Ascorbate regulates haematopoietic stem cell function and leukaemogenesis

Abstract

Stem-cell fate can be influenced by metabolite levels in culture, but it is not known whether physiological variations in metabolite levels in normal tissues regulate stem-cell function in vivo. Here we describe a metabolomics method for the analysis of rare cell populations isolated directly from tissues and use it to compare mouse haematopoietic stem cells (HSCs) to restricted haematopoietic progenitors. Each haematopoietic cell type had a distinct metabolic signature. Human and mouse HSCs had unusually high levels of ascorbate, which decreased with differentiation. Systemic ascorbate depletion in mice increased HSC frequency and function, in part by reducing the function of Tet2, a dioxygenase tumour suppressor. Ascorbate depletion cooperated with Flt3 internal tandem duplication (Flt3ITD) leukaemic mutations to accelerate leukaemogenesis, through cell-autonomous and possibly non-cell-autonomous mechanisms, in a manner that was reversed by dietary ascorbate. Ascorbate acted cell-autonomously to negatively regulate HSC function and myelopoiesis through Tet2-dependent and Tet2-independent mechanisms. Ascorbate therefore accumulates within HSCs to promote Tet activity in vivo, limiting HSC frequency and suppressing leukaemogenesis.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: HSCs have high ascorbate levels and ascorbate depletion increases HSC frequency.
Figure 2: Ascorbate depletion reduces Tet2 activity in HSCs and progenitors in vivo.
Figure 3: Low ascorbate levels cooperate with Flt3ITD to promote myelopoiesis, in part by reducing Tet2 function, and cell-autonomously promote HSC function.
Figure 4: Low ascorbate levels accelerate leukaemogenesis.
Figure 5: The effect of ascorbate depletion on leukaemogenesis is reversible and ascorbate levels are high in human HSCs.

Accession codes

Primary accessions

BioProject

Sequence Read Archive

References

  1. 1

    Garaycoechea, J. I. et al. Genotoxic consequences of endogenous aldehydes on mouse haematopoietic stem cell function. Nature 489, 571–575 (2012)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  2. 2

    Yan, H. et al. IDH1 and IDH2 mutations in gliomas. N. Engl. J. Med. 360, 765–773 (2009)

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3

    Mihaylova, M. M., Sabatini, D. M. & Yilmaz, O. H. Dietary and metabolic control of stem cell function in physiology and cancer. Cell Stem Cell 14, 292–305 (2014)

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Zhang, H. et al. NAD+ repletion improves mitochondrial and stem cell function and enhances life span in mice. Science 352, 1436–1443 (2016)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

    Agathocleous, M. et al. Metabolic differentiation in the embryonic retina. Nat. Cell Biol. 14, 859–864 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Wang, J. et al. Dependence of mouse embryonic stem cells on threonine catabolism. Science 325, 435–439 (2009)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  7. 7

    Blaschke, K. et al. Vitamin C induces Tet-dependent DNA demethylation and a blastocyst-like state in ES cells. Nature 500, 222–226 (2013)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  8. 8

    Carey, B. W., Finley, L. W., Cross, J. R., Allis, C. D. & Thompson, C. B. Intracellular α-ketoglutarate maintains the pluripotency of embryonic stem cells. Nature 518, 413–416 (2015)

    ADS  CAS  Google Scholar 

  9. 9

    Naka, K. et al. Dipeptide species regulate p38MAPK–Smad3 signalling to maintain chronic myelogenous leukaemia stem cells. Nat. Commun. 6, 8039 (2015)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Takubo, K. et al. Regulation of glycolysis by Pdk functions as a metabolic checkpoint for cell cycle quiescence in hematopoietic stem cells. Cell Stem Cell 12, 49–61 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11

    Simsek, T. et al. The distinct metabolic profile of hematopoietic stem cells reflects their location in a hypoxic niche. Cell Stem Cell 7, 380–390 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12

    May, J. M. The SLC23 family of ascorbate transporters: ensuring that you get and keep your daily dose of vitamin C. Br. J. Pharmacol. 164, 1793–1801 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13

    Seita, J. et al. Gene Expression Commons: an open platform for absolute gene expression profiling. PLoS ONE 7, e40321 (2012)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  14. 14

    Manning, J. et al. Vitamin C promotes maturation of T-cells. Antioxid. Redox Signal. 19, 2054–2067 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15

    Young, J. I., Züchner, S. & Wang, G. Regulation of the epigenome by vitamin C. Annu. Rev. Nutr. 35, 545–564 (2015)

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    Wang, T. et al. The histone demethylases Jhdm1a/1b enhance somatic cell reprogramming in a vitamin-C-dependent manner. Cell Stem Cell 9, 575–587 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17

    Kivirikko, K. I., Myllylä, R. & Pihlajaniemi, T. Protein hydroxylation: prolyl 4-hydroxylase, an enzyme with four cosubstrates and a multifunctional subunit. FASEB J. 3, 1609–1617 (1989)

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18

    Rebouche, C. J. Ascorbic acid and carnitine biosynthesis. Am. J. Clin. Nutr. 54, 1147S–1152S (1991)

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19

    Knowles, H. J., Raval, R. R., Harris, A. L. & Ratcliffe, P. J. Effect of ascorbate on the activity of hypoxia-inducible factor in cancer cells. Cancer Res. 63, 1764–1768 (2003)

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20

    Vukovic, M. et al. Adult hematopoietic stem cells lacking Hif-1α self-renew normally. Blood 127, 2841–2846 (2016)

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21

    Yin, R. et al. Ascorbic acid enhances Tet-mediated 5-methylcytosine oxidation and promotes DNA demethylation in mammals. J. Am. Chem. Soc. 135, 10396–10403 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22

    Chen, J. et al. Vitamin C modulates TET1 function during somatic cell reprogramming. Nat. Genet. 45, 1504–1509 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    Minor, E. A., Court, B. L., Young, J. I. & Wang, G. Ascorbate induces ten-eleven translocation (Tet) methylcytosine dioxygenase-mediated generation of 5-hydroxymethylcytosine. J. Biol. Chem. 288, 13669–13674 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24

    Ito, S. et al. Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science 333, 1300–1303 (2011)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  25. 25

    Tahiliani, M. et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324, 930–935 (2009)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  26. 26

    Jan, M. et al. Clonal evolution of preleukemic hematopoietic stem cells precedes human acute myeloid leukemia. Sci. Transl. Med. 4, 149ra118 (2012)

    PubMed  PubMed Central  Google Scholar 

  27. 27

    Moran-Crusio, K. et al. Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation. Cancer Cell 20, 11–24 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28

    Ko, M. et al. Ten-eleven-translocation 2 (TET2) negatively regulates homeostasis and differentiation of hematopoietic stem cells in mice. Proc. Natl Acad. Sci. USA 108, 14566–14571 (2011)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  29. 29

    Quivoron, C. et al. TET2 inactivation results in pleiotropic hematopoietic abnormalities in mouse and is a recurrent event during human lymphomagenesis. Cancer Cell 20, 25–38 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30

    Abdel-Wahab, O. et al. Genetic characterization of TET1, TET2, and TET3 alterations in myeloid malignancies. Blood 114, 144–147 (2009)

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31

    Shih, A. H. et al. Mutational cooperativity linked to combinatorial epigenetic gain of function in acute myeloid leukemia. Cancer Cell 27, 502–515 (2015)

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32

    Delhommeau, F. et al. Mutation in TET2 in myeloid cancers. N. Engl. J. Med. 360, 2289–2301 (2009)

    PubMed  PubMed Central  Google Scholar 

  33. 33

    Schleicher, R. L., Carroll, M. D., Ford, E. S. & Lacher, D. A. Serum vitamin C and the prevalence of vitamin C deficiency in the United States: 2003–2004 National Health and Nutrition Examination Survey (NHANES). Am. J. Clin. Nutr. 90, 1252–1263 (2009)

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    Pronier, E. et al. Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors. Blood 118, 2551–2555 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

    Patel, J. P. et al. Prognostic relevance of integrated genetic profiling in acute myeloid leukemia. N. Engl. J. Med. 366, 1079–1089 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36

    Cimmino, L. et al. TET1 is a tumor suppressor of hematopoietic malignancy. Nat. Immunol. 16, 653–662 (2015)

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37

    An, J. et al. Acute loss of TET function results in aggressive myeloid cancer in mice. Nat. Commun. 6, 10071 (2015)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Loria, C. M., Klag, M. J., Caulfield, L. E. & Whelton, P. K. Vitamin C status and mortality in US adults. Am. J. Clin. Nutr. 72, 139–145 (2000)

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39

    Khaw, K. T. et al. Relation between plasma ascorbic acid and mortality in men and women in EPIC-Norfolk prospective study: a prospective population study. Lancet 357, 657–663 (2001)

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40

    Liu, M. et al. Vitamin C increases viral mimicry induced by 5-aza-2′-deoxycytidine. Proc. Natl Acad. Sci. USA 113, 10238–10244 (2016)

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41

    Huijskens, M. J., Wodzig, W. K., Walczak, M., Germeraad, W. T. & Bos, G. M. Ascorbic acid serum levels are reduced in patients with hematological malignancies. Results Immunol. 6, 8–10 (2016)

    PubMed  PubMed Central  Google Scholar 

  42. 42

    Moertel, C. G. et al. High-dose vitamin C versus placebo in the treatment of patients with advanced cancer who have had no prior chemotherapy—a randomized double-blind comparison. N. Engl. J. Med. 312, 137–141 (1985)

    CAS  PubMed  Google Scholar 

  43. 43

    Yun, J. et al. Vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting GAPDH. Science 350, 1391–1396 (2015)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  44. 44

    Chen, Q. et al. Pharmacologic doses of ascorbate act as a prooxidant and decrease growth of aggressive tumor xenografts in mice. Proc. Natl Acad. Sci. USA 105, 11105–11109 (2008)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  45. 45

    Busque, L. et al. Recurrent somatic TET2 mutations in normal elderly individuals with clonal hematopoiesis. Nat. Genet. 44, 1179–1181 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46

    Genovese, G. et al. Clonal hematopoiesis and blood-cancer risk inferred from blood DNA sequence. N. Engl. J. Med. 371, 2477–2487 (2014)

    PubMed  PubMed Central  Google Scholar 

  47. 47

    Jaiswal, S. et al. Age-related clonal hematopoiesis associated with adverse outcomes. N. Engl. J. Med. 371, 2488–2498 (2014)

    PubMed  PubMed Central  Google Scholar 

  48. 48

    Maeda, N. et al. Aortic wall damage in mice unable to synthesize ascorbic acid. Proc. Natl Acad. Sci. USA 97, 841–846 (2000)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  49. 49

    Lee, B. H. et al. FLT3 mutations confer enhanced proliferation and survival properties to multipotent progenitors in a murine model of chronic myelomonocytic leukemia. Cancer Cell 12, 367–380 (2007)

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50

    Sotiriou, S. et al. Ascorbic-acid transporter Slc23a1 is essential for vitamin C transport into the brain and for perinatal survival. Nat. Med. 8, 514–517 (2002)

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51

    Kühn, R., Schwenk, F., Aguet, M. & Rajewsky, K. Inducible gene targeting in mice. Science 269, 1427–1429 (1995)

    ADS  PubMed  Google Scholar 

  52. 52

    Washko, P. W., Welch, R. W., Dhariwal, K. R., Wang, Y. & Levine, M. Ascorbic acid and dehydroascorbic acid analyses in biological samples. Anal. Biochem. 204, 1–14 (1992)

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53

    Mullen, A. R. et al. Oxidation of alpha-ketoglutarate is required for reductive carboxylation in cancer cells with mitochondrial defects. Cell Reports 7, 1679–1690 (2014)

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54

    Xia, J., Sinelnikov, I. V., Han, B. & Wishart, D. S. MetaboAnalyst 3.0—making metabolomics more meaningful. Nucleic Acids Res. 43, W251–W257 (2015)

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55

    Doulatov, S. et al. Revised map of the human progenitor hierarchy shows the origin of macrophages and dendritic cells in early lymphoid development. Nat. Immunol. 11, 585–593 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 56

    Kogan, S. C. et al. Bethesda proposals for classification of nonlymphoid hematopoietic neoplasms in mice. Blood 100, 238–245 (2002)

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57

    Noguchi, K., Gel, Y. R., Brunner, E. & Konietschke, F. NparLD: an R software package for the nonparametric analysis of longitudinal data in factorial experiments. J. Stat. Softw. 50, 1–23 (2012)

    Google Scholar 

  58. 58

    Acar, M. et al. Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal. Nature 526, 126–130 (2015)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

S.J.M. is a Howard Hughes Medical Institute (HHMI) Investigator, the Mary McDermott Cook Chair in Pediatric Genetics, the Kathryn and Gene Bishop Distinguished Chair in Pediatric Research, the director of the Hamon Laboratory for Stem Cells and Cancer, and a Cancer Prevention and Research Institute of Texas Scholar. M.A. was a Royal Commission for the Exhibition of 1851 Research Fellow. We thank F. Harrison for sharing the Slc23a2−/− mice, N. Loof and the Moody Foundation Flow Cytometry Facility for flow cytometry, K. Correll, A. Leach and A. Gross for mouse colony management and BioHPC at UT Southwestern for providing high-performance computing. This work was supported by the Cancer Prevention and Research Institute of Texas and the National Institutes of Health (R37 AG024945 and R01 DK100848).

Author information

Affiliations

Authors

Contributions

M.A. conceived and performed most experiments. C.E.M. performed experiments with Tet2fl and Tet2fl;Flt3ITD mice in Extended Data Figs 5, 8. R.J.B. and E.P. performed the histone methylation analysis. E.P. and Z.Z. performed RNA-sequencing analysis. Z.Z. performed the statistical analyses. G.M.C. assessed haematopathology in Figs 4, 5. E.B. and B.L.C. provided technical assistance. M.M.M. performed collagen staining. W.C. provided human bone marrow specimens. G.J.S. provided some of the data from Slc23a2−/− mice. Z.H., M.A., and R.J.D. developed the metabolomics methods and R.J.D. helped to interpret metabolomics results. M.A. and S.J.M. designed experiments, interpreted results and wrote the manuscript.

Corresponding author

Correspondence to Sean J. Morrison.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks H. Christofk, R. Levine and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Figure 1 Stability of metabolites during cell isolation.

a, Diagram of the isolation procedure. bf, Fold changes in the levels of representative metabolites in 20,000 bone marrow cells before and after each step of the cell isolation procedure. Metabolites were extracted before and after bone marrow cells were kept on ice for 7 h (b), centrifuged at 4 °C at 300g for 5 min (c), stained with the antibodies used for HSC isolation (d), cells underwent positive selection with anti-CD45 and anti-Ter119 beads (e) or were sorted by flow cytometry for CD45 and Ter119 (f). G1P, glucose/fructose-1-phosphate; GPC, glycerophosphocholine; GSH, glutathione; NAD, nicotinamide adenine dinucleotide; SAM, S-adenosylmethionine; SMTA, S-methyl-5′-thioadenosine. Although most bone marrow cells are CD45+ or Ter119+, unfractionated samples are different in cellular composition from samples after selection and sorting, perhaps contributing to the changes observed in some metabolites in e and f. Data were normalized to the median metabolite signal intensity of each sample. Statistical significance was assessed with t-tests performed on log2-transformed data. We accounted for multiple comparisons by controlling the false discovery rate. n = 3 mice; *P < 0.05, **P < 0.01, ***P < 0.001. g, Ascorbate levels were compared in whole bone marrow cells from which metabolites were extracted before the cell purification procedure, or after flow cytometric purification (a total of n = 4 mice from two independent experiments). Statistical significance was assessed using a paired t-test. All data represent mean ± s.d. ns, not significant. Source data

Extended Data Figure 2 Differences in metabolite levels among haematopoietic stem and progenitor cell populations in the bone marrow.

a, Types of metabolites detected in 10,000 HSCs. b, Unsupervised clustering of metabolomic data from HSCs, MPPs and CD45+ bone marrow haematopoietic cells isolated from six independent experiments. BM, bone marrow. c, Metabolites that significantly differed between HSCs and CD45+ bone marrow cells (six independent experiments with a total of 6 HSC samples and 16 CD45+ bone marrow samples). GSH, glutathione; IMP, inosine monophosphate. Statistical significance was assessed using t-tests performed on log2-transformed data. We accounted for multiple comparisons by controlling the false discovery rate. All data represent mean ± s.d. d, The metabolites that we measured in stem and progenitor cell populations. The display is autoscaled for each metabolite to illustrate changes across samples. In total, 52 out of 64 metabolites show statistically significant changes among at least some cell populations (one experiment is shown, representative of three independent experiments; *P < 0.05, **P < 0.01, ***P < 0.001) assessed using a one-way ANOVA for each metabolite followed by Fisher’s LSD tests corrected for multiple comparisons by controlling the false discovery rate. Source data

Extended Data Figure 3 The ascorbate content in haematopoietic stem and progenitor cells correlates with ascorbate transporter expression level and the phenotype of ascorbate-depleted Gulo−/− mice.

a, Ascorbate content versus slc23a2 expression in haematopoietic stem and progenitor cell populations. The plotted data are from Fig. 1c, d. bs, u, Analysis of ascorbate-depleted Gulo−/− mice and littermate controls (a total of n = 5–11 mice per genotype per time point were analysed from 3–6 independent experiments per time point). t, The percentage of cells that incorporated a three-day pulse of 5-bromodeoxyuridine (BrdU) at 7–8 weeks of age (a total of n = 4–6 mice per genotype from three independent experiments). Statistical significance was assessed with a two-way ANOVA followed by Fisher’s LSD tests (bl) or Mann–Whitney U-tests (p, u). We corrected for multiple comparisons by controlling the false discovery rate. *P < 0.05, **P < 0.01, ***P < 0.001. All data represent mean ± s.d. Source data

Extended Data Figure 4 Ascorbate depletion did not have any detectable effects on global histone methylation, collagen levels, ROS levels, carnitine metabolism or Tet1–Tet3 expression in HSCs or other haematopoietic progenitors.

a, Western blots with antibodies against the indicated histone modifications were performed using haematopoietic cells from eight-week-old ascorbate-depleted Gulo−/− mice and littermate controls (a total of n = 3–6 mice per genotype from three independent experiments). Bar graphs show band intensity relative to the band intensity of the wild-type sample of each cell type. We did not observe an increase in histone methylation for any of the modifications, as would be expected if ascorbate was promoting global histone demethylase activity. We observed reduced H3K27me3 in HPCs (note that the data in Fig. 3e show that the effects of ascorbate depletion on Flt3ITD-driven myelopoiesis were mediated mainly by reduced Tet2 function). For gel source data, see Supplementary Fig. 1. MyP, myeloid progenitor; WBM, whole bone marrow. b, Histochemical staining of collagen with Sirius red (bright field or polarized light) or Masson’s trichrome (blue) of bone sections from 8–16-week-old Gulo−/− mice and littermate controls (photographs are representative of 6–8 mice per genotype from two independent experiments). c, Carnitine and acetylcarnitine levels were measured by LC–MS/MS for the indicated cell populations obtained from 7–8-week-old Gulo−/− and littermate-control mice (a total of n = 4–7 mice per genotype from four independent experiments). Data show carnitine/acetylcarnitine levels relative to the average levels in wild-type whole bone marrow samples. d, ROS levels were measured in 12-week-old Gulo−/− and littermate-control mice using CellRox Deep Red and Enzo Total ROS dyes (a total of n = 3–4 mice per genotype from three independent experiments). Data show ROS levels relative to wild-type whole bone marrow samples. e, Ascorbate levels were measured in the plasma and bone marrow (BM) of 4–6-month-old Gulo−/− mice or controls (a total of n = 12–17 mice per genotype for plasma and n = 5 mice per genotype for bone marrow were analysed from three independent experiments). f, Tet1Tet3 transcript levels did not change in Gulo−/− versus littermate control mice, suggesting that the effect of ascorbate depletion on Tet activity was not due to reduced Tet1Tet3 transcription (n = 3 mice). Statistical significance was assessed with two-way ANOVAs (a-d, f) followed by Fisher’s LSD tests or Welch’s tests (e), or Mann–Whitney test (a- H3K36me2-HPC). We corrected for multiple comparisons by controlling the false discovery rate. All data represent mean ± s.d. Source data

Extended Data Figure 5 Tet2 deletion increases HSC frequency and function.

ag, Mx1-Cre;Tet2fl/fl mice and littermate controls (+/+) were injected with poly(I:C) at 6–8 weeks of age. The frequencies of HSCs and other haematopoietic progenitors were analysed three weeks later (a total of n = 10–24 mice per genotype from 9–14 independent experiments). h, Percentage of donor-derived haematopoietic cells after competitive transplantation of 200,000 donor Tet2+/+, or Tet2Δ/+ or Tet2Δ/Δ bone marrow cells along with 500,000 wild-type recipient competitor cells in irradiated recipient mice (a total of five donors and 15–25 recipients per treatment from five independent experiments). All data represent mean ± s.d. Statistical significance was assessed with Kruskal–Wallis tests (ag), or a non-parametric mixed model followed by a Kruskal–Wallis test for individual time points (h). We corrected for multiple comparisons by controlling the false discovery rate. *P < 0.05, **P < 0.01, ***P < 0.001. Source data

Extended Data Figure 6 Ascorbate regulates HSC function in vivo and HSC differentiation in culture.

a, Ascorbate levels in sorted cell populations from transplanted recipient mice (a total of n = 2–4 mice per genotype from two independent experiments). b, Percentage of donor cells that are myeloid, B or T cells in wild-type or Gulo−/− recipients 16 weeks after transplantation (a total of n = 13–18 mice per condition were analysed from four independent experiments). c, d, Competitive transplantation of 500,000 donor bone marrow cells from Tet2fl/fl;Mx1-cre mice or littermate controls along with 1,500,000 competitor wild-type cells in irradiated wild-type (ascorbate-replete) or Gulo−/− (ascorbate-depleted) mice (a total of two donor mice and 5–10 recipient mice per treatment from two independent experiments). e, Plasma ascorbate levels in wild-type mice, Gulo−/− mice or Gulo−/− mice fed an ascorbate-supplemented diet (a total of n = 6–17 mice per condition were analysed from four independent experiments). f, Ascorbate content in bone marrow cells from wild-type and Gulo−/− mice fed normal mouse chow or an ascorbate-supplemented diet (a total of n = 3–11 mice per condition were analysed from three independent experiments). gm, Myeloid and erythroid differentiation were assessed eight days after culturing HSCs in the presence or absence of ascorbate or its more stable derivative, 2-phospho-ascorbate (a total of n = 48 wells for myeloid and 24 wells for erythroid differentiation from two independent experiments). All data represent mean ± s.d. Statistical significance was assessed with Welch’s tests (a, b), Kruskal–Wallis tests (em), or a non-parametric mixed model followed by a Kruskal–Wallis test for individual time points (d). We corrected for multiple comparisons by controlling the false discovery rate. *P < 0.05, **P < 0.01, ***P < 0.001. Source data

Extended Data Figure 7 Gene expression changes in HSCs/MPPs from Gulo−/−, Tet2Δ/Δ and Tet2Δ/Δ;Gulo−/− mice relative to control HSCs/MPPs.

ac, HSCs/MPPs were isolated from 4–5-month-old mice of the indicated genotypes. Mice from all treatments were maintained on low ascorbate water and treated with poly(I:C) to induce Tet2 deletion by Mx1-Cre two months before HSC/MPP isolation. a, Fold-change values for all genes that significantly differed among any combination of genotypes (DESeq2 likelihood ratio tests for differential expression, corrected for multiple comparisons by controlling the false discovery rate). The value for each gene in the control cells was set to one. Red and green indicate increases and decreases in gene expression, respectively. The number of genes in Gulo−/− and Tet2Δ/Δ HSCs/MPPs that changed in expression in the same direction in both genotypes relative to control HSCs/MPPs was significantly higher than would be expected by chance (P = 0.01, binomial test). b, Fold change (log2) for HSCs/MPPs of each genotype relative to control HSCs/MPPs. All genes that changed in the same direction in all three genotypes relative to control are shown and for which the log2 fold change was >0.7 or <−0.7 and fragments per kilobase of transcript per million mapped reads (FPKM) > 1. Negative fold change values indicate that the expression of the indicated gene decreased in the indicated genotypes compared to control and positive values indicate that the expression of the gene increased. c, Gene set enrichment analysis showed that of the top 20 gene sets (based on normalized enrichment score) that were downregulated in Gulo−/− compared to control HSCs/MPPs, 4 were also among the top 20 gene sets downregulated in Tet2Δ/Δ compared to control HSCs/MPPs (n = 3 mice per genotype, except for the Tet2Δ/Δ treatment, for which n = 2). Source data

Extended Data Figure 8 Collaboration between Flt3ITD and either Tet2 deficiency or ascorbate depletion.

af, Analysis of the frequencies of haematopoietic stem and progenitor cell populations in the bone marrow of 10–12-week-old Mx1-Cre;Tet2fl/fl;Flt3ITD mice and littermate controls three weeks after poly(I:C) treatment (12–17 independent experiments; the numbers of mice per treatment in ac are shown across the top of a and for df across the top of d). g, Percentage of donor-derived haematopoietic cells after competitive transplantation of 300,000 donor bone marrow cells of the indicated genotypes along with 300,000 competing recipient cells in irradiated recipient mice (a total of 2–5 donor mice and 6–20 recipient mice per treatment from five independent experiments). h, Secondary transplantation of 5 million bone marrow cells from primary Gulo−/− (ascorbate-depleted) recipients of Flt3ITD/+ cells in irradiated wild-type or ascorbate-depleted Gulo−/− recipient mice (a total of three donor mice and 10–18 recipient mice per treatment from three independent experiments). *Comparison to wild type and #comparison to Flt3ITD/+. i, Ascorbate levels in donor cells of the indicated genotypes sorted from transplant recipients (a total of n = 4 mice per condition from 2 independent experiments). 1°, primary transplantation; 2°, secondary transplantation. j, Frequencies of donor cells of the indicated genotypes in the blood of transplant recipients described in Fig. 3f, g. k, Analysis of HSC frequency in the fetal liver of embryonic day (E)17.5 mice of the indicated genotypes (five independent experiments; the numbers of mice per treatment are shown across the top). All data represent mean ± s.d. Statistical significance was assessed with one-way ANOVAs followed by Fisher’s LSD tests (d, k) or Kruskal–Wallis tests (ac, e). In other cases, we used two-way ANOVAs followed by Fisher’s LSD tests (i), Kruskal–Wallis tests (j) or a non-parametric mixed model followed by Kruskal–Wallis tests for individual time points (g, h). We corrected for multiple comparisons by controlling the false discovery rate. *P < 0.05, **P < 0.01, ***P < 0.001; #P < 0.05, ##P < 0.01, ###P < 0.001; +P < 0.05, ++P < 0.01, +++P < 0.001. *Comparison to the wild-type (+/+) condition, #comparison to the Flt3ITD condition and +comparison to the Tet2Δ/+ or Tet2Δ/Δ conditions. To simplify the representation of statistical significance in g, statistical significance was only noted when both Tet2Δ/+ and Tet2Δ/Δ conditions, or both Tet2Δ/+;Flt3ITD and Tet2Δ/Δ;Flt3ITD conditions, were significantly different from other conditions). Source data

Extended Data Figure 9 Analysis of Tet2Δ/+;Flt3ITD leukaemias.

ag, Analysis of recipient mice described in Fig. 4a–d. Statistical significance was assessed with one-way ANOVAs followed by Fisher’s LSD tests (f, g) or Kruskal–Wallis tests (e). d, Representative images from the experiments quantified in Fig. 4a. All data represent mean ± s.d. We corrected for multiple comparisons by controlling the false discovery rate. *P < 0.05, **P < 0.01, ***P < 0.001.

Extended Data Figure 10 Analysis of Tet2Δ/+;Flt3ITD and Tet2Δ/Δ;Flt3ITD leukaemias.

a, b, Analysis of recipient mice described in Fig. 4e–j. Statistical significance was assessed with one-way ANOVAs followed by Fisher’s LSD tests (b) or Kruskal–Wallis tests (a). c, Western blots with antibodies against the indicated histone modifications were performed using protein extracted from Tet2Δ/+;Flt3ITD/+ leukaemia cells isolated by flow cytometry from wild-type or ascorbate-depleted Gulo−/− transplant recipients (results are representative of two independent experiments). d, Diff-Quik-stained blood smears from Gulo−/− recipients of Tet2Δ/+;Flt3ITD/+ cells fed with an ascorbate-supplemented diet before and after the engraftment of leukaemia cells (representative images from the experiments described in Fig. 5a and quantified in Fig. 5c). Cells with an immature blast-like morphology were more abundant in the blood of ascorbate-depleted Gulo−/− recipients compared to ascorbate-fed Gulo−/− recipients. e, f, WBCs in recipient mice from the experiment described in Fig. 5a. The statistical significance of differences among treatments was assessed with Kruskal–Wallis tests (a, e, f) or a one-way ANOVA (b) or a two-way ANOVA followed by Fisher’s LSD tests (c). All data represent mean ± s.d. We corrected for multiple comparisons by controlling the false discovery rate. *P < 0.05, **P < 0.01, ***P < 0.001. Source data

Supplementary information

Supplementary Information

This file contains the uncropped gels, representative flow cytometry plots and a list of antibodies used. (PDF 5215 kb)

Reporting Summary (PDF 88 kb)

PowerPoint slides

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Agathocleous, M., Meacham, C., Burgess, R. et al. Ascorbate regulates haematopoietic stem cell function and leukaemogenesis. Nature 549, 476–481 (2017). https://doi.org/10.1038/nature23876

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Sign up for the Nature Briefing newsletter for a daily update on COVID-19 science.
Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing