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Cited by 94Acute Myeloid Leukemia ResearchEpigenetics and DNA MethylationChromatin Remodeling and Cancer

Vitamin C-induced epigenomic remodelling in IDH1 mutant acute myeloid leukaemia

Open access
Mingay, M, Chaturvedi, A, Bilenky, M, Cao, Q, Jackson, L, Hui, T, Moksa, M, Heravi-Moussavi, A +3 more·2018·Leukemia
DOI 10.1038/leu.2017.171PMC PMC5770587
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Abstract

The genomes of myeloid malignancies are characterized by epigenomic abnormalities. Heterozygous, inactivating ten-eleven translocation 2 (TET2) mutations and neomorphic isocitrate dehydrogenase (IDH) mutations are recurrent and mutually exclusive in acute myeloid leukaemia genomes. Ascorbic acid (vitamin C) has been shown to stimulate the catalytic activity of TET2 in vitro and thus we sought to explore its effect in a leukaemic model expressing IDH1 R132H . Vitamin C treatment induced an IDH1 R132H -dependent reduction in cell proliferation and an increase in expression of genes involved in leukocyte differentiation. Vitamin C induced differentially methylated regions that displayed a significant overlap with enhancers implicated in myeloid differentiation and were enriched in sequence elements for the haematopoietic transcription factors CEBPβ, HIF1α, RUNX1 and PU.1. Chromatin immunoprecipitation sequencing of PU.1 and RUNX1 revealed a significant loss of PU.1 and increase of RUNX1-bound DNA elements accompanied by their demethylation following vitamin C treatment. In addition, vitamin C induced an increase in H3K27ac flanking sites bound by RUNX1. On the basis of these data we propose a model of vitamin C-induced epigenetic remodelling of transcription factor-binding sites driving differentiation in a leukaemic model.

AI snapshot

Written from the paper’s full text.

TL;DR

In a murine HOXA9-immortalized bone marrow model expressing IDH1R132H, vitamin C increased 5hmC, reduced proliferation, and promoted myeloid differentiation. Vitamin C induced DNA demethylation and histone changes at enhancers and transcription factor-binding sites, including loss of PU.1 and gain of RUNX1 binding, suggesting epigenomic remodelling.

Key findings

  • Vitamin C (0.345 mM 2-phosphate ascorbic acid) increased 5hmC in IDH1R132H cells but not IDH1WT cells, reduced proliferation dose-dependently, and increased Mac1+F4/80+ (+13%) and Mac1+Gr1+ (+34%) populations while decreasing Sca1+ (−4%) and cKit+ (−30.8%) populations in IDH1R132H but not IDH1WT cells.
  • RNA-seq after vitamin C treatment identified 450 upregulated and 221 downregulated genes; upregulated genes were enriched in 47 Gene Ontology terms (Bonferroni P<0.01), including positive regulation of leukocyte differentiation, chemotaxis, migration, and negative regulation of cell proliferation. TET2 knockdown reduced vitamin C-induced 5hmC gain and reduced upregulation of all five tested target genes.
  • Vitamin C induced 3,789 deMRs and 4,182 iMRs by meDIP/hmeDIP-seq, and 6,496 deMRs and 8,374 iMRs by whole-genome bisulfite sequencing; DxMRs numbered 712. Top de novo motifs in deMRs were HIF1α (68%), PU.1 (34%), RUNX1 (30%), and CEBPβ (15%); 1,584 of 48,415 haematopoietic enhancers (24.4%) overlapped vitamin C deMRs, and 43% of these contained PU.1-binding sites.
  • PU.1-enriched regions decreased from 62,770 in untreated cells to 23,781 after vitamin C, with 95% of vitamin C PU.1 sites also found in untreated cells; RUNX1-enriched regions increased from 4,047 to 6,587, with 48% specific to vitamin C-treated cells. Vitamin C-specific RUNX1 sites showed significant gains in flanking H3K4me1 and H3K27ac, and H3K27ac increased at deMRs, iHRs, and DxMRs (t-test; P<1.34e−9) but not iMRs (t-test; P=0.472).

Methods

  • Murine bone marrow cells were immortalized by HOXA9 overexpression and transduced with IDH1WT or IDH1R132H; cells were treated with vitamin C (0.345 mM added daily unless otherwise indicated) and assessed by proliferation assays, lineage staining/FACS, colony-forming cell assays, and immunofluorescence for 5hmC.
  • Molecular profiling included RNA-seq, meDIP-seq, hmeDIP-seq, whole-genome bisulfite sequencing, histone ChIP-seq (H3K27ac, H3K4me1, H3K4me3), and transcription factor ChIP-seq for PU.1 and RUNX1, with TET2 knockdown and quantitative PCR validation.
  • Bioinformatics used MACS, MEDIPS, GREAT, HOMER, and intersections with haematopoietic enhancers, DNase I hypersensitive sites, and transcription factor-binding datasets.

Limitations

  • The paper states that prior randomized trials used oral vitamin C, which induces transient micromolar plasma levels significantly below the 0.345 mM concentration used here, and that the lack of an appropriate genotypically stratified patient cohort may have contributed to previously observed patient-dependent responses.
  • Evident from the text: the main findings come from a murine HOXA9-immortalized bone marrow model expressing IDH1R132H and are largely in vitro; no human primary AML patient samples or in vivo therapeutic validation are reported in this study.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1038/leu.2017.171

Cites

59 of 59 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 46 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Genes downregulated upon vitC treatment revealed no significantly enriched GO terms.

    “In contrast, genes downregulated upon vitC treatment revealed no significantly enriched GO terms.”
    Quote in the paperNot a substantial claim
  • Findingown data

    Multiple genes expressed in differentiated myeloid cells, including CSF2RA, ITGAM (Mac1), CEBPE and ELANE, were significantly upregulated (FDR<0.05) upon vitC treatment.

    “Further examination revealed significant upregulation (false discovery rate <0.05) of multiple genes expressed in differentiated myeloid cells, including CSF2RA,23 ITGAM (Mac1), CEBPE24 and ELANE,25 and increased expression of genes encoding cell surface markers that define mature myeloid cells (Supplementary Figure S2b, upper).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [23] de Groot RP, Coffer PJ, Koenderman L . Regulation of proliferation, differentiation and survival by the IL-3/IL-5/GM-CSF receptor family. Cell Signal 1998; 10: 619–628. source

      Not checked in the free run.

    • cites [24] Tavor S, Vuong PT, Park DJ, Gombart AF, Cohen AH, Koeffler HP . Macrophage functional maturation and cytokine production are impaired in C/EBP epsilon-deficient mice. Blood 2002; 99: 1794–1801. source

      Not checked in the free run.

    • cites [25] Wang Q, Li N, Wang X, Shen J, Hong X, Yu H et al. Membrane protein hMYADM preferentially expressed in myeloid cells is up-regulated during differentiation of stem cells and myeloid leukemia cells. Life Sci 2007; 80: 420–429. source

      Not checked in the free run.

  • Findingown data

    Whole-genome shotgun bisulfite sequencing identified 6496 deMRs and 8374 iMRs induced by vitC in IDH1R132H cells.

    “We identified 6496 deMRs and 8374 iMRs (Table 1; Materials and Methods) induced by vitC in IDH1R132H cells.”
    Quote in the paperSubstantial claim
  • Findingown data

    No de novo motifs were present in >1% of iMR regions.

    “In contrast, no de novo motifs were present in >1% of our iMR regions, suggesting vitC-induced increases in methylation are not associated with transcription factor-binding motifs.”
    Quote in the paperSubstantial claim
  • Findingown data

    VitC treatment decreased Sca1+ (−4%) and cKit+ (−30.8%) populations in IDH1R132H but not IDH1WT cells.

    “vitC treatment increased the Mac1+F4/80+ (+13%) and Mac1+Gr1+ (+34%) populations and decreased the Sca1+ (−4%) and cKit+ (−30.8%) populations in IDH1R132H but not in IDH1WT cells (Figure 1c; Supplementary Figure S1e).”
    Quote in the paperSubstantial claim
  • May combine two claims. 38
  • Classified as probably not a substantial claim. 16

  • May combine two claims.

    Findingown data

    VitC treatment induced a marked increase in 5hmC signal in IDH1R132H cells, but not in IDH1WT cells.

    “VitC treatment (0.345 mm; throughout unless otherwise indicated) induced a marked increase in 5hmC signal in IDH1R132H cells, but not in IDH1WT cells (Figure 1a; Supplementary Figure S1a).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    VitC treatment increased Mac1+F4/80+ (+13%) and Mac1+Gr1+ (+34%) populations in IDH1R132H but not IDH1WT cells.

    “vitC treatment increased the Mac1+F4/80+ (+13%) and Mac1+Gr1+ (+34%) populations and decreased the Sca1+ (−4%) and cKit+ (−30.8%) populations in IDH1R132H but not in IDH1WT cells (Figure 1c; Supplementary Figure S1e).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Restricting analysis to DxMRs increased the proportion of regions with de novo motifs for PU.1 (41%) and CEBPβ (41%) while the proportion containing a RUNX1 motif decreased to 5%.

    “Restricting our analysis to DxMRs increased the proportion of regions with de novo motifs for PU.1 (41%) and CEBPβ (41%) motifs while the proportion containing a RUNX1 motif decreased to 5%.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Following vitC treatment, remaining viable IDH1R132H cells differentiated into monocytes and macrophages, but IDH1WT cells did not.

    “Morphological assessment following vitC treatment revealed an increase in apoptosis (Supplementary Figure S1d) with the remaining viable cells differentiated into monocytes and macrophage cells (Supplementary Figure S1c) in IDH1R132H but not in IDH1wt cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    VitC treatment induced 5hmC gain in IDH1R132H cells treated with scrambled small interfering RNA, but this effect was significantly reduced in cells treated with small interfering RNA targeting the TET2 transcript.

    “As expected vitC treatment induced 5hmC gain in IDH1R132H cells treated with scrambled small interfering RNA but this effect was significantly reduced in cells treated with small interfering RNA targeting the TET2 transcript (Supplementary Figures S2f and g).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    A significant proportion (226; 31%) of iMR containing CpG islands overlap haematopoietic progenitor DHSs, 95% of which become condensed during differentiation to macrophages.

    “A significant proportion (226; 31%) of iMR containing CpG islands overlap haematopoietic progenitor DHSs, 95% of which become condensed during differentiation to macrophages.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    VitC-induced transcriptional alterations were dependent on TET2, as quantitative PCR on a set of genes upregulated following vitC treatment showed reduced upregulation in all five targets tested.

    “To confirm whether the vitC-induced transcriptional alterations were dependent on TET2 we performed quantitative PCR on a set of genes upregulated following vitC treatment and observed reduced upregulation in all five targets tested (Supplementary Figure S2h; Supplementary Table 1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    A majority of vitC deMRs and iMRs were distal (>5 kb) to gene transcription start sites and iMRs were enriched at CpG islands.

    “A majority of vitC deMRs and iMRs were distal (>5 kb) to gene transcription start sites and iMRs were enriched at CpG islands (Supplementary Figure S3b).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingcitation

    Both TET2 and TET3 are highly expressed in this model and in human AML.

    “both TET2 and TET3 are highly expressed in our model and in human AML27 (Supplementary Figure S2d).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [27] Network CGAR. Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. N Engl J Med 2013; 368: 2059. source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Colony-forming cell assays revealed a vitC-induced, dose-dependent reduction in colony-forming cells, more pronounced in IDH1R132H compared to IDH1WT cells.

    “Furthermore, colony-forming cell assays revealed a vitC-induced, dose-dependent reduction in colony-forming cells, an effect that was more pronounced in IDH1R132H compared to IDH1WT cells (Supplementary Figure S1f).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    MEIS1 showed increased promoter methylation and downregulated expression (1.8-fold) in response to vitC.

    “Interestingly, MEIS1, a gene previously described to increase expression in the presence of IDH2R140Q,31 showed an increased promoter methylation and downregulated expression (1.8-fold) in response to vitC.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [31] Ogawara Y, Katsumoto T, Aikawa Y, Shima Y, Kagiyama Y, Soga T et al. IDH2 and NPM1 mutations cooperate to activate Hoxa9/Meis1 and hypoxia pathways in acute myeloid leukemia. Cancer Res 2015; 75: 2005–2016. source

      Not checked in the free run.

  • May combine two claims.

    Findingcitation

    MEIS1 and CDK1 are downregulated by vitC.

    “including MEIS1(ref. 32) and CDK1,33 both of which are downregulated by vitC.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [32] Argiropoulos B, Yung E, Humphries RK . Unraveling the crucial roles of Meis1 in leukemogenesis and normal hematopoiesis. Genes Dev 2007; 21: 2845–2849. source

      Not checked in the free run.

    • cites [33] Radomska HS, Alberich-Jorda M, Will B, Gonzalez D, Delwel R, Tenen DG . Targeting CDK1 promotes FLT3-activated acute myeloid leukemia differentiation through C/EBPalpha. J Clin Invest 2012; 122: 2955–2966. source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Top enriched GO terms for upregulated genes included positive regulation of leukocyte differentiation, chemotaxis and migration, and negative regulation of cell proliferation.

    “The top enriched GO terms for upregulated genes included terms for positive regulation of leukocyte differentiation, chemotaxis and migration, negative regulation of cell proliferation (Figure 1e)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    VitC deMRs significantly overlapped mature myeloid (macrophage) DHSs (1019; 16%) and PU.1 (1846; 28%)- and CEBPβ (1428; 22%)-binding sites individually, with 768 deMRs (12%) overlapping regions bound by both transcription factors in macrophages.

    “VitC deMRs significantly overlapped mature myeloid (macrophage) DHSs (1019; 16%) and PU.1 (1846; 28%)- and CEBPβ (1428; 22%)-binding sites individually, with 768 deMRs (12%) overlapping regions bound by both transcription factors in macrophages.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Average 5mC level was decreased in response to vitC treatment at macrophage PU.1-binding sites (t-test; P=3.7e−162), CEBPβ-binding sites (t-test; P=1.35e−119) and DHS (t-test; P=7.95e−47), but not progenitor DHS (t-test; P=0.447).

    “Furthermore, the average 5mC level was decreased in response to vitC treatment at macrophage PU.1-binding sites (t-test; P=3.7e−162), CEBPβ-binding sites (t-test; P=1.35e−119) and DHS (t-test; P=7.95e−47), but not progenitor DHS (t-test; P=0.447; Supplementary Figure S3e).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Functional annotation of genes associated with vitC iMRs shows little association to haematopoiesis but reveals various terms related to nervous system function and development.

    “Functional annotation of genes associated with vitC iMRs show little association to haematopoiesis but reveal various terms related to nervous system function and development (Supplementary Figure S3d).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Within vitC-specific RUNX1-enriched regions, there was significant demethylation flanking RUNX1-enriched summits (±100 bp) within enhancers, but not at CpG islands.

    “Examining DNA methylation values within vitC-specific RUNX1-enriched regions revealed significant demethylation flanking RUNX1-enriched summits (±100 bp) within enhancers (K–S test; P=0.0024), but not at CpG islands (K–S test; P=0.2372; Figure 5b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    A majority of vitamin C-specific RUNX1-binding events were within CpG islands, with a subset localizing to IDH1R132H cell enhancers.

    “A majority of vitamin C-specific RUNX1-binding events were within CpG islands (2128/3134; 68%) with a subset localizing to IDH1R132H cell enhancers (214/3134; 7%).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Intersection of 48,415 haematopoietic enhancers with vitC deMRs revealed a significant overlap (1584; 24.4% t-test P~0), 43% of which contained PU.1-binding sites.

    “Strikingly, intersection of 48 415 haematopoietic enhancers with vitC deMRs revealed a significant overlap (1584; 24.4% t-test P~0), 43% of which contained PU.1-binding sites.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Chromatin immunoprecipitation sequencing of PU.1 and RUNX1 revealed a significant loss of PU.1 and increase of RUNX1-bound DNA elements accompanied by their demethylation following vitamin C treatment.

    “Chromatin immunoprecipitation sequencing of PU.1 and RUNX1 revealed a significant loss of PU.1 and increase of RUNX1-bound DNA elements accompanied by their demethylation following vitamin C treatment.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Upon vitC treatment, H3K4me3 increased at deMRs and DxMRs but not at iHRs or iMRs.

    “We also observed increased H3K4me1 at all DMRs (t-test; P<3.37e−6) and increased H3K4me3 signal at deMRs (t-test; P=1.34e−4) and DxMRs (t-test; P=0.03) but not at iHRs (t-test; P=0.42) or iMRs (t-test; P=0.81).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The 'adult tissue stem module' represents a set of genes that are coordinately upregulated in somatic stem and leukaemic cells.

    “the ‘adult tissue stem module’ (false discovery rate q=1.72e−40), which represents a set of genes that are coordinately upregulated in somatic stem and leukaemic cells”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [32] Argiropoulos B, Yung E, Humphries RK . Unraveling the crucial roles of Meis1 in leukemogenesis and normal hematopoiesis. Genes Dev 2007; 21: 2845–2849. source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    DNA methylation has been shown to influence the binding affinities of transcription factors.

    “DNA methylation has been shown to influence the binding affinities of transcription factors34 and thus we sought to examine the link between vitC deMRs and transcription factors by identifying enriched motifs within vitC deMRs.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [34] Domcke S, Bardet AF, Ginno PA, Hartl D, Burger L, Schbeler D . Competition between DNA methylation and transcription factors determines binding of NRF1. Nature 2015; 528: 575–579. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    VitC reduces proliferation and promotes differentiation of myeloid progenitor cells in the context of this model.

    “Taken together these findings suggest that IDH1R132H promotes a more primitive phenotype and that vitC reduces proliferation and promotes the differentiation of myeloid progenitor cells in the context of our model.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Together, these observations suggest that in IDH1R132H-expressing cells, vitC treatment leads to a specific loss of DNA methylation at regulatory regions implicated in the control of haematopoiesis, which are associated with genes that are repressed in AML and upregulated in mature myeloid populations.

    “Together, these observations suggest that in IDH1R132H-expressing cells, vitC treatment leads to a specific loss of DNA methylation at regulatory regions implicated in the control of haematopoiesis, which are associated with genes that are repressed in AML and upregulated in mature myeloid populations.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Hypermethylation at enhancer elements coincides with leukaemogenesis in haematopoietic progenitor cells of TET2−/− mice expressing the AML-ETO9a fusion.

    “Hypermethylation at enhancer elements coincides with leukaemogenesis in haematopoietic progenitor cells of TET2−/− mice expressing the AML–ETO9a fusion.40”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [40] Rasmussen KD, Jia G, Johansen JV, Pedersen MT, Rapin N, Bagger FO et al. Loss of TET2 in hematopoietic cells leads to DNA hypermethylation of active enhancers and induction of leukemogenesis. Genes Dev 2015; 29: 910–922. source

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    The presence of DNA methylation negatively correlates with enhancer activity and alters the ability of tissue-specific DNA-binding proteins to bind their target sites.

    “In addition, the presence of DNA methylation has been shown to negatively correlate with enhancer activity41 and alters the ability of tissue-specific DNA-binding proteins to bind their target sites.34”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [41] Wiench M, John S, Baek S, Johnson TA, Sung MH, Escobar T et al. DNA methylation status predicts cell type-specific enhancer activity. EMBO J 2011; 30: 3028–3039. source

      Not checked in the free run.

    • cites [34] Domcke S, Bardet AF, Ginno PA, Hartl D, Burger L, Schbeler D . Competition between DNA methylation and transcription factors determines binding of NRF1. Nature 2015; 528: 575–579. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The observed differential expression was validated on a subset of genes by quantitative PCR.

    “We validated the observed differential expression on a subset of genes by quantitative PCR (Supplementary Figure S2c).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    PU.1 guides TET2 to a specific subset of genomic regions to facilitate the oxidation of 5mC to 5hmC, a reaction inhibited by 2-hydroxyglutarate and enhanced by vitamin C.

    “These observations support a model in which PU.1 guides TET2 to a specific subset of genomic regions to facilitate the oxidation of 5mC to 5hmC, a reaction that is inhibited by 2-hydroxyglutarate47 and enhanced by vitamin C13 (Figure 5d).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [46] de la Rica L, Rodríguez-Ubreva J, García M, Islam AB, Urquiza JM, Hernando H et al. PU. 1 target genes undergo Tet2-coupled demethylation and DNMT3b-mediated methylation in monocyte-to-osteoclast differentiation. Genome Biol 2013; 14: 1. source

      Not checked in the free run.

    • cites [47] Xu W, Yang H, Liu Y, Yang Y, Wang P, Kim S et al. Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of α-ketoglutarate-dependent dioxygenases. Cancer Cell 2011; 19: 17–30. source

      Not checked in the free run.

    • cites [13] Yin R, Mao S, Zhao B, Chong Z, Yang Y, Zhao C et al. Ascorbic acid enhances Tet-mediated 5-methylcytosine oxidation and promotes DNA demethylation in mammals. J Am Chem Soc 2013; 135: 10396–10403. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Observational studies using oral and intravenous vitamin C suggested therapeutic benefit for a subset of cancer patients, but subsequent randomized, placebo-controlled trials revealed insignificant efficacy.

    “While observational studies using oral and intravenous vitamin C suggested therapeutic benefit for a subset of cancer patients50, 51, 52 subsequent randomized, placebo-controlled trials revealed insignificant efficacy.53, 54”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [50] Cameron E, Campbell A . The orthomolecular treatment of cancer II. Clinical trial of high-dose ascorbic acid supplements in advanced human cancer. Chem Biol Interact 1974; 9: 285–315. source

      Not checked in the free run.

    • cites [51] Cameron E, Pauling L . Supplemental ascorbate in the supportive treatment of cancer: prolongation of survival times in terminal human cancer. Proc Natl Acad Sci USA 1976; 73: 3685–3689. source

      Not checked in the free run.

    • cites [52] Cameron E, Pauling L . Supplemental ascorbate in the supportive treatment of cancer: reevaluation of prolongation of survival times in terminal human cancer. Proc Natl Acad Sci USA 1978; 75: 4538–4542. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    vitC elicits multifaceted, myeloid-specific, epigenomic reprogramming that is not restricted to changes in 5mC and 5hmC.

    “This suggests that vitC elicits multifaceted, myeloid-specific, epigenomic reprogramming that is not restricted to changes in 5mC and 5hmC.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The presence of H3K4me1 and H3K27ac are hallmarks of active enhancers.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    RUNX1 is a critical regulator of haematopoiesis that frequently participates in leukaemia-associated gene aberrations, including RUNX1-RUNX1T1 fusion recurrent in myeloid malignancies.

    “RUNX1 is a critical regulator of haematopoiesis that frequently participates in leukaemia-associated gene aberrations, including the RUNX1–RUNX1T1 (AML1–ETO9a) fusion recurrent in myeloid malignancies.44”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [44] Okuda T, Nishimura M, Nakao M, Fujitaa Y . RUNX1/AML1: a central player in hematopoiesis. Int J Hematol 2001; 74: 252–257. source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Heterozygous, inactivating TET2 mutations and neomorphic IDH mutations are recurrent and mutually exclusive in acute myeloid leukaemia genomes.

    “Heterozygous, inactivating ten-eleven translocation 2 (TET2) mutations and neomorphic isocitrate dehydrogenase (IDH) mutations are recurrent and mutually exclusive in acute myeloid leukaemia genomes.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [5] Figueroa ME, Abdel-Wahab O, Lu C, Ward PS, Patel J, Shih A et al. Leukemic IDH1 and IDH2 mutations result in a hypermethylation phenotype, disrupt TET2 function, and impair hematopoietic differentiation. Cancer Cell 2010; 18: 553–567. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Overexpression of HOXA9 dysregulates MAPK signalling and collaborates with IDH1R132H to induce a short-latency AML in mice.

    “in vivo transplantation experiments have shown that the overexpression of HOXA9 dysregulates MAPK signalling and collaborates with IDH1R132H to induce a short-latency AML in mice.9”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [9] Chaturvedi A, Cruz MMA, Jyotsana N, Sharma A, Yun H, Grlich K et al. Mutant IDH1 promotes leukemogenesis in vivo and can be specifically targeted in human AML. Blood 2013; 122: 2877–2887. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    vitC treatment increases RUNX1 binding and increases H3K27ac and H3K4me1 in the flanking genomic regions.

    “Taken together these results suggest that vitC treatment increases RUNX1 binding and increases H3K27ac and H3K4me1 in the flanking genomic regions.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    2-phosphate ascorbic acid (vitC) is an oxidatively stable form of vitamin C that does not contribute extracellular H2O2 formation and is converted to ascorbic acid during transport across the cell membrane.

    “2-phosphate ascorbic acid (vitC) is an oxidatively stable form of vitamin C that does not contribute extracellular H2O2 formation and is converted to ascorbic acid during transport across the cell membrane.18”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [18] Takamizawa S, Maehata Y, Imai K, Senoo H, Sato S, Hata R . Effects of ascorbic acid and ascorbic acid 2-phosphate, a long-acting vitamin C derivative, on the proliferation and differentiation of human osteoblast-like cells. Cell Biol Int 2004; 28: 255–265. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Native vitamin C is toxic in culture through the formation of extracellular H2O2, potentially masking its more biologically relevant epigenetic effects.

    “Multiple lines of evidence have suggested that vitamin C, in its native form, is toxic in culture through the formation of extracellular H2O2(refs 16, 17) potentially masking its more biologically relevant epigenetic effects.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [16] Chen Q, Espey MG, Krishna MC, Mitchell JB, Corpe CP, Buettner GR et al. Pharmacologic ascorbic acid concentrations selectively kill cancer cells: action as a pro-drug to deliver hydrogen peroxide to tissues. Proc Natl Acad Sci USA 2005; 102: 13604–13609. source

      Not checked in the free run.

    • cites [17] Kawada H, Kaneko M, Sawanobori M, Uno T, Matsuzawa H, Nakamura Y et al. High concentrations of L-ascorbic acid specifically inhibit the growth of human leukemic cells via downregulation of HIF-1α transcription. PLoS One 2013; 8: e62717. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    De novo neomorphic IDH mutations promote aberrant DNA methylation by increased production of 2-hydroxyglutarate (2HG), which inhibits the hydroxylation of 5-methylcytosine (5mC), a reaction catalysed by TET2.

    “One unifying notion is that de novo neomorphic IDH mutations promote aberrant DNA methylation by increased production of 2-hydroxyglutarate (2HG),2 which inhibits the hydroxylation of 5-methylcytosine (5mC), a reaction catalysed by TET2.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [2] Losman J, Kaelin WG . What a difference a hydroxyl makes: mutant IDH,(R)-2-hydroxyglutarate, and cancer. Genes Dev 2013; 27: 836–852. source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    HOXA9 is frequently overexpressed in human AMLs, notably in patients harbouring the IDH1R132H mutation.

    “The homeobox protein HOXA9 contributes to haematopoiesis and is frequently overexpressed in human AMLs,8 notably in patients harbouring the IDH1R132H mutation.9”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [8] Argiropoulos B, Humphries R . Hox genes in hematopoiesis and leukemogenesis. Oncogene 2007; 26: 6766–6776. source

      Not checked in the free run.

    • cites [9] Chaturvedi A, Cruz MMA, Jyotsana N, Sharma A, Yun H, Grlich K et al. Mutant IDH1 promotes leukemogenesis in vivo and can be specifically targeted in human AML. Blood 2013; 122: 2877–2887. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    PU.1 is a pioneering transcription factor essential for haematopoiesis that can bind chromatin in the presence of restrictive epigenetic modifications.

    “PU.1 is a pioneering transcription factor that is essential for haematopoiesis and, unlike most transcription factors, can bind chromatin in the presence of restrictive epigenetic modifications.42”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [42] Feng R, Desbordes SC, Xie H, Tillo ES, Pixley F, Stanley ER et al. PU.1 and C/EBPalpha/beta convert fibroblasts into macrophage-like cells. Proc Natl Acad Sci USA 2008; 105: 6057–6062. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The oncogenic t(8;21) AML-ETO9a translocation is recurrent in AML and fuses RUNX1 to RUNX1T1 leading to aberrant HDAC2 recruitment and histone deacetylation.

    “The oncogenic t(8;21) AML–ETO9a translocation is recurrent in AML and fuses RUNX1 to RUNX1T1 leading to aberrant HDAC2 recruitment and histone deacetylation.49”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [49] Hu Z, Gu X, Baraoidan K, Ibanez V, Sharma A, Kadkol S et al. RUNX1 regulates corepressor interactions of PU.1. Blood 2011; 117: 6498–6508. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    RUNX1 acts in concert with epigenetic modifiers, most notably histone acetyl-transferases, and perturbation of this ability can facilitate transformation.

    “RUNX1 acts in concert with epigenetic modifiers, most notably histone acetyl-transferases, and perturbation of this ability can facilitate transformation.45”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [45] Wang L, Gural A, Sun XJ, Zhao X, Perna F, Huang G et al. The leukemogenicity of AML1-ETO is dependent on site-specific lysine acetylation. Science 2011; 333: 765–769. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Reduced PU.1 expression in haematopoietic cells leads to reduced growth and transformative capability in several leukaemic mouse models including AML.

    “Reduced PU.1 expression in haematopoietic cells leads to reduced growth and transformative capability in several leukaemic mouse models including AML.43”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [43] Zhou J, Wu J, Li B, Liu D, Yu J, Yan X et al. PU. 1 is essential for MLL leukemia partially via crosstalk with the MEIS/HOX pathway. Leukemia 2014; 28: 1436–48v. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    A recent report in a small cohort of patients (n=24) with haematological malignancies showed that >90% had decreased plasma vitamin C concentrations and 58% were classified as vitamin C deficient.

    “Interestingly, a recent report in a small cohort of patients (n=24) with haematological malignancies showed that >90% had decreased plasma vitamin C concentrations and 58% were classified as vitamin C deficient.56”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [56] Liu M, Ohtani H, Zhou W, Orskov AD, Charlet J, Zhang YW et al. Vitamin C increases viral mimicry induced by 5-aza-2'-deoxycytidine. Proc Natl Acad Sci USA 2016; 113: 10238–10244. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationreasoning

    The lack of an appropriate, genotypically stratified cohort of patients may have contributed to the previously observed patient dependent response to vitamin C treatment.

    “Indeed the lack of an appropriate, genotypically stratified cohort of patients may have contributed to the previously observed patient dependent response to vitamin C treatment.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Only 7.1% of the general US population are vitamin C deficient.

    “In contrast, only 7.1% of the general US population are vitamin C deficient.57”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [57] Schleicher RL, Carroll MD, Ford ES, Lacher DA . 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 2009; 90: 1252–1263. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The latter randomized trials utilized oral vitamin C, which induces transient micromolar increases in plasma levels significantly below the concentration used in this study (0.345 mm).

    “However, these latter studies utilized oral vitamin C, which induces transient micromolar increases in plasma levels55 significantly below the concentration used in our study (0.345 mm).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [55] Levine M, Padayatty SJ, Espey MG . Vitamin C: a concentration-function approach yields pharmacology and therapeutic discoveries. Adv Nutr 2011; 2: 78–88. source

      Not checked in the free run.

47 of 47

  • The paper relies on this without support.

    IDH1R132H inhibits TET activity through the production of 2-hydroxyglutarate.

    “Consistent with the notion that IDH1R132H inhibits TET activity through the production of 2-hydroxyglutarate (data not shown)”

  • The paper relies on this without support.

    PU.1 has been implicated in the control of the expression of several genes that were differentially expressed upon vitC treatment (e.g., KIT, CSF1, CSF2RA).

    “In addition, PU.1 has been implicated in the control of the expression of several genes that were differentially expressed upon vitC treatment (for example, KIT, CSF1 and CSF2RA).”

  • The paper relies on this without support.

    The presence of H3K4me1 and H3K27ac are hallmarks of active enhancers.

    “In addition to the absence of 5mC, the presence of H3K4me1 and H3K27ac are hallmarks of active enhancers.”
3 of 3

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • In murine bone marrow cells with mutant IDH1, ascorbate in vitro raised DNA demethylation at myeloid differentiation gene enhancers and promoters and increased key haematopoietic gene expression.

    “ascorbate treatment in vitro increased DNA demethylation at enhancers and promoters of genes associated with myeloid differentiation and increased the expression of several key haematopoietic genes in murine bone marrow cells expressing mutant IDH1 (REF.68).”

    From Targeting cancer vulnerabilities with high-dose vitamin C (2019) · cites this paper as [68]

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Authors
Mingay, M., Chaturvedi, A., Bilenky, M., Cao, Q., Jackson, L., Hui, T., Moksa, M., Heravi-Moussavi, A., Humphries, R. K., Heuser, M., & Hirst, M.
Affiliations
  • University of British Columbia (CA)
  • Medizinische Hochschule Hannover (DE)
  • BC Cancer Agency (CA)
Corresponding author
Hirst, M
ORCID
Venue
Leukemia
Publisher
Springer Science and Business Media LLC
Published
2017-06-02
Volume
32
Issue
1
Pages
11-20
ISSN
  • 0887-6924 (ISSN-L)
  • 1476-5551
DOI
10.1038/leu.2017.171
PMC
PMC5770587
OpenAlex
W2618381598
Semantic Scholar
23c0e0754da690c190a88ee5c6778b90dcc10c54
Cited by
94
References
59
Citations by year
2026: 6 · 2025: 12 · 2024: 14 · 2023: 4 · 2022: 17 · 2021: 12 · 2020: 9 · 2019: 10 · 2018: 7 · 2017: 3
FWCI
6.11
Type
journal article
Language
en
Open access
Licence
Locations
  • Leukemia (publishedVersion, cc-by-nc-sa)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-sa)
  • PubMed Central (publishedVersion, cc-by-nc-sa)
  • PubMed (publishedVersion)
Funders
  • Deutsche Forschungsgemeinschaft (HE 5240/6‐1, HE 5240/5-1)
  • Terry Fox Research Institute
  • Deutsche Krebshilfe
  • Canadian Cancer Society Research Institute (CCSRI #703489)
Topics
  • Health Sciences › Medicine › Hematology › Acute Myeloid Leukemia Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Epigenetics and DNA Methylation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chromatin Remodeling and Cancer
Keywords
vitamin C, acute myeloid leukemia, IDH1 R132H mutation, TET2, DNA methylation, differentially methylated regions, enhancers, PU.1, RUNX1, H3K27ac, myeloid differentiation, transcription factor binding sites, C/EBPβ, HIF-1α
MeSH terms
Animals, Ascorbic Acid, Cell Differentiation, DNA-Binding Proteins, Isocitrate Dehydrogenase, Mice, Inbred C57BL, Mutation, Transcription Factors, Leukemia, Myeloid, Acute, Epigenesis, Genetic, Cell Proliferation, Mice, Epigenomics

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-08.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-08.

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