Leukemic IDH1 and IDH2 mutations result in a hypermethylation phenotype, disrupt TET2 function, and impair hematopoietic differentiation - PubMed (original) (raw)
. 2010 Dec 14;18(6):553-67.
doi: 10.1016/j.ccr.2010.11.015. Epub 2010 Dec 9.
Omar Abdel-Wahab, Chao Lu, Patrick S Ward, Jay Patel, Alan Shih, Yushan Li, Neha Bhagwat, Aparna Vasanthakumar, Hugo F Fernandez, Martin S Tallman, Zhuoxin Sun, Kristy Wolniak, Justine K Peeters, Wei Liu, Sung E Choe, Valeria R Fantin, Elisabeth Paietta, Bob Löwenberg, Jonathan D Licht, Lucy A Godley, Ruud Delwel, Peter J M Valk, Craig B Thompson, Ross L Levine, Ari Melnick
Affiliations
- PMID: 21130701
- PMCID: PMC4105845
- DOI: 10.1016/j.ccr.2010.11.015
Leukemic IDH1 and IDH2 mutations result in a hypermethylation phenotype, disrupt TET2 function, and impair hematopoietic differentiation
Maria E Figueroa et al. Cancer Cell. 2010.
Abstract
Cancer-associated IDH mutations are characterized by neomorphic enzyme activity and resultant 2-hydroxyglutarate (2HG) production. Mutational and epigenetic profiling of a large acute myeloid leukemia (AML) patient cohort revealed that IDH1/2-mutant AMLs display global DNA hypermethylation and a specific hypermethylation signature. Furthermore, expression of 2HG-producing IDH alleles in cells induced global DNA hypermethylation. In the AML cohort, IDH1/2 mutations were mutually exclusive with mutations in the α-ketoglutarate-dependent enzyme TET2, and TET2 loss-of-function mutations were associated with similar epigenetic defects as IDH1/2 mutants. Consistent with these genetic and epigenetic data, expression of IDH mutants impaired TET2 catalytic function in cells. Finally, either expression of mutant IDH1/2 or Tet2 depletion impaired hematopoietic differentiation and increased stem/progenitor cell marker expression, suggesting a shared proleukemogenic effect.
Copyright © 2010 Elsevier Inc. All rights reserved.
Figures
Figure 1. IDH1 and IDH2 mutant AML cases tend to cluster based on their DNA methylation profiles
(A) Heatmap representation of a correlation matrix in which each patient’s DNA methylation profile is correlated with that of the other patients in the dataset. Patients are ordered according to the unsupervised analysis (hierarchical clustering) results, so that highly correlated patients are located next to each other. Parallel bars on the right of the heatmap have been used to indicate, from left to right: cluster membership, IDH1 mutational status (Green: WT, Dark red: Mutant), IDH2 mutational status (Green: WT, Dark red: Mutant) and combined IDH1/2 mutational status (Green: WT, Dark red: Mutant). (B) Heatmap representation of a correlation matrix in which each patient’s gene expression profile is correlated with that of the other patients in the dataset. Patients are ordered according to the unsupervised analysis (hierarchical clustering) results, so that highly correlated patients are located next to each other. Parallel bars on the right of the heatmap have been used to indicate, from left to right: IDH1 mutational status (Green: WT, Dark red: Mutant), IDH2 mutational status (Green: WT, Dark red: Mutant) and combined IDH1/2 mutational status (Green: WT, Dark red: Mutant). (See also Figure S1 and Table S1).
Figure 2. IDH1/2 mutant AMLs have a markedly aberrant hypermethylated DNA profile
(A)Left: Heatmap representation of a two-dimensional hierarchical clustering of genes identified as differentially methylated between IDH1/2 mutant primary AML cases (indicated by the red bar) and IDH1/2 wild-type cases (indicated by the purple bar). Each row represents a probe set and each column represents a patient. Right: Dot plot of methylation difference between IDH-mutant and IDH-wild-type AMLs (biological significance) vs. statistical significance (-log10 (T+BH p value)). Red points indicate probe sets identified as differentially methylated between the two types of AML. (B)Left: Heatmap representation of a two-dimensional hierarchical clustering of genes identified as differentially methylated between IDH1/2 mutant primary AML cases (Mut; red bar) and normal CD34+ bone marrow cells (NBM; blue bar). Each row represents a probe set and each column represents a patient. Right: Dot plot of methylation difference between IDH1/2 mutant AMLs and normal CD34+ bone marrow cells (biological significance) vs. statistical significance (-log10 (T+BH p value)). Red points indicate probe sets identified as differentially methylated between the two groups. (C) Boxplot illustrating average methylation difference between IDH1/2 mutant AMLs vs. normal CD34+ cells (left) and average gene expression difference between IDH1/2 mutant AMLs vs. normal CD34+ cells (right) of genes aberrantly methylated in IDH1/2 mutant AMLs. (D) Heatmap illustrating the validation of the IDH1/2 mutant methylation signature in an independent cohort of 344 AMLs (IDH1/2 mutant AML = Mut; red bar; normal CD34+ bone marrow cells = NBM; blue bar). (See also Figures S2 and S3 and Table S2A–B).
Figure 3. Expression of 2HG-producing IDH proteins increases global 5-methylcytosine levels
(A) 293T cells were transiently transfected with empty vector, wild-type or R132H mutant IDH1, or wild-type or R172K mutant IDH2. After 3 days, cells were lysed and assessed for IDH1 expression levels by Western blot, and then re-probed for IDH2. β-actin antibody was used as a control. (B) Cells transfected in parallel to those lysed in (A) were extracted for intracellular metabolites. Metabolites were then derivatized with MTBSTFA and analyzed by GC-MS. Shown is the quantitation of 2HG signal intensities relative to the intrasample glutamate signals for a representative experiment. (C) Global DNA methylation levels in cells were analyzed 3 days following transfection by immunofluorescence using antibody against 5-methylcytosine. Quantification of fluorescence intensities from one experiment is shown. Data is representative of three independent experiments. (D) 32D cells were transduced with empty retroviral vector or with wild-type or R172K mutant IDH2, selected in 2.5 µg/ml puromycin for 7 days, and then lysed to confirm stable expression of IDH2. Tubulin antibody was used as a control. (E) Cells were extracted for their intracellular metabolites which were then derivatized with MTBSTFA and analyzed by GC-MS. Shown are representative gas chromatographs from wild-type and mutant IDH2 expressing cells depicting the derivatized metabolites eluting between 31.3 and 33.5 min, including 4-oxoproline (4-oxo Pro), glutamate (Glu), and 2HG. Metabolite abundance refers to GC-MS signal intensity. (F) DNA was extracted from cells with stable wild-type or mutant IDH2 expression, and global DNA methylation levels were measured by slot blot using antibody against 5-methylcytosine. Relative intensity of signals of three independent experiments was quantified. Error bars: +/− SD for triplicate experiments. (See also Figure S4)
Figure 4. IDH1/2 mutations are mutually exclusive with mutations in TET2 in de novo AML
(A) Circos diagram revealing relative frequency and pairwise co-occurrences of mutations in IDH1 and IDH2 in de novo AML. (B) Circos diagram revealing relative frequency and pairwise co-occurrences of mutations in TET2 in de novo AML. (C) Two-by-two table showing that mutations in IDH1/2 and TET2 were mutually exclusive in AML (Left-tailed Fisher p value: 0.009).
Figure 5. Mutant IDH1 expression inhibits hydroxylation of 5-methylcytosine by TET2
(A) 293T cells were transiently transfected with FLAG-tagged TET2 in the absence or presence of wild-type or R132H mutant IDH1. Three days following transfection, global levels of 5-methylcytosine hydroxylation were analyzed by immunofluorescence using antibody against 5-hydroxy-methylcytosine (5-OH-MeC). Representative images from mock-transfected, TET2-transfected, TET2 + IDH1 WT co-transfected, and TET2 + IDH1 R132H co-transfected cells are shown. Scale bar: 100 µM. (B) Transfected cells were analyzed by flow cytometry and gated as TET2 positive or negative by FLAG antibody. Representative gating is shown. Intensities of 5-OH-methylcytosine staining within the TET2 positive and negative populations are shown as histogram overlays. Data in (A) and (B) are representative of three independent experiments. (See also Figure S5)
Figure 6. _TET2_-mutant AML is associated with a hypermethylation phenotype
(A) Heatmap representation of a two-dimensional hierarchical clustering of genes identified as differentially methylated between TET2 mutant primary AML cases (Mut; red bar) and normal CD34+ bone marrow cells (NBM; blue bar). Each row represents a probe set and each column represents a patient. (B) Boxplot illustrating average methylation difference between TET2 mutant AMLs vs. normal CD34+ cells (left) and average gene expression difference between TET2 mutant AMLs vs. normal CD34+ cells (right) of genes aberrantly methylated in TET2 mutant AMLs. (C) Dot plot of methylation difference between _TET2_-mutant AMLs and normal CD34+ bone marrow cells (biological significance) vs. statistical significance (-log10 (BH p value)). Red points indicate probe sets identified as differentially methylated between the two groups. (D) Dot plot of methylation difference between _TET2_-mutant AMLs and TET2 and _IDH1/2_- wild-type AMLs (biological significance) vs. statistical significance (-log10 (T+BH)). Red points indicate probe sets statistically significant between the two groups. (
See also table S3A–C).
Figure 7. IDH2 mutant expression and TET2 knockdown in hematopoietic cells impairs differentiation
(A) 32D cells retrovirally transduced with empty vector, IDH2 WT, IDH2 R140Q, IDH2 R172K or three independent shRNAs against mouse TET2 were analyzed for C-Kit expression by flow cytometry. Intensities of fluorescence signals are depicted as histograms. (B) Primary mouse bone marrow cells were retrovirally transduced with MIGR1 vector, IDH2 WT, IDH2 R140Q, or two shRNAs against mouse TET2. GFP-positive cells were sorted and expanded in methylcellulose media for 14 days Cells were analyzed for Mac-1 and C-Kit expression by flow cytometry. (C) Cells treated as in (B) were analyzed for Mac-1 and Gr-1 expression by flow cytometry. (D) Murine primary bone marrow cells were retrovirally transduced with MIGR1 vector, IDH2 WT, IDH2 R140Q, or two shRNAs against mouse TET2. Cells were grown in liquid culture for 5 days ex-vivo and assessed for the percentage of LSK cells out of the total lineage-negative, GFP-positive cell population. (See also Figure S6)
Similar articles
- 5-Hydroxymethylcytosine correlates with epigenetic regulatory mutations, but may not have prognostic value in predicting survival in normal karyotype acute myeloid leukemia.
Ahn JS, Kim HJ, Kim YK, Lee SS, Ahn SY, Jung SH, Yang DH, Lee JJ, Park HJ, Choi SH, Jung CW, Jang JH, Kim HJ, Moon JH, Sohn SK, Won JH, Kim SH, Michael S, Minden MD, Kim DD. Ahn JS, et al. Oncotarget. 2017 Jan 31;8(5):8305-8314. doi: 10.18632/oncotarget.14171. Oncotarget. 2017. PMID: 28039446 Free PMC article. - Genetic Polymorphism Study of IDH 1/2 and TET2 Genes in Acute Myeloid leukemia Patients.
Atef M, Shafik NF, H A Hassan N, Allam RM, El-Meligui YM, Abdelaziz H. Atef M, et al. Asian Pac J Cancer Prev. 2023 Sep 1;24(9):3169-3182. doi: 10.31557/APJCP.2023.24.9.3169. Asian Pac J Cancer Prev. 2023. PMID: 37774069 Free PMC article. - DNA hydroxymethylation profiling reveals that WT1 mutations result in loss of TET2 function in acute myeloid leukemia.
Rampal R, Alkalin A, Madzo J, Vasanthakumar A, Pronier E, Patel J, Li Y, Ahn J, Abdel-Wahab O, Shih A, Lu C, Ward PS, Tsai JJ, Hricik T, Tosello V, Tallman JE, Zhao X, Daniels D, Dai Q, Ciminio L, Aifantis I, He C, Fuks F, Tallman MS, Ferrando A, Nimer S, Paietta E, Thompson CB, Licht JD, Mason CE, Godley LA, Melnick A, Figueroa ME, Levine RL. Rampal R, et al. Cell Rep. 2014 Dec 11;9(5):1841-1855. doi: 10.1016/j.celrep.2014.11.004. Epub 2014 Dec 4. Cell Rep. 2014. PMID: 25482556 Free PMC article. - Clinical implications of novel mutations in epigenetic modifiers in AML.
Abdel-Wahab O, Patel J, Levine RL. Abdel-Wahab O, et al. Hematol Oncol Clin North Am. 2011 Dec;25(6):1119-33. doi: 10.1016/j.hoc.2011.09.013. Epub 2011 Oct 29. Hematol Oncol Clin North Am. 2011. PMID: 22093580 Review. - IDH mutations in acute myeloid leukemia.
Rakheja D, Konoplev S, Medeiros LJ, Chen W. Rakheja D, et al. Hum Pathol. 2012 Oct;43(10):1541-51. doi: 10.1016/j.humpath.2012.05.003. Epub 2012 Aug 20. Hum Pathol. 2012. PMID: 22917530 Review.
Cited by
- RNA m5C oxidation by TET2 regulates chromatin state and leukaemogenesis.
Zou Z, Dou X, Li Y, Zhang Z, Wang J, Gao B, Xiao Y, Wang Y, Zhao L, Sun C, Liu Q, Yu X, Wang H, Hong J, Dai Q, Yang FC, Xu M, He C. Zou Z, et al. Nature. 2024 Oct 2. doi: 10.1038/s41586-024-07969-x. Online ahead of print. Nature. 2024. PMID: 39358506 - Clonal hematopoiesis and hematological malignancy.
Dunn WG, McLoughlin MA, Vassiliou GS. Dunn WG, et al. J Clin Invest. 2024 Oct 1;134(19):e180065. doi: 10.1172/JCI180065. J Clin Invest. 2024. PMID: 39352393 Free PMC article. Review. - TCA metabolism regulates DNA hypermethylation in LPS and _Mycobacterium tuberculosis_-induced immune tolerance.
Abhimanyu, Longlax SC, Nishiguchi T, Ladki M, Sheikh D, Martinez AL, Mace EM, Grimm SL, Caldwell T, Portillo Varela A, Sekhar RV, Mandalakas AM, Mlotshwa M, Ginidza S, Cirillo JD, Wallis RS, Netea MG, van Crevel R, Coarfa C, DiNardo AR. Abhimanyu, et al. Proc Natl Acad Sci U S A. 2024 Oct 8;121(41):e2404841121. doi: 10.1073/pnas.2404841121. Epub 2024 Sep 30. Proc Natl Acad Sci U S A. 2024. PMID: 39348545 - Ollier Disease, Acute Myeloid Leukemia, and Brain Glioma: IDH as the Common Denominator.
Corvino S, Somma T, Certo F, Bonomo G, Grasso E, Esposito F, Berardinelli J, Barbagallo G. Corvino S, et al. Cancers (Basel). 2024 Sep 11;16(18):3125. doi: 10.3390/cancers16183125. Cancers (Basel). 2024. PMID: 39335096 Free PMC article. Review. - Role of epigenetic in cancer biology, in hematologic malignancies and in anticancer therapy.
Nwabo Kamdje AH, Dongmo Fogang HP, Mimche PN. Nwabo Kamdje AH, et al. Front Mol Med. 2024 Sep 6;4:1426454. doi: 10.3389/fmmed.2024.1426454. eCollection 2024. Front Mol Med. 2024. PMID: 39308891 Free PMC article. Review.
References
- Abbas S, Lugthart S, Kavelaars FG, Schelen A, Koenders J, Zeilemaker A, van Putten WJ, Rijneveld A, Lowenberg B, Valk PJ. Acquired mutations in the genes encoding IDH1 and IDH2 both are recurrent aberrations in acute myeloid leukemia (AML): prevalence and prognostic value. Blood. 2010 - PubMed
- Delhommeau F, Dupont S, Della Valle V, James C, Trannoy S, Masse A, Kosmider O, Le Couedic JP, Robert F, Alberdi A, et al. Mutation in TET2 in myeloid cancers. N Engl J Med. 2009;360:2289–2301. - PubMed
- Dickins RA, Hemann MT, Zilfou JT, Simpson DR, Ibarra I, Hannon GJ, Lowe SW. Probing tumor phenotypes using stable and regulated synthetic microRNA precursors. Nat Genet. 2005;37:1289–1295. - PubMed
Publication types
MeSH terms
Substances
Grants and funding
- R01 CA173636/CA/NCI NIH HHS/United States
- U54 CA143798/CA/NCI NIH HHS/United States
- CA 114737/CA/NCI NIH HHS/United States
- U10 CA021115/CA/NCI NIH HHS/United States
- HHMI/Howard Hughes Medical Institute/United States
- U54CA143798-01/CA/NCI NIH HHS/United States
- R01 HL082950/HL/NHLBI NIH HHS/United States
- R01 CA105463/CA/NCI NIH HHS/United States
- CA21115/CA/NCI NIH HHS/United States
- U24 CA114737/CA/NCI NIH HHS/United States
LinkOut - more resources
Full Text Sources
Other Literature Sources
Medical
Molecular Biology Databases
Miscellaneous