2-hydroxyglutarate inhibits MyoD-mediated differentiation by preventing H3K9 demethylation

Abstract

Oncogenic IDH1/2 mutations produce 2-hydroxyglutarate (2HG), resulting in competitive inhibition of DNA and protein demethylation. IDH-mutant cancer cells show an inability to differentiate but whether 2HG accumulation is sufficient to perturb differentiation directed by lineage-specifying transcription factors is unknown. A MyoD-driven model was used to study the role of IDH mutations in the differentiation of mesenchymal cells. The presence of 2HG produced by oncogenic IDH2 blocks the ability of MyoD to drive differentiation into myotubes. DNA 5mC hypermethylation is dispensable while H3K9 hypermethylation is required for this differentiation block. IDH2-R172K mutation results in H3K9 hypermethylation and impaired accessibility at myogenic chromatin regions but does not result in genome-wide decrease in accessibility. The results demonstrate the ability of the oncometabolite 2HG to block transcription factor-mediated differentiation in a molecularly defined system.

Keywords: 2-hydroxyglutarate; differentiation; isocitrate dehydrogenase; myogenesis; sarcoma.

Fig. 1.

2HG-producing IDH2 mutations block the ability of MyoD to drive myotube differentiation. (A) 10T1/2 cells were transduced with wild-type MyoD (M) or MyoD-ER (MER) fusion cDNA-encoding lentiviral vectors and grown in the presence or absence of 4-OH-T. Western blots were probed with indicated antibodies (Top shows exogenous MyoD-ER and endogenous MyoD; second blot shows only endogenous MyoD; MyoG, myogenin; MHC, myosin heavy chain; Vinc, vinculin). Right are representative images of myotubes by phase contrast microscopy (Top), GFP (Middle), and immunohistochemistry for MHC (Bottom). (B) 10T1/2 cells were transduced with constructs containing doxycycline inducible (Tet-ON) vector, IDH2-WT, or IDH2-R172K in the presence or absence of doxycycline. 2HG levels shown were measured by GCMS and normalized to spiked-in D5-2HG. Data are mean ± SD of three biological replicates. P values were determined using unpaired Student’s t test. (C) Western blot of 10T1/2 cells transduced with doxycycline-inducible constructs as indicated in B. (D) Fluorescence microscopy of cells as described in C. MHC is in green; DAPI is in blue. Quantification of MHC stained areas is shown on Right. Data are mean ± SD of MHC signal from three representative areas. P values were determined using unpaired Student’s t test.

Fig. 2.

Continuous 2HG production is required for the 2HG-dependent differentiation block. (A, Upper) Strategy for experiments shown in A and B; details indicated in the text. (A, Lower) 10T1/2 cells were passaged four times and differentiated for 2 days with 4-OH-T with 0.5 mM or 2 mM 2HG added throughout the experiment (^+/+) or only during the differentiation step (^−/+). Western blots were probed with antibodies indicated at Left. The line that appears in the reproduced panel quantitating MyoD represents the top edge of the membrane that was probed. The membrane was cut after transfer to allow the upper part of the western blot to be probed independently. (B) Western blot of 10T1/2 cells expressing doxycycline-inducible IDH2-WT or IDH2-R172K passaged and/or differentiated in the presence or absence of doxycycline.

Fig. 3.

2HG blocks differentiation by impairing H3K9 demethylation. (A) Phase contrast and GFP fluorescence microscopy of 10T1/2 MyoD-ER IDH2-WT (Upper Left) and IDH2-R172K (all others) transduced cells passaged with indicated inhibitors [DMSO (D), UNC0638 (U), GSK287 (G1), GSK126 (G2), GSK343 (G3)] and triggered to differentiate with 4-OH-T. (B) Western blot corresponding to cells indicated in A. (C and D) Western blot and phase contrast images of 10T1/2 MyoD-ER cells expressing IDH2-WT or IDH2-R172K and transduced with CRISPR Cas9/guide vectors targeting control Rosa26 (R) or EHMT1 using two different guides (E1.1 and E1.2). Blotted proteins indicated at C, Left).

Fig. 4.

Oncogenic IDH mutations impair H3K9me2 demethylation at myogenic sites. (A) H3K9me2 MetaPeaks (mean read count per million mapped reads) for indicated samples centered around ChIP-seq peaks (delineated by gray vertical lines) for MyoG in 7-d myotubes; for clarity, IDH2-WT samples shown at Left and IDH2-R172K samples shown at Right. (B) Integrated normalized read counts for peak-flanking regions (±3 kb) shown in A; black dots indicate mean read count. *****P < 1 × 10e-50; NS: not significant with P > 0.5 based on Welch’s one-sided t test with expectation of decrease compared with control condition. Cont, no MyoD Control; Ei, EHMT1 inhibitor; M, MyoD. (C) Example H3K9me2 ChIP-seq tracks for indicated samples centered around the myogenic gene Pvalb.

Fig. 5.

Oncogenic IDH mutations impair a MyoD-dependent increase in chromatin accessibility at myogenic regions but do not have a global effect on chromatin accessibility. (A) ATAC-seq signal tracks of IDH2-WT and IDH2-R172K cells before and after MyoD activation at a myogenic locus (Linc-md1). Differential accessibility peaks are highlighted. Also shown is the myogenin ChIP-seq track for C2C12 differentiated myotubes (MT) for orientation. (B) Gene set enrichment analysis plots for two myogenesis gene sets for genes with increased MyoD-dependent accessibility between IDH2-WT and IDH2-R172K. Vertical black line indicates position right of which genes are negatively correlated. (C) Superimposed differential accessibility (difference in MyoD-dependent accessibility change between IDH2-WT and IDH2-R172K samples) measured by differences in normalized read counts in regions flanking ChIP-seq peaks corresponding to MyoD or CTCF in C2C12 myoblasts and myogenin in differentiating C2C12 cells at 60 h or 7 d. All differential accessibility traces have nonoverlapping 99% confidence intervals with respect to CTCF and shuffled control. (D) H3K9me2 MetaPeaks (mean read count per million mapped reads) for indicated samples centered around differential ATAC peaks (peaks with 1.5× or higher increased MyoD-dependent accessibility in IDH2-WT but not in IDH2-R172K). (E) Integrated normalized read counts for peak-flanking regions (±3 kb) shown in D; black dots indicate mean read count. *****P < 1 × 10e-50; NS: not significant with P > 0.5 based on Welch’s one-sided t test with expectation of decrease compared with control condition.