IDH1 mutation identified in one human melanoma metastasis, but not correlated with metastases to the brain

Isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) are enzymes which convert isocitrate to α-ketoglutarate while reducing nicotinamide adenine dinucleotide phosphate (NADP + to NADPH). IDH1/2 were recently identified as mutated in a large percentage of progressive gliomas. These mutations occur at IDH1^R132 or the homologous IDH2^R172. Melanomas share some genetic features with IDH1/2-mutated gliomas, such as frequent TP53 mutation. We sought to test whether melanoma is associated with IDH1/2 mutations. Seventy-eight human melanoma samples were analyzed for IDH1^R132 and IDH2^R172 mutation status. A somatic, heterozygous IDH1 c.C394T (p.R132C) mutation was identified in one human melanoma metastasis to the lung. Having identified this mutation in one metastasis, we sought to test the hypothesis that certain selective pressures in the brain environment may specifically favor the cell growth or survival of tumor cells with mutations in IDH1/2, regardless of primary tumor site. To address this, we analyzed IDH1^R132 and IDH2^R172 mutation status 53 metastatic brain tumors, including nine melanoma metastases. Results revealed no mutations in any samples. This lack of mutations would suggest that mutations in IDH1^R132 or IDH2^R172 may be necessary for the formation of tumors in a cell-lineage dependent manner, with a particularly strong selective pressure for mutations in progressive gliomas; this also suggests the lack of a particular selective pressure for growth in brain tissue in general. Studies on the cell-lineages of tumors with IDH1/2 mutations may help clarify the role of these mutations in the development of brain tumors.     

Identification and functional characterization of isocitrate dehydrogenase 1 (IDH1) mutations in thyroid cancer

Mutations in the genes for isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) have been recently identified in glioblastoma. In the present study, we investigated IDH1 and IDH2 mutations in follicular thyroid cancer (FTC) and anaplastic thyroid cancer (ATC), with the latter, like glioblastoma, having a rapidly aggressive and lethal clinical course. By direct genomic DNA sequencing, we analyzed exon 4 of the IDH1 and IDH2 genes that harbored the mutation hot spots codon 132 and 172 of the two genes in glioblastoma, respectively, in 12 thyroid cancer cell lines, 20 FTC, and 18 ATC tumor samples. A novel homozygous G367A IDH1 mutation, resulting in a G123R amino acid change in codon 123, was identified in a case of ATC. A previously described IDH1 V71I mutation was found in a case of FTC and a case of ATC and no mutations were found in the cell lines. The overall prevalence of mutations was thus 1/20 (5%) in FTC and 2/18 (11%) in ATC. We did not find mutation in the IDH2 gene in these thyroid cancer cell lines and tumor samples. Sequence alignment analysis of 16 species revealed that the novel IDH1 G123R mutation was located in a highly conserved region, raising the possibility of a serious functional consequence as could also be predicted by the occurrence of a positively charged amino acid from this mutation. To test this, we created a G123R mutant by site-directed mutagenesis and demonstrated a decreased enzymatic activity of IDH1, similar to the expected reduction in the enzymatic activity of the previously described R132H IDH1 mutant measured as a control. Thus, functionally relevant IDH1 mutations can also occur in thyroid cancer, particularly ATC, suggesting a potential tumorigenic role of the IDH1 system that could represent a new therapeutic target for thyroid cancer.     

The Frequency and Clinical Significance of IDH1 Mutations in Chinese Acute Myeloid Leukemia Patients

Objective

Mutations in the gene encoding isocitrate dehydrogenease 1 (IDH1) occur in various hematopoietic tumors including acute myeloid leukemia (AML), myeloproliferative neoplasms and myelodysplastic syndromes. IDH1 mutations are significant in both diagnosis and prognosis of these conditions. In the present study we determined the prevalence and clinical significance of IDH1 mutations in 349 samples from newly diagnosed AML patients.

Results

Of the 349 AML patient specimens analyzed, 35 (10.03%) were found to have IDH1 mutations including 4 IDH1 R132 mutations and 31 non-R132 mutations. IDH1 non-R132 mutations were largely concentrated within AML-M₁ (35.72%, p<0.01). We identified five IDH1 mutations that were novel to AML: (1) c.299 G>A, p.R100Q; (2) c.311G>T, p.G104V; (3) c.322T>C, p.F108L; (4) c.356G>A, p.R119Q; and (5) c.388A>G, p.I130V. In addition, we identified three IDH1 mutations that were previously described in AML. The frequency of IDH1 mutations in AML patients with normal karyotype was 9.9%. IDH1 non-R132 mutations were concurrent with mutations in FLT3-ITD (p<0.01), CEBPA (p<0.01), and NRAS (p<0.01), as well as the overexpression of MN1 (p<0.01) and WT1(p<0.01). The overall survival (OS) in the patients with IDH1 non-R132 mutations compared to patients without IDH1 mutations don''t reach statistically significance (median 521 days vs median: not reached; n.s.).

Conclusion

IDH1 non-R132 mutations occurred frequently in newly diagnosed adult Chinese AML patients, and these mutations were associated with genetic alterations. The OS was not influenced by IDH1 non-R132 mutations in the present study.     

Isocitrate dehydrogenase 1 R132C mutation occurs exclusively in microsatellite stable colorectal cancers with the CpG island methylator phenotype

The CpG Island Methylator Phenotype (CIMP) is fundamental to an important subset of colorectal cancer; however, its cause is unknown. CIMP is associated with microsatellite instability but is also found in BRAF mutant microsatellite stable cancers that are associated with poor prognosis. The isocitrate dehydrogenase 1 (IDH1) gene causes CIMP in glioma due to an activating mutation that produces the 2-hydroxyglutarate oncometabolite. We therefore examined IDH1 alteration as a potential cause of CIMP in colorectal cancer. The IDH1 mutational hotspot was screened in 86 CIMP-positive and 80 CIMP-negative cancers. The entire coding sequence was examined in 81 CIMP-positive colorectal cancers. Forty-seven cancers varying by CIMP-status and IDH1 mutation status were examined using Illumina 450K DNA methylation microarrays. The R132C IDH1 mutation was detected in 4/166 cancers. All IDH1 mutations were in CIMP cancers that were BRAF mutant and microsatellite stable (4/45, 8.9%). Unsupervised hierarchical cluster analysis identified an IDH1 mutation-like methylation signature in approximately half of the CIMP-positive cancers. IDH1 mutation appears to cause CIMP in a small proportion of BRAF mutant, microsatellite stable colorectal cancers. This study provides a precedent that a single gene mutation may cause CIMP in colorectal cancer, and that this will be associated with a specific epigenetic signature and clinicopathological features.     

IDH1/IDH2 Mutations Define the Prognosis and Molecular Profiles of Patients with Gliomas: A Meta-Analysis

Background

Isocitrate dehydrogenase isoforms 1 and 2 (IDH1 and IDH2) mutations have received considerable attention since the discovery of their relation with human gliomas. The predictive value of IDH1 and IDH2 mutations in gliomas remains controversial. Here, we present the results of a meta-analysis of the associations between IDH mutations and both progression-free survival (PFS) and overall survival (OS) in gliomas. The interrelationship between the IDH mutations and MGMT promoter hypermethylation, EGFR amplification, codeletion of chromosomes 1p/19q and TP53 gene mutation were also revealed.

Methodology and Principal Findings

An electronic literature search of public databases (PubMed, Embase databases) was performed. In total, 10 articles, including 12 studies in English, with 2,190 total cases were included in the meta-analysis. The IDH mutations were frequent in WHO grade II and III glioma (59.5%) and secondary glioblastomas (63.4%) and were less frequent in primary glioblastomas (7.13%). Our study provides evidence that IDH mutations are tightly associated with MGMT promoter hypermethylation (P<0.001), 1p/19q codeletion (P<0.001) and TP53 gene mutation (P<0.001) but are mutually exclusive with EGFR amplification (P<0.001). This meta-analysis showed that the combined hazard ratio (HR) estimate for overall survival and progression-free survival in patients with IDH mutations was 0.33 (95% CI: 0.25–0.42) and 0.38 (95% CI: 0.21–0.68), compared with glioma patients whose tumours harboured the wild-type IDH. Subgroup analyses based on tumour grade also revealed that the presence of IDH mutations was associated with a better outcome.

Conclusion

Our study suggests that IDH mutations, which are closely linked to the genomic profile of gliomas, are potential prognostic biomarkers for gliomas.     

Glioma-derived mutations in IDH: From mechanism to potential therapy

Heterozygous mutations in either the R132 residue of isocitrate dehydrogenase I (IDH1) or the R172 residue of IDH2 in human gliomas were recently highlighted. Heterozygous mutations in the IDH1 occur in the majority of grade II and grade III gliomas and secondary glioblastomas and change the structure of the enzyme, which diminishes its ability to convert isocitrate (ICT) to α-ketoglutarate (α-KG) and provides it with a newly acquired ability to convert α-KG to R(-)-2-hydroxyglutarate [R(-)-2HG]. The IDH1 and IDH2 mutations are relevant to the progression of gliomas, the prognosis and treatment of the patients with gliomas harboring the mutation. In this paper, we reviewed these recent findings which were essential for the further exploration of human glioma cancer and might be responsible for developing a newer and more effective therapeutic approach in clinical treatment of this cancer.     

PCR-Based Simple Subgrouping Is Validated for Classification of Gliomas and Defines Negative Prognostic Copy Number Aberrations in IDH Mutant Gliomas

Genetic subgrouping of gliomas has been emphasized recently, particularly after the finding of isocitrate dehydrogenase 1 (IDH1) mutations. In a previous study, we investigated whole-chromosome copy number aberrations (CNAs) of gliomas and have described genetic subgrouping based on CNAs and IDH1 mutations. Subsequently, we classified gliomas using simple polymerase chain reaction (PCR)-based methods to improve the availability of genetic subgrouping. We selected IDH1/2 and TP53 as markers and analyzed 237 adult supratentorial gliomas using Sanger sequencing. Using these markers, we classified gliomas into three subgroups that were strongly associated with patient prognoses. These included IDH mutant gliomas without TP53 mutations, IDH mutant gliomas with TP53 mutations, and IDH wild-type gliomas. IDH mutant gliomas without TP53 mutations, which mostly corresponded to gliomas carrying 1p19q co-deletions, showed lower recurrence rates than the other 2 groups. In the other high-recurrence groups, the median progression-free survival (PFS) and overall survival (OS) of patients with IDH mutant gliomas with TP53 mutations were significantly longer than those of patients with IDH wild-type gliomas. Notably, most IDH mutant gliomas with TP53 mutations had at least one of the CNAs +7q, +8q, −9p, and −11p. Moreover, IDH mutant gliomas with at least one of these CNAs had a significantly worse prognosis than did other IDH mutant gliomas. PCR-based mutation analyses of IDH and TP53 were sufficient for simple genetic diagnosis of glioma that were strongly associated with prognosis of patients and enabled us to detect negative CNAs in IDH mutant gliomas.     

Establishment of a novel monoclonal antibody SMab-1 specific for IDH1-R132S mutation

Isocitrate dehydrogenase 1 (IDH1) mutations, which are early and frequent genetic alterations in gliomas, are specific to a single codon in the conserved and functionally important Arginine 132 (R132) in IDH1. We earlier established a monoclonal antibody (mAb), IMab-1, which is specific for R132H-containing IDH1 (IDH1-R132H), the most frequent IDH1 mutation in gliomas. To establish IDH1-R132S-specific mAb, we immunized mice with R132S-containing IDH1 (IDH1-R132S) peptide. After cell fusion using Sendai virus envelope, IDH1-R132S-specific mAbs were screened in ELISA. One mAb, SMab-1, reacted with the IDH1-R132S peptide, but not with other IDH1 mutants. Western-blot analysis showed that SMab-1 reacted only with the IDH1-R132S protein, not with IDH1-WT protein or IDH1 mutants, indicating that SMab-1 is IDH1-R132S-specific. Furthermore, SMab-1 specifically stained the IDH1-R132S-expressing glioblastoma cells in immunocytochemistry and immunohistochemistry, but did not react with IDH1-WT or IDH1-R132H-containing glioblastoma cells. We newly established an anti-IDH1-R132S-specific mAb SMab-1 for use in diagnosis of mutation-bearing gliomas.     

A monoclonal antibody IMab-1 specifically recognizes IDH1, the most common glioma-derived mutation

IDH1 (isocitrate dehydrogenase 1) mutations have been identified as early and frequent genetic alterations in astrocytomas, oligodendrogliomas, and oligoastrocytomas as well as secondary glioblastomas. In contrast, primary glioblastomas very rarely contain IDH1 mutations, although primary and secondary glioblastomas are histologically indistinguishable. The IDH1 mutations are remarkably specific to a single codon in the conserved and functionally important Arg132 in IDH1. In gliomas, the most frequent IDH1 mutations (>90%) were G395A (R132H). In this study, we immunized mice with R132H-containing IDH1 (IDH1^R132H) peptide. After cell fusion using Sendai virus envelope, the monoclonal antibodies (mAbs), which specifically reacted with IDH1^R132H, were screened in ELISA. One of the mAbs, IMab-1 reacted with the IDH1^R132H peptide, but not with wild type IDH1 (IDH1^wt) peptide in ELISA. In Western-blot analysis, IMab-1 reacted with only the IDH1^R132H protein, not IDH1^wt protein or the other IDH1 mutants, indicating that IMab-1 is IDH1^R132H-specific. Furthermore, IMab-1 specifically stained the IDH1^R132H-expressing cells in astrocytomas in immunohistochemistry, whereas it did not react with IDH1^R132H-negative primary glioblastoma sections. In conclusion, we established an anti-IDH1^R132H-specific monoclonal antibody IMab-1, which should be significantly useful for diagnosis and biological evaluation of mutation-bearing gliomas.     

Licochalcone A suppresses the proliferation of sarcoma HT-1080 cells,as a selective R132C mutant IDH1 inhibitor

IDH1 mutations are closely related to the development and progression of various human cancers, such as glioblastoma, sarcoma, and acute myeloid leukemia. By screening dozens of reported natural compounds using both wild-type and mutant IDH1 enzymatic assays, we discovered Licochalcone A is a selective inhibitor to the R132C-mutant IDH1 with an IC₅₀ value of 5.176 μM, and inhibits the proliferation of sarcoma HT-1080 cells with an IC₅₀ value of 10.75 μM. Suggested by the molecular docking results, Licochalcone A might occupy the allosteric pocket between the two monomers of IDH1 homodimer, and the R132H mutation was unfavorable for the binding of Licochalcone A with the IDH1 protein, as compared to the R132C mutation. Revealed by the RNA-Seq data analysis, the Cell Cycle pathway was the most over-represented pathway for HT-1080 cells treated with Licochalcone A. Consistent with these results, Licochalcone A induced apoptosis and cell cycle arrest of HT-1080 cells, while it showed minimal effect against the proliferation of normal RCTEC cells. The discovery of Licochalcone A as a mutation-selective IDH1 inhibitor can serve as a promising starting point for the development of mutation-selective anti-tumor lead compounds targeting IDH1.     

Mutation Analysis of IDH1 in Paired Gliomas Revealed IDH1 Mutation Was Not Associated with Malignant Progression but Predicted Longer Survival

Recurrence and progression to higher grade lesions are characteristic behaviorsof gliomas. Though IDH1 mutation frequently occurs and is considered as an early event in gliomagenesis, little is known about its role in the recurrence and progression of gliomas. We therefore analysed IDH1 and IDH2 statusat codon 132 of IDH1 and codon 172 of IDH2 by direct sequencing and anti-IDH1-R132H immunohistochemistry in 53 paired samples and their recurrences, including 29 low- grade gliomas, 16 anaplastic gliomas and 8 Glioblastomas. IDH1/IDH2 mutation was detected in 32 primarytumors, with 25 low- grade gliomas and 6 anaplastic gliomas harboring IDH1 mutation and 1 low- grade glioma harboring IDH2 mutation. All of the paired tumors showed consistent IDH1 and IDH2 status. Patients were analyzed according to IDH1 status and tumor-related factors. Malignant progression at recurrence was noted in 22 gliomas and was not associated with IDH1 mutation. Survival analysis revealed patients with IDH1 mutated gliomas had a significantly longer progression-free survival (PFS) and overall survival (OS). In conclusion, this study demonstrated a strong tendency of IDH1/IDH2 status being consistent during progression of glioma. IDH1 mutation was not a predictive marker for malignant progression and it was a potential prognostic marker for gliomas of Chinese patients.     

Discovery and structure-activity-relationship study of novel conformationally restricted indane analogues for mutant isocitric dehydrogenase 1 (IDH1) inhibitors

The discovery and optimization of various of indane amides as mutant IDH1 inhibitors via structure-based rational design were reported. The optimal compounds demonstrated both potent inhibition in IDH1^R132H enzymatic activity and 2HG production in IDH1 mutant HT1080 cell line, favorable PK properties and great selectivity against IDH1^wt and IDH2^R140Q.     

The mechanisms of IDH mutations in tumorigenesis

Tumor-associated mutations in the isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) genes result in the loss of normal catalytic activity, the production of α-ketoglutarate (α-KG), and gain of a new activity, the production of an oncometabolite, R-2-hydroxylglutarate (R-2-HG). New evidence supports previous findings that R-2-HG acts as an antagonist of α-KG to competitively inhibit the activity of multiple α-KG-dependent dioxygenases, including both histones and DNA demethylases involved in epigenetic control of gene expression and cell differentiation, and also reveals an intriguing new facet of R-2-HG in tumorigenesis.The NADP⁺-dependent isocitrate dehydrogenase IDH1 and IDH2 catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG). IDH1 and IDH2 are localized in the cytoplasm and mitochondria, respectively, and represent by far the most frequently mutated metabolic enzymes in human cancer¹. The tumor-derived mutants of both IDH1 and IDH2 lose their activity in producing α-KG^2,3, and gain a surprising new catalytic activity, the production of R-2-hydroxyglutarate (R-2-HG) by reduction of α-KG⁴. Previous studies have shown that R-2-HG acts as an antagonist of α-KG to competitively inhibit a number of α-KG-dependent dioxygenases, including the JmjC domain-containing histone demethylases (KDMs) and the TET (ten-eleven translocation) family of DNA hydroxylases that catalyze the sequential oxidation of 5-methlycytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), leading to eventual DNA demethylation (Figure 1)^5,6. Three papers recently published in Nature provide additional evidence that α-KG-dependent dioxygenases are the pathophysiological targets of mutant IDH1/2, and further underscore the presumptive role of R-2-HG as the first oncometabolite in contributing to tumorigenesis after IDH1/2 mutations.Open in a separate windowFigure 1Summarization of reported mechanisms linking IDH mutation to tumorigensis. Regulation of α-KG-dependent dioxygenases by R-2-HG is likely to play a major role in the pathophysiology of tumors with IDH mutation.A subset of glioblastoma, known as the proneural subgroup, has previously found to display hypermethylation at a large number of loci and is enriched with IDH1 mutations⁷. In one of the three Nature papers, Turcan et al.⁸ determined whether IDH1 mutation alone is sufficient to cause the hypermethylation phenotype by ectopic expression of IDH1^R132H mutant in immortalized primary human astrocytes, a cell type from which glioblastoma is believed to develop. The authors found that introduction of mutant IDH1 induced extensive DNA hypermethylation, altered the methylation of specific histones, and reshaped the methylome in a fashion that mirrors the changes observed in IDH1-mutated low-grade gliomas. The observed hypermethylation of DNA and histones can be explained by the direct inhibition of TET methylcytosine hydroxylases and JmjC family histone demethylases by R-2-HG, respectively. In keeping with the notion that TET hydroxylases directly regulate genomic DNA methylation levels and can be inhibited by the R-2-HG accumulated in IDH1/2-mutated cells, Turcan et al. also showed that ectopic expression of TET2 in cultured astrocytes decreased 5mC and increased 5hmC, and that both changes were inhibited by the co-expression of TET2 with mutant IDH1. These results are consistent with the findings made in acute myeloid leukemia (AML) in which IDH1/2 and TET2 genes are mutated in a mutually exclusive manner⁹. Moreover, Turcan et al. found that expression of wild-type IDH1 decreased the average DNA methylation level in the genome, supporting the notion that the concentration of α-KG may be a rate-limiting factor of TET-catalyzed DNA demethylation⁵.In the second paper, Lu et al.¹⁰ reported that ectopic expression of tumor-derived mutant IDH1/2 or feeding cells with cell-permeable R-2-HG increases histone demethylation and results in blockade of the differentiation of 3T3-L1 adipoblasts to adipocytes. These results indicate that mutation of IDH1/2 and accumulation of R-2-HG can broadly impair cell differentiation beyond the cell types in which IDH1/2 mutations are found to associate with tumorigenesis. The authors further confirmed that IDH1-mutated gliomas have elevated levels of histone methylation compared with gliomas retaining the wild-type IDH1^5,6. As previously reported^5,6, multiple KDMs that are inhibited by 2-HG, including KDM4C/JMJD2C, which causes repressive histone H3K9 di- and trimethylation and, when suppressed by RNA interference, blocks the 3T3-L1 adipogenesis. It remains to be determined whether collective inhibition of multiple KDMs or a few individual ones, such as KDM4C, is responsible for altering cell differentiation in IDH1/2-mutated cells. The authors also noted that expression of mutant IDH1 increased histone methylation prior to the increase of DNA methylation, raising an intriguing possibility that histone methylation status may affect DNA methylation.In the third paper, Koivunen et al.¹¹ proposed an enantiomer-specific mechanism of 2-HG in tumorigenesis. The authors reported two surprising findings. They showed first that immortalized human astrocytes stably expressing tumor-derived IDH1^R132H mutant proliferate faster during late passages than those expressing either wild-type IDH1 or IDH1^R132H/3DN mutant that lacks 2-HG-producing activity. Ectopic expression of R132H mutant IDH1 has previously been reported to decrease the growth of D54 glioblastoma cells¹², raising an intriguing possibility that the mutation of IDH1/2 may exhibit different effects on cell growth in a cell context-dependent manner. More surprisingly, they found that R-2-HG, but not its enantiomer S-2-HG, substitutes for α-KG as a co-substrate, as opposed to an inhibitor, of EGLN, an α-KG-dependent prolylhydroxylase responsible for promoting the degradation of hypoxia inducible factor 1α (HIF-1α) (Figure 1). As the result of stimulating EGLN, accumulation of R-2-HG was found to associate with diminished, instead of increased, HIF-1α levels in cells expressing mutant IDH1/2. At first glance, these observations appear to be at odds with the generally accepted role of both enantiomers of 2-HG as inhibitors of α-KG-dependent dioxygenases, and HIF-1α as an oncogene in tumorigenesis, but may at least in part explain the apparent selection for IDH mutations to produce R-, but not S-2-HG in cancer. This data, also for the first time, reveals a qualitatively different property of two 2-HG enantiomers with respect to α-KG-dependent dioxygenases. It will be interesting to determine the strutural basis of this enantimoer-specific effect of 2-HG toward different α-KG-dependent dixoygenases. The observation that ectopic increase of R-2-HG reduces HIF-1α suggests that endogenous α-KG is limiting for HIF-1α hydroxylation by EGLN. The study by Koivunen et al. also suggests the complexity of EGLN regulation by R-2-HG and subsequent downregulation of HIF-1α. It remains to be determined genetically whether a reduction or fluctuation of HIF-1α levels contributes to gliomagenesis in IDH1/2-mutated cells, because elevated HIF-1α generally contributes to cancer development. The only piece of genetic evidece—IDH1/2 mutation occurs in a mutually exclusive manner with TET2 mutation in AML—supports the notion that epigenetic alteration plays a direct and perhaps a key role in IDH1/2 mutation-associated tumorigenesis.IDH1/2 mutation has rapidly emerged as a favorable diagnostic and prognostic marker for certain tumors, such as low-grade gliomas and benign cartilaginous tumors. While the full mechanism linking IDH mutation to tumorigenesis is incompletely understood, regulation of α-KG-dependent dioxygenases by 2-HG is likely to play a major role in the pathophysiology of tumors with IDH mutation. These recent reports also highlight the impact of altered metabolism and metabolites on the epigenetic modification of cell differentiation and tumorigenesis.     

Biochemical,Cellular, and Biophysical Characterization of a Potent Inhibitor of Mutant Isocitrate Dehydrogenase IDH1

Two mutant forms (R132H and R132C) of isocitrate dehydrogenase 1 (IDH1) have been associated with a number of cancers including glioblastoma and acute myeloid leukemia. These mutations confer a neomorphic activity of 2-hydroxyglutarate (2-HG) production, and 2-HG has previously been implicated as an oncometabolite. Inhibitors of mutant IDH1 can potentially be used to treat these diseases. In this study, we investigated the mechanism of action of a newly discovered inhibitor, ML309, using biochemical, cellular, and biophysical approaches. Substrate binding and product inhibition studies helped to further elucidate the IDH1 R132H catalytic cycle. This rapidly equilibrating inhibitor is active in both biochemical and cellular assays. The (+) isomer is active (IC₅₀ = 68 nm), whereas the (−) isomer is over 400-fold less active (IC₅₀ = 29 μm) for IDH1 R132H inhibition. IDH1 R132C was similarly inhibited by (+)-ML309. WT IDH1 was largely unaffected by (+)-ML309 (IC₅₀ >36 μm). Kinetic analyses combined with microscale thermophoresis and surface plasmon resonance indicate that this reversible inhibitor binds to IDH1 R132H competitively with respect to α-ketoglutarate and uncompetitively with respect to NADPH. A reaction scheme for IDH1 R132H inhibition by ML309 is proposed in which ML309 binds to IDH1 R132H after formation of the IDH1 R132H NADPH complex. ML309 was also able to inhibit 2-HG production in a glioblastoma cell line (IC₅₀ = 250 nm) and had minimal cytotoxicity. In the presence of racemic ML309, 2-HG levels drop rapidly. This drop was sustained until 48 h, at which point the compound was washed out and 2-HG levels recovered.     

Glioma-derived mutations in isocitrate dehydrogenase 2 beneficial to traditional chemotherapy

Heterozygous mutations in either the R132 residue of isocitrate dehydrogenase I (IDH1) or the R172 residue of IDH2 in human gliomas were recently highlighted. In the present study, we report that mutations of IDH1 and IDH2 are not detected in the rat C6 glioma cell line model, which suggests that these mutations are not required for the development of glioblastoma induced by N,N′-nitroso-methylurea. The effects of IDH2 and IDH2^R172G on C6 cells proliferation and sensitivity to chemotherapy and the possible mechanism are analyzed at the cellular level. IDH1 and IDH2 mutations lead to simultaneous loss and gain of activities in the production of α-ketoglutarate (α-KG) and 2-hydroxyglutarate (2HG), respectively, and result in lowering NADPH levels even further. The low NADPH levels can sensitize tumors to chemotherapy, and account for the prolonged survival of patients harboring the mutations. Our data extrapolate potential importance of the in vitro rat C6 glioma cell model, show that the IDH2^R172G mutation in gliomas may give a benefit to traditional chemotherapy of this cancer and serve as an important complement to existing research on this topic.     

Mutation and expression analysis of the IDH1, IDH2, DNMT3A, and MYD88 genes in colorectal cancer

Colorectal cancer (CRC) is one of the leading causes of death around the world. Its genetic mechanism was intensively investigated in the past decades with findings of a number of canonical oncogenes and tumor-suppressor genes such as APC, KRAS, and TP53. Recent genome-wide association and sequencing studies have identified a series of promising oncogenes including IDH1, IDH2, DNMT3A, and MYD88 in hematologic malignancies. However, whether these genes are involved in CRC remains unknown. In this study, we screened the hotspot mutations of these four genes in 305 CRC samples from Han Chinese by direct sequencing. mRNA expression levels of these genes were quantified by quantitative real-time PCR (RT-qPCR) in paired cancerous and paracancerous tissues. Association analyses between mRNA expression levels and different cancerous stages were performed. Except for one patient harboring IDH1 mutation p.I99M, we identified no previously reported hotspot mutations in colorectal cancer tissues. mRNA expression levels of IDH1, DNMT3A, and MYD88, but not IDH2, were significantly decreased in the cancerous tissues comparing with the paired paracancerous normal tissues. Taken together, the hotspot mutations of IDH1, IDH2, DNMT3A, and MYD88 gene were absent in CRC. Aberrant mRNA expression of IDH1, DNMT3A, and MYD88 gene might be actively involved in the development of CRC.     

Impact of Chemotherapy Delay on Overall Survival for AML with IDH1/2 Mutations: A Study in Adult Chinese Patients

The effect of time from diagnosis to treatment (TDT) on overall survival of patients with acute myeloid leukemia (AML) remains obscure. Furthermore, whether chemotherapy delay impacts overall survival (OS) of patients with a special molecular subtype has not been investigated. Here, we enrolled 364 cases of AML to assess the effect of TDT on OS by fractional polynomial regression in the context of clinical parameters and genes of FLT3ITD, NPM1, CEBPA, DNMT3a, and IDH1/2 mutations. Results of the current study show IDH1/2 mutations are associated with older age, M0 morphology, an intermediate cytogenetic risk group, and NPM1 mutations. TDT associates with OS for AML patients in a nonlinear pattern with a J shape. Moreover, adverse effect of delayed treatment on OS was observed in patients with IDH1/2 mutations, but not in those with IDH1/2 wildtype. Therefore, initiating chemotherapy as soon as possible after diagnosis might be a potential strategy to improve OS in AML patients with IDH1/2 mutations.     

Selective Inhibition of Mutant Isocitrate Dehydrogenase 1 (IDH1) via Disruption of a Metal Binding Network by an Allosteric Small Molecule

Cancer-associated point mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) confer a neomorphic enzymatic activity: the reduction of α-ketoglutarate to d-2-hydroxyglutaric acid, which is proposed to act as an oncogenic metabolite by inducing hypermethylation of histones and DNA. Although selective inhibitors of mutant IDH1 and IDH2 have been identified and are currently under investigation as potential cancer therapeutics, the mechanistic basis for their selectivity is not yet well understood. A high throughput screen for selective inhibitors of IDH1 bearing the oncogenic mutation R132H identified compound 1, a bis-imidazole phenol that inhibits d-2-hydroxyglutaric acid production in cells. We investigated the mode of inhibition of compound 1 and a previously published IDH1 mutant inhibitor with a different chemical scaffold. Steady-state kinetics and biophysical studies show that both of these compounds selectively inhibit mutant IDH1 by binding to an allosteric site and that inhibition is competitive with respect to Mg²⁺. A crystal structure of compound 1 complexed with R132H IDH1 indicates that the inhibitor binds at the dimer interface and makes direct contact with a residue involved in binding of the catalytically essential divalent cation. These results show that targeting a divalent cation binding residue can enable selective inhibition of mutant IDH1 and suggest that differences in magnesium binding between wild-type and mutant enzymes may contribute to the inhibitors'' selectivity for the mutant enzyme.     

Mutation analyses in pedigrees and sporadic cases of ethnic Han Chinese Kallmann syndrome patients

Kallmann syndrome, a form of idiopathic hypogonadotropic hypogonadism, is characterized by developmental abnormalities of the reproductive system and abnormal olfaction. Despite association of certain genes with idiopathic hypogonadotropic hypogonadism, the genetic inheritance and expression are complex and incompletely known. In the present study, seven Kallmann syndrome pedigrees in an ethnic Han Chinese population were screened for genetic mutations. The exons and intron–exon boundaries of 19 idiopathic hypogonadotropic hypogonadism (idiopathic hypogonadotropic hypogonadism)-related genes in seven Chinese Kallmann syndrome pedigrees were sequenced. Detected mutations were also tested in 70 sporadic Kallmann syndrome cases and 200 Chinese healthy controls. In pedigrees 1, 2, and 7, the secondary sex characteristics were poorly developed and the patients’ sense of smell was severely or completely lost. We detected a genetic mutation in five of the seven pedigrees: homozygous KAL1 p.R191ter (pedigree 1); homozygous KAL1 p.C13ter (pedigree 2; a novel mutation); heterozygous FGFR1 p.R250W (pedigree 3); and homozygous PROKR2 p.Y113H (pedigrees 4 and 5). No genetic change of the assayed genes was detected in pedigrees 6 and 7. Among the 70 sporadic cases, we detected one homozygous and one heterozygous PROKR2 p.Y113H mutation. This mutation was also detected heterozygously in 2/200 normal controls and its pathogenicity is likely questionable. The genetics and genotype–phenotype relationships in Kallmann syndrome are complicated. Classical monogenic inheritance does not explain the full range of genetic inheritance of Kallmann syndrome patients. Because of stochastic nature of genetic mutations, exome analyses of Kallmann syndrome patients may provide novel insights.