RESUMO
Mitochondrial aldehyde dehydrogenase 2 (ALDH2) in the liver removes toxic aldehydes including acetaldehyde, an intermediate of ethanol metabolism. Nearly 40% of East Asians inherit an inactive ALDH2*2 variant, which has a lysine-for-glutamate substitution at position 487 (E487K), and show a characteristic alcohol flush reaction after drinking and a higher risk for gastrointestinal cancers. Here we report the characterization of knockin mice in which the ALDH2(E487K) mutation is inserted into the endogenous murine Aldh2 locus. These mutants recapitulate essentially all human phenotypes including impaired clearance of acetaldehyde, increased sensitivity to acute or chronic alcohol-induced toxicity, and reduced ALDH2 expression due to a dominant-negative effect of the mutation. When treated with a chemical carcinogen, these mutants exhibit increased DNA damage response in hepatocytes, pronounced liver injury, and accelerated development of hepatocellular carcinoma (HCC). Importantly, ALDH2 protein levels are also significantly lower in patient HCC than in peritumor or normal liver tissues. Our results reveal that ALDH2 functions as a tumor suppressor by maintaining genomic stability in the liver, and the common human ALDH2 variant would present a significant risk factor for hepatocarcinogenesis. Our study suggests that the ALDH2*2 allele-alcohol interaction may be an even greater human public health hazard than previously appreciated.
Assuntos
Aldeído Desidrogenase/genética , Carcinoma Hepatocelular/enzimologia , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , Mutação/genética , Intoxicação Alcoólica/enzimologia , Intoxicação Alcoólica/patologia , Aldeído-Desidrogenase Mitocondrial , Substituição de Aminoácidos , Animais , Sequência de Bases , Carcinogênese/patologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Etanol/efeitos adversos , Técnicas de Introdução de Genes , Técnicas de Genotipagem , Hepatócitos/enzimologia , Hepatócitos/patologia , Humanos , Hiperpigmentação/patologia , Imuno-Histoquímica , Fígado/enzimologia , Fígado/patologia , Neoplasias Hepáticas/patologia , Camundongos Endogâmicos C57BL , Proteínas Mutantes/metabolismo , Polimorfismo Genético , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , Pele/patologia , Análise de SobrevidaRESUMO
Mutations of IDH1 and IDH2, which produce the oncometabolite 2-hydroxyglutarate (2HG), have been identified in several tumors, including acute myeloid leukemia. Recent studies have shown that expression of the IDH mutant enzymes results in high levels of 2HG and a block in cellular differentiation that can be reversed with IDH mutant-specific small-molecule inhibitors. To further understand the role of IDH mutations in cancer, we conducted mechanistic studies in the TF-1 IDH2 R140Q erythroleukemia model system and found that IDH2 mutant expression caused both histone and genomic DNA methylation changes that can be reversed when IDH2 mutant activity is inhibited. Specifically, histone hypermethylation is rapidly reversed within days, whereas reversal of DNA hypermethylation proceeds in a progressive manner over the course of weeks. We identified several gene signatures implicated in tumorigenesis of leukemia and lymphoma, indicating a selective modulation of relevant cancer genes by IDH mutations. As methylation of DNA and histones is closely linked to mRNA expression and differentiation, these results indicate that IDH2 mutant inhibition may function as a cancer therapy via histone and DNA demethylation at genes involved in differentiation and tumorigenesis.
Assuntos
Metilação de DNA/genética , Inibidores Enzimáticos/farmacologia , Histonas/genética , Isocitrato Desidrogenase/genética , Mutação , Transcriptoma/efeitos dos fármacos , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Cromatografia Líquida , Histonas/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/genética , Compostos de Fenilureia/farmacologia , Análise de Componente Principal , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfonamidas/farmacologia , Espectrometria de Massas em TandemRESUMO
Mutations in the metabolic enzymes isocitrate dehydrogenase-1 (IDH1) and IDH2 that produce the oncometabolite D-2-hydroxyglutarate (2-HG) occur frequently in human acute myeloid leukemia (AML). 2-HG modulates numerous biological pathways implicated in malignant transformation, but the contribution of mutant IDH proteins to maintenance and progression of AML in vivo is currently unknown. To answer this crucial question we have generated transgenic mice that express IDH2(R140Q) in an on/off- and tissue-specific manner using a tetracycline-inducible system. We found that IDH2(R140Q) can cooperate with overexpression of HoxA9 and Meis1a and with mutations in FMS-like tyrosine kinase 3 (FLT3) to drive acute leukemia in vivo. Critically, we show that genetic deinduction of mutant IDH2 in leukemic cells in vivo has profound effects on their growth and/or maintenance. Our data demonstrate the proto-oncogenic role of mutant IDH2 and support its relevance as a therapeutic target for the treatment of human AML.
Assuntos
Carcinogênese/patologia , Isocitrato Desidrogenase/genética , Leucemia Mieloide Aguda/patologia , Mutação/genética , Oncogenes , Animais , Medula Óssea/patologia , Carcinogênese/genética , Diferenciação Celular , Proliferação de Células , Transformação Celular Neoplásica/patologia , Modelos Animais de Doenças , Células Eritroides/metabolismo , Células Eritroides/patologia , Hematopoese , Células-Tronco Hematopoéticas/patologia , Proteínas de Homeodomínio/metabolismo , Isocitrato Desidrogenase/metabolismo , Leucemia Mieloide Aguda/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Meis1 , Proteínas de Neoplasias/metabolismo , Baço/patologia , Transcrição Gênica , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
The recent discovery of mutations in metabolic enzymes has rekindled interest in harnessing the altered metabolism of cancer cells for cancer therapy. One potential drug target is isocitrate dehydrogenase 1 (IDH1), which is mutated in multiple human cancers. Here, we examine the role of mutant IDH1 in fully transformed cells with endogenous IDH1 mutations. A selective R132H-IDH1 inhibitor (AGI-5198) identified through a high-throughput screen blocked, in a dose-dependent manner, the ability of the mutant enzyme (mIDH1) to produce R-2-hydroxyglutarate (R-2HG). Under conditions of near-complete R-2HG inhibition, the mIDH1 inhibitor induced demethylation of histone H3K9me3 and expression of genes associated with gliogenic differentiation. Blockade of mIDH1 impaired the growth of IDH1-mutant--but not IDH1-wild-type--glioma cells without appreciable changes in genome-wide DNA methylation. These data suggest that mIDH1 may promote glioma growth through mechanisms beyond its well-characterized epigenetic effects.
Assuntos
Benzenoacetamidas/farmacologia , Diferenciação Celular , Inibidores Enzimáticos/farmacologia , Glioma/enzimologia , Glioma/patologia , Imidazóis/farmacologia , Isocitrato Desidrogenase/antagonistas & inibidores , Isocitrato Desidrogenase/genética , Animais , Benzenoacetamidas/administração & dosagem , Benzenoacetamidas/toxicidade , Diferenciação Celular/efeitos dos fármacos , Transformação Celular Neoplásica , Inibidores Enzimáticos/toxicidade , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioma/tratamento farmacológico , Glioma/genética , Glutaratos/metabolismo , Histonas/metabolismo , Imidazóis/administração & dosagem , Imidazóis/toxicidade , Isocitrato Desidrogenase/química , Isocitrato Desidrogenase/metabolismo , Metilação , Camundongos , Camundongos SCID , Proteínas Mutantes/antagonistas & inibidores , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Multimerização Proteica , Interferência de RNA , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A number of human cancers harbor somatic point mutations in the genes encoding isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2). These mutations alter residues in the enzyme active sites and confer a gain-of-function in cancer cells, resulting in the accumulation and secretion of the oncometabolite (R)-2-hydroxyglutarate (2HG). We developed a small molecule, AGI-6780, that potently and selectively inhibits the tumor-associated mutant IDH2/R140Q. A crystal structure of AGI-6780 complexed with IDH2/R140Q revealed that the inhibitor binds in an allosteric manner at the dimer interface. The results of steady-state enzymology analysis were consistent with allostery and slow-tight binding by AGI-6780. Treatment with AGI-6780 induced differentiation of TF-1 erythroleukemia and primary human acute myelogenous leukemia cells in vitro. These data provide proof-of-concept that inhibitors targeting mutant IDH2/R140Q could have potential applications as a differentiation therapy for cancer.
Assuntos
Inibidores Enzimáticos/farmacologia , Hematopoese/efeitos dos fármacos , Isocitrato Desidrogenase/antagonistas & inibidores , Isocitrato Desidrogenase/genética , Leucemia Mieloide Aguda/enzimologia , Compostos de Fenilureia/farmacologia , Sulfonamidas/farmacologia , Sítio Alostérico , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Domínio Catalítico , Linhagem Celular Tumoral , Proliferação de Células , Células Cultivadas , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Eritropoese/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica , Glutaratos/metabolismo , Humanos , Isocitrato Desidrogenase/química , Isocitrato Desidrogenase/metabolismo , Leucemia Eritroblástica Aguda , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Terapia de Alvo Molecular , Proteínas Mutantes/antagonistas & inibidores , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Compostos de Fenilureia/química , Compostos de Fenilureia/metabolismo , Mutação Puntual , Multimerização Proteica , Estrutura Secundária de Proteína , Bibliotecas de Moléculas Pequenas , Sulfonamidas/química , Sulfonamidas/metabolismoRESUMO
Proliferating tumor cells use aerobic glycolysis to support their high metabolic demands. Paradoxically, increased glycolysis is often accompanied by expression of the lower activity PKM2 isoform, effectively constraining lower glycolysis. Here, we report the discovery of PKM2 activators with a unique allosteric binding mode. Characterization of how these compounds impact cancer cells revealed an unanticipated link between glucose and amino acid metabolism. PKM2 activation resulted in a metabolic rewiring of cancer cells manifested by a profound dependency on the nonessential amino acid serine for continued cell proliferation. Induction of serine auxotrophy by PKM2 activation was accompanied by reduced carbon flow into the serine biosynthetic pathway and increased expression of high affinity serine transporters. These data support the hypothesis that PKM2 expression confers metabolic flexibility to cancer cells that allows adaptation to nutrient stress.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Serina/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Hormônios Tireóideos/metabolismo , Sítio Alostérico/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Proteínas de Ligação a Hormônio da TireoideRESUMO
Isocitrate dehydrogenase-1 (IDH1) R132 mutations occur in glioma, but their physiological significance is unknown. Here we describe the generation and characterization of brain-specific Idh1 R132H conditional knock-in (KI) mice. Idh1 mutation results in hemorrhage and perinatal lethality. Surprisingly, intracellular reactive oxygen species (ROS) are attenuated in Idh1-KI brain cells despite an apparent increase in the NADP(+)/NADPH ratio. Idh1-KI cells also show high levels of D-2-hydroxyglutarate (D2HG) that are associated with inhibited prolyl-hydroxylation of hypoxia-inducible transcription factor-1α (Hif1α) and up-regulated Hif1α target gene transcription. Intriguingly, D2HG also blocks prolyl-hydroxylation of collagen, causing a defect in collagen protein maturation. An endoplasmic reticulum (ER) stress response induced by the accumulation of immature collagens may account for the embryonic lethality of these mutants. Importantly, D2HG-mediated impairment of collagen maturation also led to basement membrane (BM) aberrations that could play a part in glioma progression. Our study presents strong in vivo evidence that the D2HG produced by the mutant Idh1 enzyme is responsible for the above effects.
Assuntos
Membrana Basal/patologia , Colágeno/metabolismo , Glutaratos/metabolismo , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Animais , Membrana Basal/metabolismo , Encéfalo/citologia , Encéfalo/patologia , Técnicas de Introdução de Genes , Genótipo , Glioma/patologia , Camundongos , Mutação , Estabilidade Proteica , Espécies Reativas de Oxigênio/metabolismo , Estresse FisiológicoRESUMO
Mutations in the IDH1 and IDH2 genes encoding isocitrate dehydrogenases are frequently found in human glioblastomas and cytogenetically normal acute myeloid leukaemias (AML). These alterations are gain-of-function mutations in that they drive the synthesis of the 'oncometabolite' R-2-hydroxyglutarate (2HG). It remains unclear how IDH1 and IDH2 mutations modify myeloid cell development and promote leukaemogenesis. Here we report the characterization of conditional knock-in (KI) mice in which the most common IDH1 mutation, IDH1(R132H), is inserted into the endogenous murine Idh1 locus and is expressed in all haematopoietic cells (Vav-KI mice) or specifically in cells of the myeloid lineage (LysM-KI mice). These mutants show increased numbers of early haematopoietic progenitors and develop splenomegaly and anaemia with extramedullary haematopoiesis, suggesting a dysfunctional bone marrow niche. Furthermore, LysM-KI cells have hypermethylated histones and changes to DNA methylation similar to those observed in human IDH1- or IDH2-mutant AML. To our knowledge, our study is the first to describe the generation and characterization of conditional IDH1(R132H)-KI mice, and also the first report to demonstrate the induction of a leukaemic DNA methylation signature in a mouse model. Our report thus sheds light on the mechanistic links between IDH1 mutation and human AML.
Assuntos
Epigênese Genética/genética , Células-Tronco Hematopoéticas/citologia , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Proteínas Mutantes/metabolismo , Mutação/genética , Envelhecimento , Animais , Medula Óssea/patologia , Linhagem da Célula , Ilhas de CpG/genética , Metilação de DNA , Modelos Animais de Doenças , Feminino , Técnicas de Introdução de Genes , Glioma/patologia , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Histonas/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Masculino , Camundongos , Proteínas Mutantes/genética , Células Mieloides/citologia , Células Mieloides/metabolismo , Baço/patologiaRESUMO
Mutations of the isocitrate dehydrogenase 1 and 2 genes (IDH1 and IDH2) are commonly found in primary brain cancers. We previously reported that a novel enzymatic activity of these mutations results in the production of the putative oncometabolite, R(-)-2-hydroxyglutarate (2-HG). Here we investigated the ability of magnetic resonance spectroscopy (MRS) to detect 2-HG production in order to non-invasively identify patients with IDH1 mutant brain tumors. Patients with intrinsic glial brain tumors (n = 27) underwent structural and spectroscopic magnetic resonance imaging prior to surgery. 2-HG levels from MRS data were quantified using LC-Model software, based upon a simulated spectrum obtained from a GAMMA library added to the existing prior knowledge database. The resected tumors were then analyzed for IDH1 mutational status by genomic DNA sequencing, Ki-67 proliferation index by immunohistochemistry, and concentrations of 2-HG and other metabolites by liquid chromatography-mass spectrometry (LC-MS). MRS detected elevated 2-HG levels in gliomas with IDH1 mutations compared to those with wild-type IDH1 (P = 0.003). The 2-HG levels measured in vivo with MRS were significantly correlated with those measured ex vivo from the corresponding tumor samples using LC-MS (r (2) = 0.56; P = 0.0001). Compared with wild-type tumors, those with IDH1 mutations had elevated choline (P = 0.01) and decreased glutathione (P = 0.03) on MRS. Among the IDH1 mutated gliomas, quantitative 2-HG values were correlated with the Ki-67 proliferation index of the tumors (r ( 2 ) = 0.59; P = 0.026). In conclusion, water-suppressed proton ((1)H) MRS provides a non-invasive measure of 2-HG in gliomas, and may serve as a potential biomarker for patients with IDH1 mutant brain tumors. In addition to 2-HG, alterations in several other metabolites measured by MRS correlate with IDH1 mutation status.
Assuntos
Biomarcadores Tumorais/metabolismo , Glioma/genética , Glioma/metabolismo , Glutaratos/metabolismo , Isocitrato Desidrogenase/genética , Espectroscopia de Ressonância Magnética , Mutação/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Cromatografia Líquida , DNA de Neoplasias/genética , Feminino , Seguimentos , Glioma/patologia , Humanos , Imageamento por Ressonância Magnética , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Prognóstico , Estudos Prospectivos , Adulto JovemRESUMO
Optimization of a series of R132H IDH1 inhibitors from a high throughput screen led to the first potent molecules that show robust tumor 2-HG inhibition in a xenograft model. Compound 35 shows good potency in the U87 R132H cell based assay and â¼90% tumor 2-HG inhibition in the corresponding mouse xenograft model following BID dosing. The magnitude and duration of tumor 2-HG inhibition correlates with free plasma concentration.
RESUMO
The systematic sequencing of glioblastoma multiforme (GBM) genomes has identified the recurrent mutation of IDH1, a gene encoding NADP(+)-dependent isocitrate dehydrogenase 1 (IDH1) that catalyzes the oxidative decarboxylation of isocitrate yielding alpha-ketoglutarate (alpha-KG). Subsequent studies have confirmed recurrent IDH1 and IDH2 mutations in up to 70% of low-grade glioma and secondary GBM, as well as in 10% of acute myeloid leukemia (AML) cases. The heterozygous somatic mutations at arginine R132 (IDH1) and at R140 or R172 (IDH2) in the enzyme active site confer a gain of function to the enzymes, which can both produce the metabolite 2-hydroxyglutarate. This review surveys the prevalence of IDH mutations in cancer and explores current mechanistic understanding of IDH mutations with implications for diagnostic and therapeutic development for the treatment of gliomas and AML.
Assuntos
Neoplasias Encefálicas/enzimologia , Glioma/enzimologia , Isocitrato Desidrogenase/genética , Leucemia Mieloide Aguda/enzimologia , Mutação , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Glioma/genética , Glioma/terapia , Humanos , Isocitrato Desidrogenase/química , Isocitrato Desidrogenase/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapiaRESUMO
The somatic mutations in cytosolic isocitrate dehydrogenase 1 (IDH1) observed in gliomas can lead to the production of 2-hydroxyglutarate (2HG). Here, we report that tumor 2HG is elevated in a high percentage of patients with cytogenetically normal acute myeloid leukemia (AML). Surprisingly, less than half of cases with elevated 2HG possessed IDH1 mutations. The remaining cases with elevated 2HG had mutations in IDH2, the mitochondrial homolog of IDH1. These data demonstrate that a shared feature of all cancer-associated IDH mutations is production of the oncometabolite 2HG. Furthermore, AML patients with IDH mutations display a significantly reduced number of other well characterized AML-associated mutations and/or associated chromosomal abnormalities, potentially implicating IDH mutation in a distinct mechanism of AML pathogenesis.
Assuntos
Glutaratos/metabolismo , Isocitrato Desidrogenase/genética , Ácidos Cetoglutáricos/metabolismo , Leucemia Mieloide Aguda/genética , Proliferação de Células , Humanos , Isocitrato Desidrogenase/química , Isocitratos/química , Isocitratos/metabolismo , Leucemia Mieloide Aguda/metabolismo , Mitocôndrias/metabolismo , Mutação , Células Tumorais CultivadasRESUMO
Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2), are present in most gliomas and secondary glioblastomas, but are rare in other neoplasms. IDH1/2 mutations are heterozygous, and affect a single arginine residue. Recently, IDH1 mutations were identified in 8% of acute myelogenous leukemia (AML) patients. A glioma study revealed that IDH1 mutations cause a gain-of-function, resulting in the production and accumulation of 2-hydroxyglutarate (2-HG). Genotyping of 145 AML biopsies identified 11 IDH1 R132 mutant samples. Liquid chromatography-mass spectrometry metabolite screening revealed increased 2-HG levels in IDH1 R132 mutant cells and sera, and uncovered two IDH2 R172K mutations. IDH1/2 mutations were associated with normal karyotypes. Recombinant IDH1 R132C and IDH2 R172K proteins catalyze the novel nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of alpha-ketoglutarate (alpha-KG) to 2-HG. The IDH1 R132C mutation commonly found in AML reduces the affinity for isocitrate, and increases the affinity for NADPH and alpha-KG. This prevents the oxidative decarboxylation of isocitrate to alpha-KG, and facilitates the conversion of alpha-KG to 2-HG. IDH1/2 mutations confer an enzymatic gain of function that dramatically increases 2-HG in AML. This provides an explanation for the heterozygous acquisition of these mutations during tumorigenesis. 2-HG is a tractable metabolic biomarker of mutant IDH1/2 enzyme activity.
Assuntos
Isocitrato Desidrogenase/genética , Leucemia Mieloide Aguda/enzimologia , Mutação , Catálise , Glutaratos/sangue , Humanos , Isocitrato Desidrogenase/metabolismo , Ácidos Cetoglutáricos/metabolismo , Leucemia Mieloide Aguda/sangue , Leucemia Mieloide Aguda/genética , NADP/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
Mutations in the enzyme cytosolic isocitrate dehydrogenase 1 (IDH1) are a common feature of a major subset of primary human brain cancers. These mutations occur at a single amino acid residue of the IDH1 active site, resulting in loss of the enzyme's ability to catalyse conversion of isocitrate to alpha-ketoglutarate. However, only a single copy of the gene is mutated in tumours, raising the possibility that the mutations do not result in a simple loss of function. Here we show that cancer-associated IDH1 mutations result in a 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. Excess accumulation of 2HG has been shown to lead to an elevated risk of malignant brain tumours in patients with inborn errors of 2HG metabolism. Similarly, in human malignant gliomas harbouring IDH1 mutations, we find markedly elevated levels of 2HG. These data demonstrate that the IDH1 mutations result in production of the onco-metabolite 2HG, and indicate that the excess 2HG which accumulates in vivo contributes to the formation and malignant progression of gliomas.