Leukemic IDH1 and IDH2 mutations result in a hypermethylation phenotype, disrupt TET2 function, and impair hematopoietic differentiation

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.

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)