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Cytosolic NADK is conditionally essential for folate-dependent nucleotide synthesis

Abstract

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.

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Fig. 1: Conditional NADK dependence is linked to folic acid availability.
Fig. 2: Cytosolic NADK activity is essential in low folate conditions.
Fig. 3: NADK is conditionally required for nucleotide synthesis and supports cell cycle progression in low folate conditions.
Fig. 4: NADK deletion alters subcellular contributions to 1C production and depletes cellular folate pools.
Fig. 5: NADK facilitates DHFR activity in low folate conditions, and folic acid influences NADK dependence in different cell types.
Fig. 6: TN can form a drug–nutrient interaction with folic acid.

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Data availability

All data needed to evaluate the findings of this study can be found within the article, extended data or supplementary information. The individual plasmids generated in this study have been deposited in Addgene (identifiers found in Supplementary Table 5). Unique reagents generated in this study are available upon reasonable request from the corresponding author. Identifiers for deposited datasets accessed in this study are found in Supplementary Table 5. Source data are provided with this paper.

Code availability

This paper does not report original code.

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Acknowledgements

We thank members of the Cantor laboratory for the upkeep of both the LC–MS system and cell sorter and for helpful discussions. We also thank J. Skotheim for helpful comments and discussion, A. Hunger for generating the pLJC2-Blast-Rap2A-3×FLAG plasmid, M. Stefely for figure assets related to the serine tracing schematic and the University of Wisconsin Carbone Cancer Center Flow Cytometry Laboratory (supported by National Institutes of Health (NIH) P30CA014520) for use of its facilities and services. This work was supported by grants from the American Cancer Society (RSG-21-170-01-TBE, to J.R.C.), the Glenn Foundation and American Federation for Aging Research (A22068, to J.A.S.), NIH (R01DK133479, to J.A.S.), the University of Wisconsin–Madison Hatch Grant (WIS04000-1024796, to J.A.S.) and the Juvenile Diabetes Research Foundation (JDRF) (JDRF201309442, to J.A.S.). Fellowship support was provided by the NIH (T32HG002760, to K.S.H.) and the University of Wisconsin–Madison Department of Biochemistry (to K.M.F., K.S.H. and G.M.W.). J.A.S. is a Howard Hughes Medical Institute (HHMI) Freeman Hrabowski Scholar. J.R.C. is a Hartwell Foundation Investigator.

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Authors and Affiliations

Authors

Contributions

K.M.F. and J.R.C. initiated the project and designed the research plan. K.M.F. performed most of the experiments, with assistance from K.S.H. and G.R.C. C.M.F. optimized and helped perform cell cycle and cell death experiments. G.M.W. performed the lipidomics with guidance from J.A.S. K.C.F. carried out the cell cycle analysis. K.M.F. and J.R.C. analysed and interpreted the experimental data. J.R.C. wrote the manuscript with assistance from K.M.F. All authors discussed the manuscript. J.R.C. supervised the studies.

Corresponding author

Correspondence to Jason R. Cantor.

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Competing interests

J.R.C. is an inventor on an issued patent for HPLM assigned to the Whitehead Institute (Patent no. US11453858). The remaining authors declare no competing interests.

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Nature Metabolism thanks Jiyeon Kim, Johannes Meiser and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Alfredo Giménez-Cassina, in collaboration with the Nature Metabolism team.

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Extended data

Extended Data Fig. 1 Related to conditional NADK dependence is linked to folic acid availability.

(a) Human cell lines ranked by probability of dependency values for NADK across CRISPR screen data cataloged in the DepMap12. Probability > 0.5 is the reference threshold for essentiality. (b) Immunoblots for expression of NADK and NADK2. RAPTOR served as the loading control in both cases. (c) Reported concentration ranges for folic acid and 5-methyl-THF (5-mTHF) in human plasma32,33,34. (d, e, h) Relative growth of NADK-knockout versus control cells (mean ± s.d., n = 3 biologically independent samples). EV, empty vector. Two-tailed Welch’s t-test comparing the respective mean ± s.d. (bar) versus mean ± s.d. (control cells) between bars. (f) Dependency phenotypes for NADK from conditional essentiality profiling in K562 cells11. (g) Extracellular levels of folic acid following 96-hr culture of NADK-knockout or control cells versus those at inoculation (mean ± s.e.m., n = 3 biologically independent samples). Two-tailed Welch’s t-test comparing the respective mean ± s.d. (bar) versus mean ± s.d. (inoculation) between bars.

Source data

Extended Data Fig. 2 Related to cytosolic NADK activity is essential in low folate conditions.

(a) Amino acid sequence alignment depicting a GGDG motif conserved among NADK proteins across different species. D184 is the putative catalytic aspartate (red) in human NADK. (b) Pseudocolor Coomassie-stained gel imaged using a LI-COR Odyssey FC. 1: M.W. standards; 2: wild-type NADK-3xFLAG; 3: NADK (D184A)-3xFLAG. (c) Immunoblots for expression of NADK and NADK2 in NADK-knockout cells. GAPDH or RAPTOR served as the loading control. (d, f) Relative NADP+ levels in NADK-knockout versus control cells (mean ± s.e.m., n = 3 biologically independent samples). EV, empty vector. MTS, mitochondrial targeting sequence. (e) Cellular NADPH abundances (mean ± s.e.m., n = 3 biologically independent samples). Two-tailed Welch’s t-test. (g) Relative NADP+ levels in NADK2-knockout versus control cells (mean ± s.e.m., n = 3 biologically independent samples). (h) Dependency phenotypes for NADK2 from conditional essentiality profiling in K562 cells11. (i) Defined proline levels in HPLM, RPMI, and DMEM. (j, k) Relative growth of NADK2-knockout versus control cells (mean ± s.d., n = 3 biologically independent samples). (d, f, g, j, k) Two-tailed Welch’s t-test comparing the respective mean ± s.d. (bar) versus mean ± s.d. (control cells) between bars.

Source data

Extended Data Fig. 3 Related to NADK is conditionally required for nucleotide synthesis and supports cell cycle progression in low folate conditions.

(a) Relative levels of various lipid classes in NADK-knockout versus control cells (mean ± s.e.m., n = 3 biologically independent samples). Parenthetical value corresponds to the total number of filtered species that comprised each lipid class for analysis (see Supplementary Table 2). Cer_NS, ceramide non-hydroxyfatty acid-sphingosine. EtherPE, ether-linked phosphatidylethanolamine. PC, phosphatidylcholine. PE, phosphatidylethanolamine. SM, sphingomyelin. PS, phosphatidylserine. Two-tailed Welch’s t-test comparing the respective mean ± s.d. (bar) versus mean ± s.d. (control cells) between bars. (b) (top) Dose-responses of control cells to treatment with hydrogen peroxide (H2O2) (mean ± s.d., n = 3 biologically independent samples). (bottom) Defined concentrations of glutathione in HPLM and RPMI. (c) Relative growth of H2O2-treated NADK-knockout and control cells versus untreated control cells (mean ± s.d., n = 3 biologically independent samples). Source data for control cells are shared from Extended Data Fig. 3b. Two-tailed Welch’s t-test comparing the respective mean ± s.d. (bar) versus mean ± s.d. (untreated control cells) between bars. **P < 0.005. (d) Specific growth rates for control cells (mean ± s.d., n = 3 biologically independent samples). Two-tailed Welch’s t-test. (e) Flow cytometric analysis of NADK-knockout and control cells stained with 7-Aminoactinomycin D (7-AAD), which is excluded from live cells. EV, empty vector. (f) (top) Immunoblot for expression of phosphorylated CHK1 (p-CHK1). ACTN1 served as the loading control. Bottom row, p-CHK1 signal normalized by ACTN1 signal in the respective lane versus lane 1. (bottom) Immunoblot for expression of p-CHK2. GAPDH served as the loading control. Bottom row, p-CHK2 signal normalized by GAPDH signal in the respective lane versus lane 1. (g) Relative levels of IMP and dTMP in NADK-knockout versus control cells at indicated time points during log growth (mean ± s.e.m., n = 3 biologically independent samples). Two-tailed Welch’s t-test versus control cells (above bars) or comparing the respective mean ± s.d. (bar) versus mean ± s.d. (control cells) between bars. *P < 0.05, **P < 0.005. (c, g) Values above brackets indicate fold-change between bars.

Source data

Extended Data Fig. 4 Related to NADK deletion alters subcellular contributions to 1 C production and depletes cellular folate pools.

(a) Fractional labeling of dTTP in control cells (mean ± s.d., n = 3 biologically independent samples). Value above bracket indicates difference in M + 1 labeling. Two-tailed Welch’s t-test for comparisons of M + 1 labeling. (b) Immunoblot for expression of SHMT2 in control and SHMT2-knockout cells. RAPTOR served as the loading control. EV, empty vector. (c) Fractional labeling of ATP in SHMT2-knockout cells (mean ± s.d., n = 3 biologically independent samples). Values above bracket indicate differences in M + 1 (bottom bracket) and M + 2 (top bracket) labeling. Only unlabeled ATP could be detected from SHMT2-knockout cells transduced with empty vector (EV). Two-tailed Welch’s t-test for comparisons of M + 1 (bottom) and M + 2 (top) labeling.

Source data

Extended Data Fig. 5 Related to NADK facilitates DHFR activity in low folate conditions.

(a) Immunoblots for expression of DHFR and NADK in control and DHFR-knockout cells. RAPTOR served as the loading control. (b) (left) Relative growth of NADK-knockout and MTX-treated control cells versus vehicle-treated control cells (mean ± s.d., n = 3 biologically independent samples). Two-tailed Welch’s t-test comparing the respective mean ± s.d. (bar) versus mean ± s.d. (vehicle-treated control cells) between bars. (right) MTX is a potent DHFR inhibitor. MTX, methotrexate. (c) Relative levels of tetrahydrofolate (THF), 10-formyl-THF (10-fTHF), and 5-methyl-THF (5-mTHF) in MTX- versus vehicle-treated control cells (mean ± s.e.m., n = 3 biologically independent samples). Two-tailed Welch’s t-test versus vehicle-treated control cells. (d) Schematic for a method to evaluate DHFR activity by measuring THF production from reactions containing recombinant DHFR and different combinations of folic acid, DHF, and NAD(P)H. (e) Pseudocolor Coomassie-stained gel imaged using a LIC-OR Odyssey FC. 1: M.W. standards; 2: DHFR-3xFLAG. (f) Schematic for a method to evaluate MTHFD1 (CD) activity by measuring 5,10-meTHF production from reactions containing recombinant MTHFD1, 10-formyl-THF, and NAD(P)H. (g) Pseudocolor Coomassie-stained gel imaged using a LIC-OR Odyssey FC. 1: M.W. standards; 2: MTHFD1-3xFLAG. (h) 5,10-meTHF levels measured from reactions that contained recombinant MTHFD1 with indicated substrate combinations (mean ± s.d., n = 3 independent reactions). Value above brackets indicates fold-change between bars. Two-tailed Welch’s t-test. (i) Dependency phenotypes for NADK from conditional essentiality profiling of the indicated cell lines using a focused sgRNA library11.

Source data

Extended Data Fig. 6 Related to TN can form a drug-nutrient interaction with folic acid.

(a) Dose-responses of control cells treated with thionicotinamide (TN) (mean ± s.d., n = 3 biologically independent samples). (b, e) Relative levels of (b) NADP+ (e) and NAD+ in TN-treated control and NADK-knockout versus untreated control cells (mean ± s.e.m., n = 3 biologically independent samples). Two-tailed Welch’s t-test versus control cells (above bars) or comparing the respective mean ± s.d. (bar) versus mean ± s.d. (control cells) between bars. *P < 0.05, **P < 0.005. EV, empty vector. (c) Relative growth of TN-treated control and NADK2-knockout versus untreated control cells (mean ± s.d., n = 3 biologically independent samples). Two-tailed Welch’s t-test comparing the respective mean ± s.d. (bar) versus mean ± s.d. (control cells) between bars. (d) Mass-to-charge ratios (m/z) for various products of TN metabolism via the NAD+ salvage pathway based on either addition (+H) or removal (-H) of a proton adduct. Only peaks corresponding to NADPS could not be detected in either ionization mode in TN-treated K562 cells. NMNS, TN mononucleotide. NADS, thio-NAD. NADPS, thio-NADP. NADPSH, thio-NADPH.

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Supplementary information

Reporting Summary

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Synthetic media construction.

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Datasets related to lipidomics.

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Datasets related to cell cycle distributions.

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Datasets related to cellular metabolomics.

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Reagents and resources.

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Gating strategy for cell cycle analysis.

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Uncropped immunoblots and Coomassie-stained gels.

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Flickinger, K.M., Mellado Fritz, C.A., Huggler, K.S. et al. Cytosolic NADK is conditionally essential for folate-dependent nucleotide synthesis. Nat Metab 7, 1150–1167 (2025). https://doi.org/10.1038/s42255-025-01272-3

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