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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., Competing Interests: Conflict of interest statement: C.B.T. is a founder of Agios Pharmaceuticals and a member of its scientific advisory board. He also previously served on the board of directors of Merck and Charles River Laboratories.

Enchondromas are benign cartilage tumors and precursors to malignant chondrosarcomas. Somatic mutations in the isocitrate dehydrogenase genes (IDH1 and IDH2) are present in the majority of these tumor types. How these mutations cause enchondromas is unclear. Here, we identified the spectrum of IDH mutations in human enchondromas and chondrosarcomas and studied their effects in mice. A broad range of mutations was identified, including the previously unreported IDH1-R132Q mutation. These mutations harbored enzymatic activity to catalyze α-ketoglutarate to d-2-hydroxyglutarate (d-2HG). Mice expressing Idh1-R132Q in one allele in cells expressing type 2 collagen showed a disordered growth plate, with persistence of type X-expressing chondrocytes. Chondrocyte cell cultures from these animals or controls showed that there was an increase in proliferation and expression of genes characteristic of hypertrophic chondrocytes with expression of Idh1-R132Q or 2HG treatment. Col2a1-Cre;Idh1-R132Q mutant knock-in mice (mutant allele expressed in chondrocytes) did not survive after the neonatal stage. Col2a1-Cre/ERT2;Idh1-R132 mutant conditional knock-in mice, in which Cre was induced by tamoxifen after weaning, developed multiple enchondroma-like lesions. Taken together, these data show that mutant IDH or d-2HG causes persistence of chondrocytes, giving rise to rests of growth-plate cells that persist in the bone as enchondromas.

Point mutations of the NADP(+)-dependent isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2) occur early in the pathogenesis of gliomas. When mutated, IDH1 and IDH2 gain the ability to produce the metabolite (R)-2-hydroxyglutarate (2HG), but the downstream effects of mutant IDH1 and IDH2 proteins or of 2HG on cellular metabolism are unknown. We profiled >200 metabolites in human oligodendroglioma (HOG) cells to determine the effects of expression of IDH1 and IDH2 mutants. Levels of amino acids, glutathione metabolites, choline derivatives, and tricarboxylic acid (TCA) cycle intermediates were altered in mutant IDH1- and IDH2-expressing cells. These changes were similar to those identified after treatment of the cells with 2HG. Remarkably, N-acetyl-aspartyl-glutamate (NAAG), a common dipeptide in brain, was 50-fold reduced in cells expressing IDH1 mutants and 8.3-fold reduced in cells expressing IDH2 mutants. NAAG also was significantly lower in human glioma tissues containing IDH mutations than in gliomas without such mutations. These metabolic changes provide clues to the pathogenesis of tumors associated with IDH gene mutations.

The runoff of a mixture of "heavy" and "light" polysomes has been reported to yield hybrid ribosomes, of intermediate density. However, we found that the dissociation of free heavy ribosomes as they advance in a sucrose gradient causes a serious artefact in the demonstration of hybrid ribosomes. This artefact is eliminated by fixation with glutaraldehyde before sedimentation. Formation of hybrid ribosomes can then be demonstrated, but it is equally complete whether the heavy and light preparations are mixed before or after runoff. Hence, free ribosomes exchange subunits rapidly in extracts. The previously reported exchange thus does not provide evidence for formation of free subunits during runoff.

Earlier studies showed that two protein components, P(I) and P(II), are concerned with the adenylylation and deadenylylation of Escherichia coli glutamine synthetase (EC 6.3.1.2). P(I) by itself catalyzes both adenylylation and deadenylylation, but its activity is modulated by the P(II)-protein and by glutamine, 2-oxoglutarate, ATP, and UTP, The P(II)-protein exists in two forms: one form, P(II)-AT, stimulates P(I)-catalyzed adenylylation activity in the absence of glutamine and makes this activity very sensitive to inhibition by 2-oxoglutarate; it does not affect deadenylylation activity. The other form, P(II)-DA, stimulates adenylylation only if glutamine is present, and also stimulates the deadenylylation activity of P(I), which is then dependent upon the presence of ATP and 2-oxoglutarate. Conversion of P(II)-AT to P(II)-DA requires the presence of UTP, ATP, and 2-oxoglutarate; it is catalyzed by an enzyme present in P(I) preparations. UTP may be directly involved in this conversion since P(II)-DA fractions reisolated by filtration through Sephadex G-100 contain small quantities of a bound uridine derivative that lacks the gamma-phosphoryl group of UTP. The activity of P(II)-DA, but not of P(II)-AT, is destroyed by treatment with snake-venom phosphodiesterase. ATP and 2-oxoglutarate apparently function as allosteric effectors for the conversion of P(II)-AT to P(I)-DA.

ALDOSE REDUCTASE (ALDITOL: NADP oxidoreductase, EC 1.1.1.21) is the enzyme responsible for the conversion of glucose to its sugar alcohol, sorbitol. In this study, aldose reductase and a closely related enzyme, L-hexonate dehydrogenase (L-gulonate: NADP oxidoreductase, EC 1.1.1.19), were purified from rat pancreas. Glutaric acid, 2,4-dimethyl glutaric acid, 3,3-tetramethylene glutaric acid, and colchicine inhibited both enzymes, albeit with different potencies. These compounds also inhibited both phases of glucose-induced release of insulin by the perfused rat pancreas. The potencies of these inhibitors in depressing the release of insulin correlated with their effectiveness in inhibiting aldose reductase. At higher concentrations of inhibitors, tolbutamide-induced release of insulin was also depressed. The addition of exogenous sorbitol to pancreases treated with glutaric acid restored their ability to respond to glucose and tolbutamide. These findings suggest that the conversion of free intracellular glucose to sorbitol in the beta cell is an essential step in the glucose-induced release mechanism.

Synthesis of extracellular ribonuclease is induced in cell cultures of Ustilago sphaerogena that are starved for nitrogen and exposed to the gratuitous inducer, 6-mercaptopurine. Cesium, ammonium, or alkylammonium ion represses ribonuclease induction. Addition of citric-acid cycle intermediates to cesium ionrepressed cultures partially restores the rate of ribonuclease synthesis to the induced level. Enzymes involved in assimilation of nitrogen from different sources are also repressed by cesium ion and derepressed by intermediates from the citric acid cycle.

Treatment of mouse skin homografts in vitro with glutaraldehyde prolonged their average survival time from 12.4 to 39.2 days, presumably because the reagent became covalently bound to the histocompatibility antigen sites (or in their close vicinity) and shielded them from the immune apparatus of the recipient. The attachment to skin of the inert polymer poly((L)-lysine) via this bifunctional reagent increased the average survival time to 52.9 days. The simplicity and versatility of this approach might make it possible to screen a large number of reagents that can bind covalently to tissue constituents under physiological conditions. It seems possible that a particular treatment leading to a new contact surface in the transplant might favor the acceptance of homografts as well as of heterografts.