Genglin Jin

IDH1 and IDH2 Mutations in Gliomas

BACKGROUND A recent genomewide mutational analysis of glioblastomas (World Health Organization [WHO] grade IV glioma) revealed somatic mutations of the isocitrate dehydrogenase 1 gene (IDH1) in a fraction of such tumors, most frequently in tumors that were known to have evolved from lower-grade gliomas (secondary glioblastomas). METHODS We determined the sequence of the IDH1 gene and the related IDH2 gene in 445 central nervous system (CNS) tumors and 494 non-CNS tumors. The enzymatic activity of the proteins that were produced from normal and mutant IDH1 and IDH2 genes was determined in cultured glioma cells that were transfected with these genes. RESULTS We identified mutations that affected amino acid 132 of IDH1 in more than 70% of WHO grade II and III astrocytomas and oligodendrogliomas and in glioblastomas that developed from these lower-grade lesions. Tumors without mutations in IDH1 often had mutations affecting the analogous amino acid (R172) of the IDH2 gene. Tumors with IDH1 or IDH2 mutations had distinctive genetic and clinical characteristics, and patients with such tumors had a better outcome than those with wild-type IDH genes. Each of four tested IDH1 and IDH2 mutations reduced the enzymatic activity of the encoded protein. CONCLUSIONS Mutations of NADP(+)-dependent isocitrate dehydrogenases encoded by IDH1 and IDH2 occur in a majority of several types of malignant gliomas.

Profiling the effects of isocitrate dehydrogenase 1 and 2 mutations on the cellular metabolome

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.

A heterozygous IDH1R132H/WT mutation induces genome-wide alterations in DNA methylation

Monoallelic point mutations of the NADP(+)-dependent isocitrate dehydrogenases IDH1 and IDH2 occur frequently in gliomas, acute myeloid leukemias, and chondromas, and display robust association with specific DNA hypermethylation signatures. Here we show that heterozygous expression of the IDH1(R132H) allele is sufficient to induce the genome-wide alterations in DNA methylation characteristic of these tumors. Using a gene-targeting approach, we knocked-in a single copy of the most frequently observed IDH1 mutation, R132H, into a human cancer cell line and profiled changes in DNA methylation at over 27,000 CpG dinucleotides relative to wild-type parental cells. We find that IDH1(R132H/WT) mutation induces widespread alterations in DNA methylation, including hypermethylation of 2010 and hypomethylation of 842 CpG loci. We demonstrate that many of these alterations are consistent with those observed in IDH1-mutant and G-CIMP+ primary gliomas and can segregate IDH wild-type and mutated tumors as well as those exhibiting the G-CIMP phenotype in unsupervised analysis of two primary glioma cohorts. Further, we show that the direction of IDH1(R132H/WT)-mediated DNA methylation change is largely dependent upon preexisting DNA methylation levels, resulting in depletion of moderately methylated loci. Additionally, whereas the levels of multiple histone H3 and H4 methylation modifications were globally increased, consistent with broad inhibition of histone demethylation, hypermethylation at H3K9 in particular accompanied locus-specific DNA hypermethylation at several genes down-regulated in IDH1(R132H/WT) knock-in cells. These data provide insight on epigenetic alterations induced by IDH1 mutations and support a causal role for IDH1(R132H/WT) mutants in driving epigenetic instability in human cancer cells.

A monoclonal antibody IMab-1 specifically recognizes IDH1R132H, 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.

2-hydroxyglutarate production, but not dominant negative function, is conferred by glioma-derived NADP-dependent isocitrate dehydrogenase mutations.

Background Gliomas frequently contain mutations in the cytoplasmic NADP+-dependent isocitrate dehydrogenase (IDH1) or the mitochondrial NADP+-dependent isocitrate dehydrogenase (IDH2). Several different amino acid substitutions recur at either IDH1 R132 or IDH2 R172 in glioma patients. Genetic evidence indicates that these mutations share a common gain of function, but it is unclear whether the shared function is dominant negative activity, neomorphic production of (R)-2-hydroxyglutarate (2HG), or both. Methodology/Principal Findings We show by coprecipitation that five cancer-derived IDH1 R132 mutants bind IDH1-WT but that three cancer-derived IDH2 R172 mutants exert minimal binding to IDH2-WT. None of the mutants dominant-negatively lower isocitrate dehydrogenase activity at physiological (40 µM) isocitrate concentrations in mammalian cell lysates. In contrast to this, all of these mutants confer 10- to 100-fold higher 2HG production to cells, and glioma tissues containing IDH1 R132 or IDH2 R172 mutations contain high levels of 2HG compared to glioma tissues without IDH mutations (54.4 vs. 0.1 mg 2HG/g protein). Conclusions Binding to, or dominant inhibition of, WT IDH1 or IDH2 is not a shared feature of the IDH1 and IDH2 mutations, and thus is not likely to be important in cancer. The fact that the gain of the enzymatic activity to produce 2HG is a shared feature of the IDH1 and IDH2 mutations suggests that this is an important function for these mutants in driving cancer pathogenesis.

HDMX regulates p53 activity and confers chemoresistance to 3-Bis(2-chloroethyl)-1- nitrosourea

Glioblastoma multiforme (GBM) is one of the deadliest tumors afflicting humans, and the mechanisms of its onset and progression remain largely undefined. Our attempts to elucidate its molecular pathogenesis through DNA copy-number analysis by genome-wide digital karyotyping and single nucleotide polymorphism arrays identified a dramatic focal amplification on chromosome 1q32 in 4 of 57 GBM tumors. Quantitative real-time PCR measurements revealed that HDMX is the most commonly amplified and overexpressed gene in the 1q32 locus. Further genetic screening of 284 low- and high-grade gliomas revealed that HDMX amplifications occur solely in pediatric and adult GBMs and that they are mutually exclusive of TP53 mutations and MDM2 amplifications. Here, we demonstrate that HDMX regulates p53 to promote GBM growth and attenuates tumor response to chemotherapy. In GBM cells, HDMX overexpression inhibits p53-mediated transcriptional activation of p21, releases cells from G0 to G1 phase, and enhances cellular proliferation. HDMX overexpression does not affect the expression of PUMA and BAX proapoptotic genes. While in GBM cells treated with the chemotherapeutic agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), HDMX appears to stabilize p53 and promote phosphorylation of the DNA double-stranded break repair protein H2AX, up-regulate the DNA repair gene VPX, stimulate DNA repair, and confer resistance to BCNU. In summary, HDMX exhibits bona fide oncogenic properties and offers a promising molecular target for GBM therapeutic intervention.

Aberrant Otx2 expression enhances migration and induces ectopic proliferation of hindbrain neuronal progenitor cells.

Dysregulation of Otx2 is a hallmark of the pediatric brain tumor medulloblastoma, yet its functional significance in the establishment of these tumors is unknown. Here we have sought to determine the functional consequences of Otx2 overexpression in the mouse hindbrain to characterize its potential role in medulloblastoma tumorigenesis and identify the cell types responsive to this lineage-specific oncogene. Expression of Otx2 broadly in the mouse hindbrain resulted in the accumulation of proliferative clusters of cells in the cerebellar white matter and dorsal brainstem of postnatal mice. We found that brainstem ectopia were derived from neuronal progenitors of the rhombic lip and that cerebellar ectopia were derived from granule neuron precursors (GNPs) that had migrated inwards from the external granule layer (EGL). These hyperplasias exhibited various characteristics of medulloblastoma precursor cells identified in animal models of Shh or Wnt group tumors, including aberrant localization and altered spatiotemporal control of proliferation. However, ectopia induced by Otx2 differentiated and dispersed as the animals reached adulthood, indicating that factors restricting proliferative lifespan were a limiting factor to full transformation of these cells. These studies implicate a role for Otx2 in altering the dynamics of neuronal progenitor cell proliferation.

Abstract 3352: The medulloblastoma oncogene Otx2 enhances migration and permits ectopic proliferation of neuronal progenitor cells of the cerebellum and brainstem

Medulloblastoma is the most common malignant brain tumor in children. Despite a thorough understanding of the genetic underpinnings of this tumor, the pathogenesis of some variants of medulloblastoma, in particular the non-Shh/non-Wnt subtypes, is poorly understood. One of the most common genetic aberrations in non-Shh/non-Wnt medulloblastomas is copy number gain of the homeobox transcription factor OTX2. Here we have characterized mice engineered to ectopically express Otx2 in the hindbrain to identify the functional consequences of this subtype-specific genetic insult. Otx2 expression induced accumulation of proliferative hyperplasias in the cerebellar white matter and dorsal brainstem that were reminiscent of preneoplastic cells identified in other animal models of medulloblastoma. These hyperplasias were comprised of neuronal progenitor cells that had migrated away from their mitogenic niches. As animals reached adulthood, ectopic cells exited the cell cycle and differentiated, indicating that factors limiting proliferative lifespan prevented full transformation by Otx2. Comparison of the effect of Otx2 with activation of the Shh or Wnt pathways, which occur in distinct subtypes of medulloblastoma, revealed overlap of responsive cell types but distinct developmental processes involved. While activation of the Shh or Wnt pathways give rise to sustained proliferation of neuronal progenitor cells at their mitogenic niches, we found that Otx2 permitted or enhanced migration of these cells away from these niches and induced ectopic proliferation. These findings identify a role for Otx2 overexpression in altering spatiotemporal control of neuronal progenitor cell proliferation and implicate a relationship between the cells of origin of non-Shh/non-Wnt medulloblastomas and those of other medulloblastoma subtypes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3352. doi:1538-7445.AM2012-3352

Abstract LB-257: Profiling the effects of IDH1 and IDH2 mutants on the glioma cell metabolome

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Somatic hotspot mutations in the NADP+-dependent isocitrate dehydrogenases, IDH1 and IDH2, arise frequently in gliomas. These gliomas include WHO grades II and III oligodendrogliomas and astrocytomas, as well as WHO grade IV secondary glioblastomas. When mutated, IDH1 and IDH2 gain the ability to produce (R)-2-hydroxyglutarate (2HG) and may dominant-negatively inhibit IDH1-WT. Aside from these specific functions, the downstream effect of these metabolic enzyme mutations on cellular metabolism is unknown. To identify metabolic alterations caused by IDH1 and IDH2 mutants, we used three mass spectrometry methods (GC-MS/MS, LC-MS/MS +ESI, and LC-MS/MS -ESI) to profile >200 biochemicals in lysates of human oligodendroglial cell line (HOG) cells that homologously express IDH1-R132H or IDH2-R172K, which are the most frequent IDH1 and IDH2 mutants in glioma, respectively. To determine whether 2HG production or inhibition of IDH1-WT could mediate similar effects on cellular metabolism as IDH mutant expression, we also analyzed cells treated cells with either 7.5mM or 30mM 2HG as well as cells with stable shRNA knockdown of endogenous IDH1-WT. Unsupervised hierarchical clustering, univariate threshold tests, correlation analysis, and principal component analysis revealed that cells expressing IDH1-R132H and IDH2-R172K have similar metabolite profiles, and that 2HG treatment also results in a similar profile. However, cell with shRNA IDH1 knockdown shared few metabolic features with cells expressing IDH mutants. Metabolite set enrichment analyses revealed that numerous amino acids, N-acetylated amino acids, TCA cycle metabolites, glutathione metabolites, and choline derivatives were markedly altered in IDH-mutant expressing cells. Strikingly, we found that N-acetyl-aspartyl-glutamate (NAAG), a common brain metabolite, is 50-fold reduced in cells expressing IDH1-R132H and >2-fold reduced in human glioma tissues with IDH1-R132H compared to glioma tissues without IDH mutations. This work shows that IDH mutants can induce widespread cellular metabolic changes. Additionally, the metabolites that were found to be altered in this study may play a role in glioma pathogenesis and may be useful for diagnosis or treatment for gliomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-257. doi:10.1158/1538-7445.AM2011-LB-257