Abstract
NADPH donates high-energy electrons for antioxidant defence and reductive biosynthesis. Cytosolic NADP is recycled to NADPH by the oxidative pentose-phosphate pathway (oxPPP), malic enzyme 1 (ME1) and isocitrate dehydrogenase 1 (IDH1). Here we show that any one of these routes can support cell growth, but the oxPPP is uniquely required to maintain a normal NADPH/NADP ratio, mammalian dihydrofolate reductase (DHFR) activity and folate metabolism. These findings are based on CRISPR deletions of glucose-6-phosphate dehydrogenase (G6PD, the committed oxPPP enzyme), ME1, IDH1 and combinations thereof in HCT116 colon cancer cells. Loss of G6PD results in high NADP, which induces compensatory increases in ME1 and IDH1 flux. But the high NADP inhibits DHFR, resulting in impaired folate-mediated biosynthesis, which is reversed by recombinant expression of Escherichia coli DHFR. Across different cancer cell lines, G6PD deletion produced consistent changes in folate-related metabolites, suggesting a general requirement for the oxPPP to support folate metabolism.
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Data availability
Source data used to generate Fig. 2a–c and Supplementary Fig. 2b–d are provided as Supplementary Data 1. Uncropped versions of blots are provided in Supplementary Figs. 8–13. Other data that support the findings of this study are available from the authors upon request. Matlab code used for matrix deconvolution for NADPH redox hydride is provided as Supplementary Note 1. R code and R package used for natural abundance correction are publicly available from GitHub (https://github.com/XiaoyangSu/Isotope-Natural-Abundance-Correction and https://github.com/lparsons/accucor)41.
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Acknowledgements
We thank G. Ducker, J. Ghergurovich and X. Teng for scientific discussion and help. This work was supported by funding from the US National Institutes of Health (grant nos. R01CA163591, P30CA072720, DP1DK113643, P30DK019525, R01DK107667 and R01DK114103) and Department of Energy (grant nos. DE-SC0018420 and DE-SC0018260).
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L.C. and J.D.R. came up with the general approach. L.C., Z.Z and H.D.Z. generated and characterized different cell lines used in this studies. L.C., A.H., M.M, Z.A. and J.D.R. designed and performed CRISPR libraries screening. L.C. and Z.Z. designed and performed isotope tracing studies. L.C. performed metabolomics studies. L.C. and J.D.R. wrote the paper with help from all authors.
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J.D.R. is a founder of Raze Therapeutics and advisor to L.E.A.F. Pharmaceuticals. J.D.R. is a co-inventor on patent application owned by Princeton University covering diagnostics and therapeutics related to NADPH production by the 10-formyl-THF pathway (US20170000769).
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Supplementary Figures 1–13, Supplementary Tables 1–3 and Supplementary Note 1
Supplementary Data 1
CRISPR-based genetic screen scores for three comparisons: HCT116 WT versus ΔG6PD cells, ΔG6PD versus ΔG6PD/ΔIDH1 cells and WT versus ΔG6PD/ΔIDH1 cells.
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Chen, L., Zhang, Z., Hoshino, A. et al. NADPH production by the oxidative pentose-phosphate pathway supports folate metabolism. Nat Metab 1, 404–415 (2019). https://doi.org/10.1038/s42255-019-0043-x
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DOI: https://doi.org/10.1038/s42255-019-0043-x
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