IDH mutation impairs histone demethylation and results in a block to cell differentiation

Abstract

Recurrent mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 have been identified in gliomas, acute myeloid leukaemias (AML) and chondrosarcomas, and share a novel enzymatic property of producing 2-hydroxyglutarate (2HG) from α-ketoglutarate. Here we report that 2HG-producing IDH mutants can prevent the histone demethylation that is required for lineage-specific progenitor cells to differentiate into terminally differentiated cells. In tumour samples from glioma patients, IDH mutations were associated with a distinct gene expression profile enriched for genes expressed in neural progenitor cells, and this was associated with increased histone methylation. To test whether the ability of IDH mutants to promote histone methylation contributes to a block in cell differentiation in non-transformed cells, we tested the effect of neomorphic IDH mutants on adipocyte differentiation in vitro. Introduction of either mutant IDH or cell-permeable 2HG was associated with repression of the inducible expression of lineage-specific differentiation genes and a block to differentiation. This correlated with a significant increase in repressive histone methylation marks without observable changes in promoter DNA methylation. Gliomas were found to have elevated levels of similar histone repressive marks. Stable transfection of a 2HG-producing mutant IDH into immortalized astrocytes resulted in progressive accumulation of histone methylation. Of the marks examined, increased H3K9 methylation reproducibly preceded a rise in DNA methylation as cells were passaged in culture. Furthermore, we found that the 2HG-inhibitable H3K9 demethylase KDM4C was induced during adipocyte differentiation, and that RNA-interference suppression of KDM4C was sufficient to block differentiation. Together these data demonstrate that 2HG can inhibit histone demethylation and that inhibition of histone demethylation can be sufficient to block the differentiation of non-transformed cells.

Figure 1. IDH mutations are associated with dysregulation of glial differentiation and global histone methylation

a, Heatmap representation of a two-dimensional hierarchical clustering of genes identified as differentially expressed between IDH mutant (mut) patient oligodendroglioma samples and IDH wild-type (WT) samples. Each row represents a gene and each column represents a specimen. IDH mutational status, tumour grade and recurrence of each sample are listed. b, 293T cells transfected with empty vector (Vec), wildtype or R132H mutant IDH1, or wild-type or R172K mutant IDH2 for 3 days were lysed and assessed for expression levels of IDH1, IDH2 and histone lysine methylation by western blotting with specific antibodies. Total H3 was used as loading control. Quantification of band intensities is shown in Supplementary Fig. 1. Bottom panel provides quantification of intracellular 2HG to glutamate ratio (2HG/glutamate) as previously reported for these transfectants. c, Immunohistochemistry staining with antibodies against H3K9me3 and H3K27me3 inIDH1/2 wild-type andIDH1mutant oligodendrogliomasamples (x40 magnification). Image quantification was performed using Metamorph software (see Methods) and shown in bottom panels. Error bars represent standard deviation (s.d.) of at least three patient samples in each group.

Figure 2. Differentiation arrest induced by mutant IDH or 2HG is associated with increased global and promoter-specific H3K9 and H3K27 methylation

a, 3T3-L1 cells stably expressing empty vector, wild-type, or R172K mutant IDH2 were lysed and assessed for expression levels of IDH2 or IDH1 by western blotting. Cells were also extracted for intracellular metabolites, which were then MTBSTFA-derivatized (see Methods) and analysed by gas chromatography–mass spectrometry (GC–MS). The quantification of 2HG signal intensity relative to the intrasample glutamate signal is shown for a representative experiment. b, Cells were induced to differentiate into mature adipocytes for 7 days. The accumulation of lipid droplets was assessed by Oil-Red-O staining. Wells from a representative experiment froma total of four independent experiments are shown. c, 3T3-L1 cells were induced to differentiate for 7 days in the absence or presence of 1mM or 2mM octyl-2HG. Oil-Red-O staining was performed and quantified by measuring absorbance at 500nm.DMSO, dimethylsulphoxide. d, Vector, wildtype or R172K mutant IDH2 transduced 3T3-L1 cells were induced to differentiate for 4 days. At days 0 and 4 (d0 and d4), RNA was extracted. Relative expression of adipocyte-specific gene and transcription factors was assessed by quantitative PCR with reverse transcription (RT–qPCR). e, 3T3-L1 cells were induced to differentiate for 4 days in the absence or presence of 1mMor 2mM octyl-2HG. RNA was extracted. Relative expression of adipocyte-specific gene and transcription factors was assessed by RT–qPCR. f, Vector, wild-type or R172K mutant IDH2 transduced 3T3-L1 cells were induced to differentiate. At days 0 and 4 (d0 and d4), ChIP analysis was performed using antibodies against H3K9me3 and H3K27me3. Immunoprecipitated Cebpa and Adipoq promoter sequences were analysed by qPCR and shown as percentage of input. g, Vector, wild-type or R172K mutant IDH2 transduced 3T3-L1 cells were induced to differentiate for 4 days. At days 0 and 4 (d0 and d4), histones were acidextracted and levels of H3K9me3, H3K9me2 and acetyl-H3 were assessed by western blotting with specific antibodies. Total H3 was used as loading control. Quantification of band intensities is shown in Supplementary Fig. 4. In f, error bars indicate s.d. from triplicate wells and a representative experiment from a total of two is shown. For all other experiments, error bars indicate s.d. from three independent experiments. *P<0.05; **P<0.01; NS, not significant.

Figure 3. IDH mutation induces histone methylation increase in CNSderived cells and can alter cell lineage gene expression

a, ImmortalizedNHA cells were retrovirally transduced with constructs containing wild-type or R132H mutant IDH1. Histones were acid-extracted from parental cells or cells expressing wild-type or mutant IDH1 at late (>40) passages. Histone lysine methylation levels were assessed by western blotting with specific antibodies. Total H3 was used as loading control. Images presented are panels from different areas of the same gel. b, Parental, IDH1 wild-type and R132H mutant NHAcells at late passages were lysed and assessed for expression levels of nestin by western blotting. p85 was used as loading control. Images presented are panels from different areas of the same gel. c, NHA cells were retrovirally transduced with constructs containing wild-type or R132H mutant IDH1. Histones were acid-extracted at different time points as cells were passaged in culture. Histone lysine methylation levels were assessed by western blotting with specific antibodies. Total H3 was used as loading control. Images presented are panels from different areas of the same gel. d, Western blot band intensities of H3K9me3 in c and two additional independent experiments were quantified using Image J. Red squares indicate IDH1 wild-type cells. Green triangles indicate IDH1 R132Hmutant cells. e, TotalCpGmethylation of IDH1 wild-type and R132H mutant NHA cells at various passages was measured by FACS using 5-methylcytosine-specific antibody and shown as normalized mean fluorescence intensity. FACS histograms from a representative experiment are shown in Supplementary Fig. 5. f, Neurosphere cultures established from the subventricular zone of brains of p16/p19^−/− mice were infected with a retroviral construct containing IDH1 R132H mutant, wild-type IDH1 or the vector alone and induced to differentiate. GFAP and β3-tubulin expression levels were assessed by western blotting. p85 was used as loading control. In d and e, error bars indicate standard error of the mean from three independent experiments. *P<0.05; **P<0.01.

Figure 4. 2HG-inhibitable H3K9 demethylase KDM4C is required for cell differentiation

a, 3T3-L1 cells were induced to differentiate (Diff) for 3 days. Before (d0) and each day after differentiation induction, cells were lysed and KDM4C protein levels were assessed by western blotting with specific antibody. Tubulin was used as loading control. b, Bulk histones were incubated with purified GST-tagged human KDM4C in the reaction mix with 1 mM αKG and increasing concentrations of d-2HG. Levels of GST tag, H3K9me3 and H3K9me2 were assessed by western blotting with specific antibodies. Total H3 was used as loading control. c, Bulk histones were incubated with purified GST-tagged human KDM4C in the reaction mix. 10mMd-2HG was added to inhibit the demethylation reaction in the presence of increasing concentrations of αKG. Levels of H3K9me3 were assessed by western blotting with specific antibody. Total H3 was used as loading control. d, 3T3-L1 cells were transfected with control siRNAor siRNA specific for KDM4C. After 3 days, cells were lysed and assessed for expression levels of KDM4C and H3K9me3 by western blotting with specific antibodies. Total H3 was used as loading control. Cells from the same treatment were induced to differentiate for 7 days. The accumulation of lipid droplets was assessed by Oil-Red-O staining. Wells from a representative experiment from a total of three independent experiments are shown.