A high-throughput fluorimetric assay for 2-hydroxyglutarate identifies Zaprinast as a glutaminase inhibitor.
ABSTRACT: Recently identified isocitrate dehydrogenase (IDH) mutations lead to the production of 2-hydroxyglutarate (2HG), an oncometabolite aberrantly elevated in selected cancers. We developed a facile and inexpensive fluorimetric microplate assay for the quantitation of 2HG and performed an unbiased small-molecule screen in live cells to identify compounds capable of perturbing 2HG production. Zaprinast, a phosphodiesterase 5 inhibitor, was identified as an efficacious modulator of 2HG production and confirmed to lower 2HG levels in vivo. The mechanism of action was not due to cGMP stabilization, but rather, profiling of metabolites upstream of mutant IDH1 pointed to targeted inhibition of the enzyme glutaminase (GLS). Zaprinast treatment reversed histone hypermethylation and soft-agar growth of IDH1-mutant cells, and treatment of glutamine-addicted pancreatic cancer cells reduced growth and sensitized cells to oxidative damage. Thus, Zaprinast is efficacious against glutamine metabolism and further establishes the therapeutic linkages between GLS and 2HG-mediated oncogenesis.Gain-of-function IDH mutations are common events in glioma, acute myelogenous leukemia, and other cancer types, which lead to the accumulation of the oncometabolite 2HG. We show that the drug Zaprinast is capable of reducing cellular 2HG levels by inhibiting the upstream enzyme GLS, thus identifying a new strategy to target 2HG production in selected IDH-mutant cancers.
Project description:Chondrosarcomas are malignant bone tumors that produce cartilaginous matrix. Mutations in isocitrate dehydrogenase enzymes (IDH1/2) were recently described in several cancers including chondrosarcomas. The IDH1 inhibitor AGI-5198 abrogates the ability of mutant IDH1 to produce the oncometabolite D-2 hydroxyglutarate (D-2HG) in gliomas. We sought to determine if treatment with AGI-5198 would similarly inhibit tumorigenic activity and D-2HG production in IDH1-mutant human chondrosarcoma cells. Two human chondrosarcoma cell lines, JJ012 and HT1080 with endogenous IDH1 mutations and a human chondrocyte cell line C28 with wild type IDH1 were employed in our study. Mutation analysis of IDH was performed by PCR-based DNA sequencing, and D-2HG was detected using tandem mass spectrometry. We confirmed that JJ012 and HT1080 harbor IDH1 R132G and R132C mutation, respectively, while C28 has no mutation. D-2HG was detectable in cell pellets and media of JJ012 and HT1080 cells, as well as plasma and urine from an IDH-mutant chondrosarcoma patient, which decreased after tumor resection. AGI-5198 treatment decreased D-2HG levels in JJ012 and HT1080 cells in a dose-dependent manner, and dramatically inhibited colony formation and migration, interrupted cell cycling, and induced apoptosis. In conclusion, our study demonstrates anti-tumor activity of a mutant IDH1 inhibitor in human chondrosarcoma cell lines, and suggests that D-2HG is a potential biomarker for IDH mutations in chondrosarcoma cells. Thus, clinical trials of mutant IDH inhibitors are warranted for patients with IDH-mutant chondrosarcomas.
Project description:Somatic mutations in the isocitrate dehydrogenase 2 gene (IDH2) contribute to the pathogenesis of acute myeloid leukaemia (AML) through the production of the oncometabolite 2-hydroxyglutarate (2HG)1-8. Enasidenib (AG-221) is an allosteric inhibitor that binds to the IDH2 dimer interface and blocks the production of 2HG by IDH2 mutants9,10. In a phase I/II clinical trial, enasidenib inhibited the production of 2HG and induced clinical responses in relapsed or refractory IDH2-mutant AML11. Here we describe two patients with IDH2-mutant AML who had a clinical response to enasidenib followed by clinical resistance, disease progression, and a recurrent increase in circulating levels of 2HG. We show that therapeutic resistance is associated with the emergence of second-site IDH2 mutations in trans, such that the resistance mutations occurred in the IDH2 allele without the neomorphic R140Q mutation. The in trans mutations occurred at glutamine 316 (Q316E) and isoleucine 319 (I319M), which are at the interface where enasidenib binds to the IDH2 dimer. The expression of either of these mutant disease alleles alone did not induce the production of 2HG; however, the expression of the Q316E or I319M mutation together with the R140Q mutation in trans allowed 2HG production that was resistant to inhibition by enasidenib. Biochemical studies predicted that resistance to allosteric IDH inhibitors could also occur via IDH dimer-interface mutations in cis, which was confirmed in a patient with acquired resistance to the IDH1 inhibitor ivosidenib (AG-120). Our observations uncover a mechanism of acquired resistance to a targeted therapy and underscore the importance of 2HG production in the pathogenesis of IDH-mutant malignancies.
Project description: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.
Project description:2-Hydroxyglutarate (2HG) exists as two enantiomers, (R)-2HG and (S)-2HG, and both are implicated in tumor progression via their inhibitory effects on ?-ketoglutarate (?KG)-dependent dioxygenases. The former is an oncometabolite that is induced by the neomorphic activity conferred by isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations, whereas the latter is produced under pathologic processes such as hypoxia. We report that IDH1/2 mutations induce a homologous recombination (HR) defect that renders tumor cells exquisitely sensitive to poly(adenosine 5'-diphosphate-ribose) polymerase (PARP) inhibitors. This "BRCAness" phenotype of IDH mutant cells can be completely reversed by treatment with small-molecule inhibitors of the mutant IDH1 enzyme, and conversely, it can be entirely recapitulated by treatment with either of the 2HG enantiomers in cells with intact IDH1/2 proteins. We demonstrate mutant IDH1-dependent PARP inhibitor sensitivity in a range of clinically relevant models, including primary patient-derived glioma cells in culture and genetically matched tumor xenografts in vivo. These findings provide the basis for a possible therapeutic strategy exploiting the biological consequences of mutant IDH, rather than attempting to block 2HG production, by targeting the 2HG-dependent HR deficiency with PARP inhibition. Furthermore, our results uncover an unexpected link between oncometabolites, altered DNA repair, and genetic instability.
Project description:Mutations in the isocitrate dehydrogenase genes IDH1 and IDH2 are among the first genetic alterations observed during the development of lower-grade glioma (LGG). LGG-associated IDH mutations confer gain-of-function activity by converting ?-ketoglutarate to the oncometabolite R-2-hydroxyglutarate (2HG). Clinical samples and gene expression data from The Cancer Genome Atlas (TCGA) demonstrate reduced expression of cytotoxic T lymphocyte-associated genes and IFN-?-inducible chemokines, including CXCL10, in IDH-mutated (IDH-MUT) tumors compared with IDH-WT tumors. Given these findings, we have investigated the impact of IDH mutations on the immunological milieu in LGG. In immortalized normal human astrocytes (NHAs) and syngeneic mouse glioma models, the introduction of mutant IDH1 or treatment with 2HG reduced levels of CXCL10, which was associated with decreased production of STAT1, a regulator of CXCL10. Expression of mutant IDH1 also suppressed the accumulation of T cells in tumor sites. Reductions in CXCL10 and T cell accumulation were reversed by IDH-C35, a specific inhibitor of mutant IDH1. Furthermore, IDH-C35 enhanced the efficacy of vaccine immunotherapy in mice bearing IDH-MUT gliomas. Our findings demonstrate a mechanism of immune evasion in IDH-MUT gliomas and suggest that specific inhibitors of mutant IDH may improve the efficacy of immunotherapy in patients with IDH-MUT gliomas.
Project description:Mutations in cytosolic isocitrate dehydrogenase 1 (IDH1) or its mitochondrial homolog IDH2 can lead to R(-)-2-hydroxyglutarate (2HG) production. To date, mutations in three active site arginine residues, IDH1 R132, IDH2 R172 and IDH2 R140, have been shown to result in the neomorphic production of 2HG. Here we report on three additional 2HG-producing IDH1 mutations: IDH1 R100, which is affected in adult glioma, IDH1 G97, which is mutated in colon cancer cell lines and pediatric glioblastoma, and IDH1 Y139. All these new mutants stereospecifically produced 2HG's (R) enantiomer. In contrast, we find that the IDH1 SNPs V71I and V178I, as well as a number of other single-sample reports of IDH non-synonymous mutation, did not elevate cellular 2HG levels in cells and retained the wild-type ability for isocitrate-dependent NADPH production. Finally, we report the existence of additional rare, but recurring mutations found in lymphoma and thyroid cancer, which while failing to elevate 2HG nonetheless displayed loss of function, indicating a possible tumorigenic mechanism for a non-2HG-producing subset of IDH mutations in some malignancies. These data broaden our understanding of how IDH mutations may contribute to cancer through either neomorphic R(-)-2HG production or reduced wild-type enzymatic activity, and highlight the potential value of metabolite screening in identifying IDH-mutated tumors associated with elevated oncometabolite levels.
Project description:Chondrosarcomas are a heterogeneous group of malignant bone tumors that produce hyaline cartilaginous matrix. Mutations in isocitrate dehydrogenase enzymes (IDH1/2) were recently described in several cancers, including conventional and dedifferentiated chondrosarcomas. These mutations lead to the inability of IDH to convert isocitrate into ?-ketoglutarate (?-KG). Instead, ?-KG is reduced into D-2-hydroxyglutarate (D-2HG), an oncometabolite. IDH mutations and D-2HG are thought to contribute to tumorigenesis due to the role of D-2HG as a competitive inhibitor of ?-KG-dependent dioxygenases. However, the function of IDH mutations in chondrosarcomas has not been clearly defined. In this study, we knocked out mutant IDH1 (IDH1mut) in two chondrosarcoma cell lines using the CRISPR/Cas9 system. We observed that D-2HG production, anchorage-independent growth, and cell migration were significantly suppressed in the IDH1mut knockout cells. Loss of IDH1mut also led to a marked attenuation of chondrosarcoma formation and D-2HG production in a xenograft model. In addition, RNA-Seq analysis of IDH1mut knockout cells revealed downregulation of several integrin genes, including those of integrin alpha 5 (ITGA5) and integrin beta 5 (ITGB5). We further demonstrated that deregulation of integrin-mediated processes contributed to the tumorigenicity of IDH1-mutant chondrosarcoma cells. Our findings showed that IDH1mut knockout abrogates chondrosarcoma genesis through modulation of integrins. This suggests that integrin molecules are appealing candidates for combinatorial regimens with IDH1mut inhibitors for chondrosarcomas that harbor this mutation.
Project description:Somatic mutations in cytosolic or mitochondrial isoforms of isocitrate dehydrogenase (IDH1 or IDH2, respectively) contribute to oncogenesis via production of the metabolite 2-hydroxyglutarate (2HG). Isoform-selective IDH inhibitors suppress 2HG production and induce clinical responses in patients with IDH1- and IDH2-mutant malignancies. Despite the promising activity of IDH inhibitors, the mechanisms that mediate resistance to IDH inhibition are poorly understood. Here, we describe four clinical cases that identify mutant IDH isoform switching, either from mutant IDH1 to mutant IDH2 or vice versa, as a mechanism of acquired clinical resistance to IDH inhibition in solid and liquid tumors. SIGNIFICANCE: IDH-mutant cancers can develop resistance to isoform-selective IDH inhibition by "isoform switching" from mutant IDH1 to mutant IDH2 or vice versa, thereby restoring 2HG production by the tumor. These findings underscore a role for continued 2HG production in tumor progression and suggest therapeutic strategies to prevent or overcome resistance.This article is highlighted in the In This Issue feature, p. 1494.
Project description:Canonical mutations in IDH1 and IDH2 produce high levels of the R-enantiomer of 2-hydroxyglutarate (R-2HG), which is a competitive inhibitor of ?-ketoglutarate (?KG)-dependent enzymes and a putative oncometabolite. Mutant IDH1 collaborates with HoxA9 to induce monocytic leukemia in vivo. We used two mouse models and a patient-derived acute myeloid leukemia xenotransplantation (PDX) model to evaluate the in vivo transforming potential of R-2HG, S-2HG and ?KG independent of the mutant IDH1 protein. We show that R-2HG, but not S-2HG or ?KG, is an oncometabolite in vivo that does not require the mutant IDH1 protein to induce hyperleukocytosis and to accelerate the onset of murine and human leukemia. Thus, circulating R-2HG acts in a paracrine manner and can drive the expansion of many different leukemic and preleukemic clones that may express wild-type IDH1, and therefore can be a driver of clonal evolution and diversity. In addition, we show that the mutant IDH1 protein is a stronger oncogene than R-2HG alone when comparable intracellular R-2HG levels are achieved. We therefore propose R-2HG-independent oncogenic functions of mutant IDH1 that may need to be targeted in addition to R-2HG production to exploit the full therapeutic potential of IDH1 inhibition.
Project description:Cancer-associated isocitrate dehydrogenase (IDH) mutations produce the metabolite 2-hydroxyglutarate (2HG), but the clinical utility of 2HG has not been established. We studied whether 2HG measurements in acute myeloid leukemia (AML) patients correlate with IDH mutations, and whether diagnostic or remission 2HG measurements predict survival. Sera from 223 de novo AML patients were analyzed for 2HG concentration by reverse-phase liquid chromatography-mass spectrometry. Pretreatment 2HG levels ranged from 10 to 30?000 ng/mL and were elevated in IDH-mutants (median, 3004 ng/mL), compared to wild-type IDH (median, 61 ng/mL) (P < .0005). 2HG levels did not differ among IDH1 or IDH2 allelic variants. In receiver operating characteristic analysis, a discriminatory level of 700 ng/mL optimally segregated patients with and without IDH mutations, and on subsequent mutational analysis of the 13 IDH wild-type samples with 2HG levels >700 ng/mL, 9 were identified to have IDH mutations. IDH-mutant patients with 2HG levels >200 at complete remission had shorter overall survival compared to 2HG ?200 ng/mL (hazard ratio, 3.9; P = .02). We establish a firm association between IDH mutations and serum 2HG concentration in AML, and confirm that serum oncometabolite measurements provide useful diagnostic and prognostic information that can improve patient selection for IDH-targeted therapies.