NADP(H), essential for redox reactions, but intriguingly, large-scale CRISPR knockout screens across hundreds of cancer cell lines have demonstrated that eliminating either enzyme does not significantly impair cell proliferation under standard laboratory media conditions. This discovery initially suggested redundancy or compensatory mechanisms at play to suffice growth demands.
However, the paradigm shifts dramatically when cells are cultured in human plasma-like medium, a formulation that simulates the nutritional composition of human blood. Under these more physiologically relevant conditions, deleting cytosolic NADK caused a pronounced impairment in cell growth. This striking observation prompted investigative efforts to unearth the metabolic underpinnings of NADK’s conditional essentiality. Delving deeper, the researchers uncovered a critical link between NADK dependency and folate metabolism, a cornerstone of nucleotide biosynthesis and cell proliferation.
Folate, a water-soluble B-vitamin, must be metabolically activated by the enzyme dihydrofolate reductase (DHFR) to support one-carbon metabolism pathways integral to DNA synthesis. DHFR requires reducing equivalents to function, conventionally supplied by NADPH, the reduced form of NADP⁺. Since NADK controls the production of NADP(H) pools within the cytosol, its impairment directly jeopardizes the availability of NADPH for DHFR activity. The study’s findings demonstrate that under low folic acid conditions, which recapitulate human physiological folate levels, cytosolic NADK becomes essential for sustaining NADPH-dependent DHFR catalysis, thereby enabling the continuation of folate-driven nucleotide synthesis.
The researchers’ methodological approach included systematic knockout of NADK using CRISPR-Cas9 gene editing in cancer cell models followed by growth assays in different media environments. Conventional culture media, often enriched with supraphysiological folate concentrations, masked the essential nature of NADK, allowing cells to utilize alternative electron donors for DHFR or circumvent its dependence altogether. Conversely, when folate availability resembled normal human plasma, the metabolic bottleneck emerged clearly, emphasizing NADK’s indispensable metabolic role.
Moreover, the study sheds light on the biochemical hierarchy and electron donor preference of DHFR. The enzyme typically favors NADPH as an electron donor due to its optimal redox potential and enzyme affinity. In the absence of sufficient cytosolic NADPH—resulting from NADK deletion—cells fail to maintain the necessary flux of activated folates, which cascades into defective nucleotide biosynthesis. Interestingly, the data suggest that under certain nutrient conditions, DHFR may attempt to utilize NADH as an alternative electron source, but this substitution is inefficient and insufficient to sustain cell growth.
These insights draw attention to the intricate interplay between nutrient availability and intracellular redox states, which collectively determine enzymatic efficiencies and, ultimately, cellular fitness. The identification of NADK’s conditional essentiality also underscores the metabolic plasticity of cancer cells and their capacity to adapt to variable microenvironmental nutrient landscapes. Since folate metabolism is a well-established target in cancer therapy, understanding the factors that influence DHFR activity holds profound implications for designing novel therapeutic strategies.
Another remarkable aspect is the subcellular compartmentalization of NADK isoforms and their distinct but potentially complementary roles. While mitochondrial NADK2 supports mitochondrial NADP(H) demand, cytosolic NADK specifically regulates cytosolic NADP(H) pools, thereby influencing biosynthetic pathways like nucleotide synthesis and antioxidant defenses—processes firmly rooted in the cytosolic compartment. This spatial segregation highlights the sophistication of redox control tailored to localized metabolic requirements.
In broader terms, the research prompts reconsideration of metabolic dependencies in cancer cells grown in vitro compared to physiological contexts. Traditional tissue culture media, often nutrient-rich and non-representative of in vivo conditions, may overlook critical vulnerabilities masked by excess metabolites, such as folates. Employing human plasma-like media to cultivate cells thus reveals metabolic liabilities with potential therapeutic relevance, including the NADK-DHFR axis described here.
The conditional essentiality of NADK linked to folate conditions also provides a cautionary note in interpreting genetic screens and drug target validations. Without replicating relevant physiological nutrient milieus, some critical dependencies may remain concealed. This study therefore advocates for integrating more physiologically accurate culture systems in preclinical research to better capture the metabolic intricacies operative in human disease settings.
Furthermore, this work opens avenues for exploring how nutritional deficiencies or therapeutic modulation of folate availability might sensitize cancer cells to NADK inhibition. Combining antifolate drugs with strategies that limit NADK function could potentiate anti-proliferative outcomes, exploiting the synthetic lethality described here. The potential to selectively target folate-dependent tumors by disrupting their cytosolic NADPH supply marks a novel therapeutic frontier.
Beyond cancer biology, these findings resonate across broader fields studying redox metabolism and nutrient interactions. NADPH serves as a linchpin in cellular antioxidant defenses and biosynthesis, linking NADK activity to diverse physiological and pathological contexts. Understanding how nutrient fluctuations modulate NAD kinase functions and downstream pathways could illuminate metabolic adaptations across health and disease.
In summary, the elucidation of cytosolic NADK as conditionally essential under folate-limited conditions reveals fundamental aspects of redox metabolism governing nucleotide biosynthesis. By connecting nutrient environment, enzyme function, and metabolic regulation, this research adds a vital piece to the puzzle of cellular metabolic control, underscoring the need for context-aware approaches in biological and therapeutic discovery.
This breakthrough study, led by Flickinger, Mellado Fritz, Huggler, and colleagues, elegantly integrates genetic, biochemical, and metabolic analyses to redefine the role of NADK in folate metabolism. The findings present a compelling case for revisiting metabolic vulnerabilities with an eye toward physiological accuracy, potentially reshaping how metabolic dependencies are targeted in cancer treatment. As scientific exploration advances, the dynamic interplay of enzymes like NADK with nutrient landscapes promises to remain a fertile ground for innovation and therapeutic development.
Subject of Research: The conditional essentiality of cytosolic nicotinamide adenine dinucleotide kinase (NADK) in supporting folate-dependent nucleotide synthesis through NADPH-driven dihydrofolate reductase (DHFR) activity.
Article Title: Cytosolic NADK is conditionally essential for folate-dependent nucleotide synthesis.
Article References:
Flickinger, K.M., Mellado Fritz, C.A., Huggler, K.S. et al. Cytosolic NADK is conditionally essential for folate-dependent nucleotide synthesis. Nat Metab (2025). https://doi.org/10.1038/s42255-025-01272-3
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