Isocitrate dehydrogenases (IDHs), comprise a family of enzymes that catalyze oxidative decarboxylation of isocitrate to alpha-ketoglutarate (2-oxoglutarate).
Figure 1. Conversion of isocitrate to alpha-ketoglutarate by IDH
Eukaryotic cells express two distinct classes of IDHs that utilize either NAD or NADP as the electron acceptor and serve diverse biological functions:
- NAD- dependent IDH, IDH3, is located at the mitochondrial matrix and is well known for its central role for energy production in the Krebs cycle.
- The two NADP- dependent forms, are primarily located either in mitochondria (IDH2) or cytoplasm (IDH1). In addition to their potential catabolic role in the Krebs cycle, both mitochondrial and cytosolic IDHs are shown to play an important role in cellular defense against oxidative damage as a source of NADPH.
The three IDH isoenzymes are important players in the exchange of metabolites between the mitochondria and the cytosol.
- IDH3 is part of the TCA cycle were it generates NADH as a fuel for energy production
- IDH1 and 2 are important for shuttling electrons between the mitochondria and the cytosol.
Moreover, all eukaryotic cytosolic IDHs contain a type 1 peroxisomal targeting sequence at their C terminus that is sufficient to direct proteins into peroxisomes. Indeed cytosolic IDHs have been found in peroxisomes of yeast, human and rat liver cells and are shown to be required for the beta-oxidation of unsaturated fatty acids as a provider of NADPH inside peroxisomes.
Metabolites entering the mitochondria can be processed for energy generation usually through the production of NADH in the TCA cycle whereas metabolites exported back to the cytosol take part in anabolic processes. The transport of metabolites is also coupled to electron exchange between mitochondrial and cytosolic NADH and NADPH both of which cannot move across the mitochondrial inner membrane.
IDH1 gene is located in cr. 2q33.3 and consists of eight exons (1245nt).
IDH2 gene is located in cr. 15q26.1 and consists of eleven exons (1359 nt).
IDH3A gene encodes the alpha subunit of IDH3. It is located in cr. 15q25.1-q25.2 and consists of eleven exons ((1101 nt).
IDH3B gene, located in cr.20p13, encodes the beta subunit of IDH3. Three alternatively spliced transcript variants encoding different isoforms have been described for this gene.
IDH3G gene, located in cr.Xq28, encodes the gamma subunit of IDH3. This gene is a candidate gene for periventricular heterotopia. Several alternatively spliced transcript variants of this gene have been described, but only some of their full length natures have been determined.
CHEMICAL STRUCTURE AND IMAGES
Each NADP (+)-dependent isozyme forms an asymemetric homodimer. Structural studies of human IDH1 in complex with NADP, isocitrate and Ca2+ reveal three biologically relevant conformational states of the enzyme that differ substantially in the structure of the active site and in the overall structure (ref.1). A structural segment at the active site that forms a conserved alpha-helix in all known NADP-IDH structures assumes a loop conformation in the open, inactive form of human IDH1; a partially unraveled alpha-helix in the semi-open, intermediate form; and an alpha-helix in the closed, active form. The side chain of Asp279 of this segment occupies the isocitrate-binding site and forms hydrogen bonds with Ser94 (the equivalent of the phosphorylation site in bacterial IDHs) in the inactive form and chelates the metal ion in the active form. The structural data suggest a novel self-regulatory mechanism for IDH1 that mimics the phosphorylation mechanism used by the bacterial homologs, consistent with biochemical and biological data. This mechanism might be applicable to other eukaryotic NADP-IDHs (Figure 2A, B and C).
Figure 2A. Crystal Structure Of Human Cytosolic Nadp(+)-Dependent Isocitrate Dehydrogenase In Complex With Nadp, Isocitrate, And Calcium(2+) [Oxidoreductase, EC: 126.96.36.199]
Figure 2B. Structure of the active site of IDH1. The crystal structure of the human cytosolic NADP–dependent IDH is shown in ribbon format (PDBID: 1T0L).The active cleft of IDH1 consists of a NADP-binding site and the isocitrate-metal ion-binding site. The alpha-carboxylate oxygen and the hydroxyl group of isocitrate chelate the Ca2+ ion. NADP is colored in orange, isocitrate in purple and Ca2+ in blue. The Arg132 residue, displayed in yellow, forms hydrophilic interactions, shown in red, with the alpha-carboxylate of isocitrate
Figure 2C. Schematic diagram of the conformational changes during the regulation of IDH activity
Protein aminoacids percentage
The IDH step of the citric acid cycle, due to its large negative free energy change, is one of the irreversible reactions in the citric acid cycle, and, therefore, must be carefully regulated to avoid unnecessary depletion of isocitrate (and therefore an accumulation of alpha-ketoglutarate). The reaction is stimulated by the simple mechanisms of substrate availability (isocitrate, NAD+ or NADP+, Mg2+ / Mn2+ ), product inhibition (by NADH (or NADPH outside the citric acid cycle) and alpha-ketoglutarate), and competitive feedback inhibition (by ATP).
IDH1: a novel glioblastoma multiforme gene
The causal link between metabolic alterarions and cancer formation was revealed only this decade. Recently mutations in nuclear genes encoding mitochondrial enzymes (Fumarate Hydratase and Succinate Dehydrogenase) have been implicated in cancer susceptibility. Over the past year two of the three isoforms of the metabolic enzyme isocitrate dehydrogenase IDH (IDH1 and IDH2), were found mutated in high proportions in gliomas (ref.2 and ref.3).
Malignant gliomas are the most frequent and lethal tumors of the central nervous system, and glioblatoma multiforme (GBM;World Health Organization (WHO) grade IV glioma) is the most aggressive subtype. GBM may arise de novo (primary GBM) or develop in the setting of a lower-grade (secondary GBM).
A genome wide mutational analysis of glioblastomas (World health Organization (WHO) grade IV glioma) revealed the R132 somatic mutations of the isocitrate dehydrogenase 1 gene (IDH1) in a fraction of such tumors (12%). Additional studies have confirmed that IDH1 is mutated in > 80% of secondary GBM, whereas <10% of primary GBMs harbour these alterations (ref.3).
IDH1 and IDH2 are early genetic alterations in astrocytomas and oligodendrogliomas
Mutation of IDH1 occurs early in glioma progression, with somatic mutations of the R132 residue of IDH1 identified in the majority (>70%) of grades II and III astrocytomas and oligodendrogliomas, as well as in secondary GBMs that develop from these lower grade lesions. In addition, mutation analysis of the closely related IDH2 has revealed recurrent somatic mutations of IDH2 residue R172, with most mutations occurring in tumors lacking IDH1 mutations (ref.3 and ref.4). Astrocytomas and oligodendrogliomas both contain frequent IDH1 or IDH2 mutations but do not share other genetic alterations that occur early in the development of these two glioma lineages. For example, the majority of low-grade diffuse astrocytomas contain both an IDH mutation and a TP53 mutation, whereas most oligodendrogliomas have both IDH mutations and 1p/19q loss. Moreover in dissected multiple biopsies from the same patients IDH1 mutations always preceded the acquisition of a TP53 mutation or loss of 1p/19q (ref.5). This genetic evidence suggests that IDH mutations are early genetic events in the development of a glioma from a cell-of-origin that can give rise to both astrocytes and oligodendrocytes. IDH mutations were not identified in any WHO grade I pilocytic astrocytomas, which rarely undergo malignant transformation, indicates that these tumors arise through a different mechanism.
IDH1 and IDH2 mutations are remarkably specific to grades II and III astrocytomas, oligodendrogliomas, and secondary GBMs. Most recently, IDH1 R132 mutation have been found in 8.5% of acute myeloid leukemia samples (AML). The only tumors other than astrocytomas, oligodendrogliomas, and AMLs in which IDH1 mutations have been reported are a single case of colorectal cancer (ref.6), two prostate carcinomas (ref.7), and a minority of analyzed cases of adult supratentorial primitive neuroectodermal tumors (ref.8 and ref.9).
IDH mutations define a specific subtype of gliomas
Gliomas with IDH1 and IDH2 mutations have dinstinctive genetic and clinical characteristics compared to gliomas with wild-type IDH1 and IDH2. Nearly all of the anaplastic astrocytomas and GBMs with mutated IDH genes were also found to have a mutation of TP53, but only 5% had alterations in any of the common GBM genes PTEN, EGFR, or CDKN2A/CDKN2B. Conversely, anaplastic astrocytomas and GBMs with wild-type IDH1 and IDH2 had relatively few TP53 mutations (20%) and extremely frequent alterations of PTEN, EGFR, or CDKN2A/CDKN2B (74%). Similarly, loss of 1p/19q was observed in 85% of the oligodendrocytic tumors with mutated IDH but in none of the patients with wild-type IDH1 and IDH2 (ref.3, ref.5, ref.8). Clinically, adult patients with anaplastic astrocytomas and GBMs with IDH mutations are significantly younger than those with wild-type IDH1 and IDH2 (median age of 34 versus 56 years for anaplastic astrocytomas and 32 versus 59 years for GBMs). However, no IDH mutations have been identified in pediatric glioblastomas, and children with IDH-mutated low-grade gliomas are older than the others as well. GBM patients with IDH mutations have a median overall survival of 31 months, significantly longer than the 15-month survival in patients with wild-type IDH1 and IDH2 (ref.3). Although both younger age and mutated TP53 are positive prognostic factors for GBM patients, this association between IDH1 mutation and improved survival is noted even in the subgroup of young patients with TP53 mutations (ref.2). Mutations of IDH are also associated with improved prognosis in patients with anaplastic astrocytomas, whose median overall survival is 65 months for patients with mutations and 20 months for those without (ref.3). A multivariate analysis has confirmed that IDH1 mutation was an independent favorable prognostic marker after adjustment for grade, age, MGMT status, genomic profile, and treatment (ref.10).
Functional studies of IDH mutations: oncogenes or tumor suppressor genes?
A spectrum of missense mutations is observed at IDH1 R132 and IDH2 R172 in cancer. For instance, common IDH1 mutations include R132H and R132C, and common IDH2 mutations include R172K and R172M. One of the most striking features of IDH1 and 2 mutations is that it is always the same residue that is mutated: R132 in IDH1 and R172 in IDH2. The residues that are substituted for arginine are wide ranging, which strongly suggests that it is not the new residue, but the replacement of the arginine, which supports tumorigenesis by impairing isocitrate binding.
Modeling studies based upon the human cytosolic IDH1 crystal structure suggest that substitution of R132 with any of one of the amino acids observed thus far in gliomas would impair interactions of the enzyme with isocitrate (Figure 3).
Figure 3 IDH1 R132 and IDH2 R172 are analogous residues that both interact with the β-Carboxyl of Isocitrate (A) Active site of crystallized human IDH1 with isocitrate.(B) Active site of human IDH2 with isocitrate, modeled based on the highly homologous and crystallized pig IDH2 structure. For (A) and (B), carbon 6 of isocitrate containing the β-carboxyl is highlighted in cyan, with remaining isocitrate carbons shown in yellow. Carbon atoms of amino acids (green), amines (blue), and oxygens (red) are also shown. Hydrogen atoms are omitted from the figure for clarity. Dashed lines depict interactions < 3.1 Å, corresponding to hydrogen and ionic bonds. Residues coming from the other monomer of the IDH dimer are denoted with a prime (′) symbol
Unlike SDH and FH, IDH mutations do not follow Knudson's two-hit model of tumor suppressor genes. Almost all reported cases of IDH1 and IDH2 mutation have been heterozygous, and inactivating alterations such as frameshifts, deletions, and nonsense mutations have not been observed for these genes in cancer. This genetic evidence led to early speculation that the IDH mutations confer the enzymes with an oncogenic gain of function.
Results from initial functional studies were in contrast to the gain-of-function hypothesis. These data revealed that the mutations reduce the ability of IDH1 and IDH2 to convert isocitrate to α-KG (ref.3). Furthermore, IDH1 R132 mutants can, in a dominant-negative manner, inhibit wild-type IDH1 activity in vitro (ref.11). This led to the speculation that IDH1 and IDH2 are tumor suppressors with a propensity to develop dominant-negative point mutations.
Recently, Dang and colleagues (ref.12) demonstrated that although IDH1 mutants lose their normal enzymatic activity in tumors, they gain a new one. They showed that cancer-associated IDH1 mutations result in the new ability of the enzyme to catalyse the NADPH-dependent reduction of alpha-ketoglutarate to R (-)-2-hydroxyglutarate (2HG). Structural studies demonstrate that when arginine 132 is mutated to histidine, residues in the active site are shifted to produce structural changes consistent with reduced oxidative decarboxylation of isocitrate and acquisition of the ability to convert alpha-ketoglutarate to 2HG. Dang and colleagues (ref.12) also demonstrated that 2HG was markedly elevated in human malignant gliomas harbouring IDH1 mutations.
Later Ward and colleagues (ref.13) showed that, like IDH1 R132 mutations, IDH2 R172, and IDH2 R140 led to the production of 2HG. All these residues form hydrogen bonds with the beta-carboxyl of isocitrate. Mutation of these residues presumably favours the convertion of alpha-KG to 2HG which resembles isocitrate but lacks this beta-carboxyl (Figure 4).
Figure 4 Mutations in the active site of IDH1 and IDH2 lead to a neomorphic enzyme activity
The fact that neomorphic enzyme activity is a shared feature of the IDH1 and IDH2 mutations points to the importance of this activity in cancer.
Though the neomorphic enzyme activity of the IDH mutants probably influences cancer or precancer cell biology, the downstream effects of this activity remain unknown. Most attention has been focused on the possibility that 2HG acts as an oncometabolite, either as a general mutagen or by modulating a specific cellular process. For instance, IDH1 R132H can upregulate the cancer-associated transcription factor HIF-1α in vitro (ref.11). On one hand, it has been speculated that 2HG could mediate this upregulation by inhibiting prolyl hydroxylases and releasing HIF-1α from prolyl hydroxylase-dependent downregulation (ref.14). On the other hand, other potential effects of neomorphic IDH1 and IDH2 enzyme activity, such as metabolic flux away from α-KG, a central cellular metabolite, and alteration of the cellular NADP+/NADPH balance, could also have far-reaching metabolic effects on the cell.
Diagnostic and therapeutical relevance
Historically, glioblastomas have been divided into cancers that arise from low-grade gliomas (secondary tumors) and those without such an antecedent (primary tumors). Secondary tumors account for only 5% of all glioblastomas. The finding that IDH1 or IDH2 is mutated in the vast majority of WHO grade II or III gliomas and in the secondary glioblastomas that develop from these precursors provides a biologic explanation for this clinical categorization: tumors with mutated NADP+-dependent isocitrate dehydrogenases comprise a specific subgroup of glioblastomas.
The localization of IDH1 and IDH2 mutations to a single amino acid (R132 and R172,
respectively) simplifies the use of this genetic alteration for diagnostic purposes. For example,
IDH mutation tests could help distinguish pilocytic astrocytomas (WHO grade I) from diffuse
astrocytomas (WHO grade II), since these lesions can sometimes be difficult to categorize
solely on the basis of histopathological criteria.
Moreover 2HG production can be used as a marker to identify samples to screen for novel IDH1 and IDH2 mutations. If 2HG levels are high in the serum, urine, or cerebrospinal fluid of patients with IDH-mutated cancers, measurement of this metabolite could be used as an adjunct to histopathological analysis or even in place of a more invasive procedure.
Several features of the IDH1 and IDH2 mutations make their study exciting for the future development of therapeutics. First, in contrast to oncogenic signaling molecules that have proven difficult to target with small compounds, the active sites of metabolic enzymes are probably amenable to such targeting. Second, IDH mutations appear early in cancer development compared to other genetic alterations, and they are found in cancers that are composed of relatively undifferentiated cells. On the basis of this, Ward and colleagues (ref.13) speculate that the mutations could mediate a block in cellular differentiation that leads to carcinogenesis. If true, a therapeutic strategy aimed at modulating cell differentiation pathways may aid in the treatment of these cancers. Finally, though the mutations occur early in cancer, the same IDH mutation is always retained as gliomas progress to higher-grade tumors (ref.3) and in relapses of AML (ref.15), indicating that the mutant enzymes could serve as stable therapeutic targets. If so, small molecules that target mutated IDH enzymes or yet-to-be-identified players in their oncogenic network may be very successful in treating these challenging cancers.
1. Structures of human cytosolic NADP-dependent isocitrate dehydrogenase reveal a novel self-regulatory mechanism of activity. 2004
2. An integrated genomic analysis of human glioblastoma multiforme. 2008
3. IDH1 and IDH2 mutations in gliomas. 2009
4. Type and frequency of IDH1 and IDH2 mutations are related to astrocytic and oligodendroglial differentiation and age:a study of 1,010 diffuse gliomas. 2009
5. IDH1 mutations are early events in the development of astrocytomas and oligodendroglioma. 2009
6. The consensus coding sequences of human breast and colorectal cancers. 2006
7. Mutational analysis of IDH1 codon 132 in glioblastomas and other common cancers. 2009
8. Analysis of the IDH1 codon 132 mutation in brain tumors. 2008
9. Frequent IDH1 mutations in supratentorial primitive neuroectodermal tumors
(sPNET) of adults but not children. 2009
10. Isocitrate dehydrogenase 1 codon 132 mutation is an important prognostic biomarker in gliomas. 2009
11. Glioma-derived mutations in IDH1 dominantly inhibit IDH1 catalytic activity and induce HIF-1alpha. 2009
12. Cancer-associated IDH1 mutations produce 2-hydroxyglutarate. 2009
13. The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate. 2010
14. IDH1 mutations in gliomas: when an enzyme loses its grip. 2010
15. Distinct clinical and biologic characteristics in adult acute myeloid leukemia bearing the isocitrate dehydrogenase 1 mutation. 2010