Normal function of IDHs and IDH1/2 mutations in gliomas

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Hypermethylated phenotypes

Epigenetics is related to DNA methylation and histone modification (acetylation or methylation). These changes only affect the regulation and level of gene expression, but do not alter the nucleotide sequence (56).
5-methylcytosine (5mC) at CpG dinucleotides are the most important DNA methylation sites. 5mC may be converted into 5-hydroxymethylcytosine (5hmC) by TET2 (TET family of 5-methylcytosine hydroxylases), resulting in the DNA demethylation. This mechanism may explain why IDH mutations are associated with CIMP phenotype in gliomas.
Several studies have reported the changes of the intermediate products of DNA methylation: (i) 5mC is elevated in IDH1R132H and IDH2R172K mutated 293T cells (57); (ii) Turcan et al. showed that 5hmC is decreased in mutant IDH1-transduced astrocytes, and increased when TET2 was expressed in the astrocytes. Importantly, they confirmed that cells with IDH1 mutation developed glioma-CpG island methylator phenotype (G-CIMP), suggesting that IDH1 mutation is the molecular basis of CIMP in glioma. (58).
Histone lysine methyltransferases and TET2 (TET family of 5-methylcytosine hydroxylases) are αKG-dependent dioxygenase enzymes. D-2-HG is a competitive inhibitor of the α-ketoglutarate-dependent histone lysine methyltransferases and TEF 5-methylcytosine hydroxylases (59,60). Thus, because of the competitive inhibition by 2HG, IDH mutation prevents DNA and histones demethylation and result in hypermethylated phenotypes. These hypermethylated phenotypes decrease globally the gene expression, and probably contribute to the formation and development of tumors.

MGMT promoter methylation

O6-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that removes alkylating lesions induced by chemotherapeutic agents. Loss or reduction of MGMT activity through promoter methylation decreases DNA repair activity, increasing sensitivity to alkylating agents. A methylated MGMT promoter is observed in low-grade gliomas (60-93%) and GBM (45%) (61,62). Methylation of MGMT was the strongest predictor of outcome and benefit from temozolomide chemotherapy (62,63).

Transcriptomic classification of gliomas

Clinically relevant subtypes of GBM can be characterized by alterations and abnormal expression of EGFR, NF1, PDGFRA, and IDH1 genes. Verhaak et al. reported four transcriptomal subclasses of GBM based on transcriptomic profile: the Proneural, Mesenchymal, Classical, and Neural (29,58) (Figure 2). Gain of chromosome 7 and chromosome 10 deletion are almost constant in the Classical subtype, with frequent EGFR gene amplifications or mutations. Proneural subtype harbors frequent mutations of TP53 and alterations (mutations and amplifications) of PDGFRA gene. IDH1 mutations are found exclusively in the proneural subclasse (65). The Mesenchymal subtype is characterized by frequent deletions/mutation of NF1 gene, with overexpression of mesenchymal genes such as YKL40, IGFBP (66).The Neural subtype is characterized by neuron-related gene expression.
These four subtypes may have different benefit from combined radio-chemotherapy (65): ie low or no benefit in the Proneural subtype and greatest benefit in the classical subtype, but this need to be confirmed.

Enzymatic reactions catalyzed by IDHs

Isocitrate dehydrogenases are important enzymes in the tricarboxylic acid cycle. They catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate while converting NAD(P)+ to NAD(P)H. The reaction is a two-step process, which involves the oxidation of isocitrate to the intermediate oxalosuccinate, reducing NAD(P)+ to NAD(P)H, followed by decarboxylation of oxalosuccinate to form α-ketoglutarate (69) (Figure 6).

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IDH1/2 mutations results in the loss of normal enzymatic activity and the gain of a new enzymatic activity

Heterozygous IDH1 mutations impair the enzyme’s affinity for the Mg2+-isocitrate substrate complex and enzymatic activity (47,46,79). Dang et al. (80) first showed that the mutant IDH1 enzymes have not only (i) a loss of enzyme’s function to convert isocitrate to alpha-KG, but also (ii) a gain of enzyme’s function to catalyze the NADPH-dependent reduction of alpha-KG to R(-)-2 hydroxyglutarate (2HG) (Figure 8).

Alteration of DNA and histone methylation patterns

The balanced activities of histone lysine methyltransferases and TET family of 5-methylcytosine hydroxylases play an important role in building and keeping the methylation of histone and DNA proteins. D-2-HG is a competitive inhibitor of the α-ketoglutarate-dependent histone lysine methyltransferases and TEF 5-methylcytosine hydroxylases (81,83). Thus, because of the 2HG competitive inhibition, IDH1/2 mutations result in an hypermethylated phenotypes, decreasing globally the gene expression, blocking the lineage-specific progenitor cell differentiation and developing glioma-CpG island methylator phenotype (G-CIMP) to the formation and development of tumors (58,84).

D-2HG and glutamate-structural similarities

High concentration of glutamate in neurons acts as a neurotoxin. Glutamate neurotoxicity plays an important role in the pathogenesis of many neurological diseases (85). D-2HG and glutamate have similar structure. 2HG could lead to excitotoxicity through the activation of the glutamate receptor (N-methyl-D-asparticacid-NMDA-receptor) in neurons (86).

Table of contents :

INTRODUCTION
Chapter I. Gliomas: Epidemiology, Histo prognostic classification and Treatment options
I. Epidemiology
II. Histoprognostic classification of gliomas
III. Treatment options
Chapter II: Molecular classification of human gliomas
I. Genomic classification of gliomas
II. Epigenomic classification of gliomas
III. Transcriptomic classification of gliomas
Chapter III: Normal function of IDHs and IDH1/2 mutations in gliomas
I. Isoenzymes and roles in normal cell metabolism
II. IDH1 and IDH2 mutations in gliomas
OBJECTIVES
RESULTS
Publication n°1 « IDH mutations: genotype-phenotype correlation and
prognostic impact”
Publication n°2 “Prognostic impact of IDH1 SNP rs11554137 in
malignant gliomas”
Publication n°3 “Isocitrate dehydrogenase 1 mutation (IDH1 R132H)
increases U87 glioma cell sensitivity to radiation therapy in hypoxia”
DISCUSSION
I. Description of IDH1/IDH2 mutations in gliomas
II. Diagnostic value of IDH1/2 mutations and new methods for IDH status determination
III. Prognostic/ predictive value of IDH1/2 mutations
PERSPECTIVES
REFERENCES
ANNEXES
Article 4 “Chromosome 7p11.2 (EGFR) variation influences glioma risk.”
Article 5 “All the 1p19q codeleted gliomas are mutated on IDH1 or IDH2.”
Article 6 “IDH1 and IDH2 mutations in gliomas.”
Article 7 “COLD PCR HRM: a highly sensitive detection method for IDH1 mutations.”
Article 8 “IDH1 or IDH2 mutations predict longer survival and response to temozolomide in low-grade gliomas.”

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