Encoding Isocitrate Dehydrogenase Research Articles

Clonal heterogeneity of acute myeloid leukemia treated with the IDH2 inhibitor enasidenib.

Mutations in the gene encoding isocitrate dehydrogenase 2 (IDH2) occur in several types of cancer, including acute myeloid leukemia (AML). In model systems, mutant IDH2 causes hematopoietic differentiation arrest. Enasidenib, a selective small-molecule inhibitor of mutant IDH2, produces a clinical response in 40% of treated patients with relapsed/refractory AML by promoting leukemic cell differentiation. Here, we studied the clonal basis of response and acquired resistance to enasidenib treatment. Using sequential patient samples, we determined the clonal structure of hematopoietic cell populations at different stages of differentiation. Before therapy, IDH2-mutant clones showed variable differentiation arrest. Enasidenib treatment promoted hematopoietic differentiation from either terminal or ancestral mutant clones; less frequently, treatment promoted differentiation of nonmutant cells. Analysis of paired diagnosis/relapse samples did not identify second-site mutations in IDH2 at relapse. Instead, relapse arose by clonal evolution or selection of terminal or ancestral clones, thus highlighting multiple bypass pathways that could potentially be targeted to restore differentiation arrest. These results show how mapping of clonal structure in cell populations at different stages of differentiation can reveal the response and evolution of clones during treatment response and relapse.

IDH1-mutated transgenic zebrafish lines: An in-vivo model for drug screening and functional analysis.

IntroductionThe gene encoding isocitrate dehydrogenase 1 (IDH1) is frequently mutated in several tumor types including gliomas. The most prevalent mutation in gliomas is a missense mutation leading to a substitution of arginine with histidine at the residue 132 (R132H). Wild type IDH1 catalyzes oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG) whereas mutant IDH1 converts α-KG into D2-hydroxyglutarate (D2HG). Unfortunately, there are few in vivo model systems for IDH-mutated tumors to study the effects of IDH1 mutations in tumor development. We have therefore created transgenic zebrafish lines that express various IDH1 mutants.Materials and methodsIDH1 mutations (IDH1R132H, IDH1R132C and loss-of-function mutation IDH1G70D), IDH1wildtype or eGFP were cloned into constructs with several brain-specific promoters (Nestin, Gfap or Gata2). These constructs were injected into fertilized zebrafish eggs at the one-cell stage.ResultsIn total more than ten transgenic zebrafish lines expressing various brain-specific IDH1 mutations were created. A significant increase in the level of D2HG was observed in all transgenic lines expressing IDH1R132C or IDH1R132H, but not in any of the lines expressing IDH1wildtype, IDH1G70D or eGFP. No differences in 5-hydroxymethyl cytosine and mature collagen IV levels were observed between wildtype and mutant IDH1 transgenic fish. To our surprise, we failed to identify any strong phenotype, despite increased levels of the oncometabolite D2HG. No tumors were observed, even when backcrossing with tp53-mutant fish which suggests that additional transforming events are required for tumor formation. Elevated D2HG levels could be lowered by treatment of the transgenic zebrafish with an inhibitor of mutant IDH1 activity.ConclusionsWe have generated a transgenic zebrafish model system for mutations in IDH1 that can be used for functional analysis and drug screening. Our model systems help understand the biology of IDH1 mutations and its role in tumor formation.

Durable Remissions with Ivosidenib in IDH1-Mutated Relapsed or Refractory AML

BackgroundMutations in the gene encoding isocitrate dehydrogenase 1 (IDH1) occur in 6 to 10% of patients with acute myeloid leukemia (AML). Ivosidenib (AG-120) is an oral, targeted, small-molecule inhibitor of mutant IDH1.MethodsWe conducted a phase 1 dose-escalation and dose-expansion study of ivosidenib monotherapy in IDH1-mutated AML. Safety and efficacy were assessed in all treated patients. The primary efficacy population included patients with relapsed or refractory AML receiving 500 mg of ivosidenib daily with at least 6 months of follow-up.ResultsOverall, 258 patients received ivosidenib and had safety outcomes assessed. Among patients with relapsed or refractory AML (179 patients), treatment-related adverse events of grade 3 or higher that occurred in at least 3 patients were prolongation of the QT interval (in 7.8% of the patients), the IDH differentiation syndrome (in 3.9%), anemia (in 2.2%), thrombocytopenia or a decrease in the platelet count (in 3.4%), and leukocytosis (in 1.7%). In the primary efficacy population (125 patients), the rate of complete remission or complete remission with partial hematologic recovery was 30.4% (95% confidence interval [CI], 22.5 to 39.3), the rate of complete remission was 21.6% (95% CI, 14.7 to 29.8), and the overall response rate was 41.6% (95% CI, 32.9 to 50.8). The median durations of these responses were 8.2 months (95% CI, 5.5 to 12.0), 9.3 months (95% CI, 5.6 to 18.3), and 6.5 months (95% CI, 4.6 to 9.3), respectively. Transfusion independence was attained in 29 of 84 patients (35%), and patients who had a response had fewer infections and febrile neutropenia episodes than those who did not have a response. Among 34 patients who had a complete remission or complete remission with partial hematologic recovery, 7 (21%) had no residual detectable IDH1 mutations on digital polymerase-chain-reaction assay. No preexisting co-occurring single gene mutation predicted clinical response or resistance to treatment.ConclusionsIn patients with advanced IDH1-mutated relapsed or refractory AML, ivosidenib at a dose of 500 mg daily was associated with a low frequency of grade 3 or higher treatment-related adverse events and with transfusion independence, durable remissions, and molecular remissions in some patients with complete remission. (Funded by Agios Pharmaceuticals; ClinicalTrials.gov number, NCT02074839.)

Mutant-IDH1-dependent chromatin state reprogramming, reversibility, and persistence.

Mutations in IDH1 and IDH2 (encoding isocitrate dehydrogenase 1 and 2) drive the development of gliomas and other human malignancies. Mutant IDH1 induces epigenetic changes that promote tumorigenesis, but the scale and reversibility of these changes are unknown. Here, using human astrocyte and glioma tumorsphere systems, we generate a large-scale atlas of mutant-IDH1-induced epigenomic reprogramming. We characterize the reversibility of the alterations in DNA methylation, the histone landscape, and transcriptional reprogramming that occur following IDH1 mutation. We discover genome-wide coordinate changes in the localization and intensity of multiple histone marks and chromatin states. Mutant IDH1 establishes a CD24+ population with a proliferative advantage and stem-like transcriptional features. Strikingly, prolonged exposure to mutant IDH1 results in irreversible genomic and epigenetic alterations. Together, these observations provide unprecedented high-resolution molecular portraits of mutant-IDH1-dependent epigenomic reprogramming. These findings have substantial implications for understanding of mutant IDH function and for optimizing therapeutic approaches to targeting IDH-mutant tumors.

Abstract 4170: A transgenic zebrafish model for gliomas with mutations in isocitrate dehydrogenase 1

Abstract The gene encoding Isocitrate dehydrogenase 1 (IDH1) is frequently mutated in gliomas, chondrosarcomas, acute myeloid leukemia and intrahepatic cholangiocarcinomas. As there are few in-vivo model systems for IDH-mutated tumors we have created a transgenic zebrafish (Danio rerio) model expressing mutant IDH1. We have chosen the zebrafish as a model because they are transparent (allowing monitoring of the transgene in-vivo) and drug screening assays are straightforward (they are simply added to the aquarium). IDH1R132H and IDH1R132C, mutations found in tumors that both produce D-2-hydroxyglutarate (D2HG) instead of alpha ketoglutarate, were cloned into an expression construct that is driven either by the Nestin or GFAP promoter. IDH1G70D (a loss of function mutation), IDH1wildtype and GFP were used as control. All IDH1 constructs were fused to GFP for visualization. These constructs were injected into fertilized zebrafish eggs at the one-cell stage. All of our transgenic zebrafish lines remain healthy and produce offspring. Transgene expression was detected in the mid/hindbrain of the central nervous system by immunohistochemistry, Western blot and RT-QPCR. A significant increase in the level of D2HG was observed in all transgenic lines expressing IDH1R132C or IDH1R132H, but not in any of the lines expressing control constructs (IDH1wildtype, IDH1G70D or GFP). In contrast to reported, we failed to detect any differences in hydroxymethyl cytosine (the first step in DNA-demethylation) and mature collagen IV levels between wildtype and mutant IDH1 transgenic fish. We also performed microinjections on fertilized eggs to screen for early developmental effects of IDH1R132H and IDH1R132C. Despite of the high expression of the transgene, no developmental effects were found. Our observations therefore suggest that elevated levels of D2HG are insufficient to initiate tumorigenesis or other phenotypic effects in our fish. Treatment of the transgenic zebrafish with an IDH1 mutant inhibitor, AGI-5198, resulted in a reduction in the D2HG level in the mutant zebrafish. The L2HG level was not affected by AGI-5198. As no tumors were formed in our transgenic zebrafish lines, we backcrossed them with Tp53 mutant fish. Analysis of these lines is currently being performed. In summary, we have generated a transgenic zebrafish model system that expresses mutated IDH1 that can be used to study effects of mutant IDH1 (or elevated levels of D2HG) in vivo and can be used for drug screening. Citation Format: Ya D. Gao, Maurice de Wit, Eduard A. Struys, Martine L.M. Lamfers, Gajja S. Salomons, Peter A.E. Sillevis Smitt, Pim J. French. A transgenic zebrafish model for gliomas with mutations in isocitrate dehydrogenase 1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4170.

World Health Organization grade II–III astrocytomas consist of genetically distinct tumor lineages

Recent investigations revealed genetic analysis provides important information in management of gliomas, and we previously reported grade II–III gliomas could be classified into clinically relevant subgroups based on the DNA copy number aberrations (CNAs). To develop more precise genetic subgrouping, we investigated the correlation between CNAs and mutational status of the gene encoding isocitrate dehydrogenase (IDH) of those tumors. We analyzed the IDH status and CNAs of 174 adult supratentorial gliomas of astrocytic or oligodendroglial origin by PCR‐based direct sequencing and comparative genomic hybridization, respectively. We analyzed the relationship between genetic subclassification and clinical features. We found the most frequent aberrations in IDH mutant tumors were the combined whole arm‐loss of 1p and 19q (1p/19q codeletion) followed by gain on chromosome arm 7q (+7q). The gain of whole chromosome 7 (+7) and loss of 10q (−10q) were detected in IDH wild‐type tumors. Kaplan–Meier estimates for progression‐free survival showed that the tumors with mutant IDH, −1p/19q, or +7q (in the absence of +7p) survived longer than tumors with wild‐type IDH, +7, or −10q. As tumors with +7 (IDH wild‐type) showed a more aggressive clinical nature, they are probably not a subtype that developed from the slowly progressive tumors with +7q (IDH mutant). Thus, tumors with a gain on chromosome 7 (mostly astrocytic) comprise multiple lineages, and such differences in their biological nature should be taken into consideration during their clinical management.

Chapter 23 - Glioblastoma

Glioblastoma is the most common and aggressive primary brain tumor in adults. Defining histopathologic features are necrosis and endothelial proliferation, resulting in the assignment of grade IV, the highest grade in the World Health Organization (WHO) classification of brain tumors. The classic clinical term "secondary glioblastoma" refers to a minority of glioblastomas that evolve from previously diagnosed WHO grade II or grade III gliomas. Specific point mutations of the genes encoding isocitrate dehydrogenase (IDH) 1 or 2 appear to define molecularly these tumors that are associated with younger age and more favorable outcome; the vast majority of glioblastomas are IDH wild-type. Typical molecular changes in glioblastoma include mutations in genes regulating receptor tyrosine kinase (RTK)/rat sarcoma (RAS)/phosphoinositide 3-kinase (PI3K), p53, and retinoblastoma protein (RB) signaling. Standard treatment of glioblastoma includes surgery, radiotherapy, and alkylating chemotherapy. Promoter methylation of the gene encoding the DNA repair protein, O(6)-methylguanyl DNA methyltransferase (MGMT), predicts benefit from alkylating chemotherapy with temozolomide and guides choice of first-line treatment in elderly patients. Current developments focus on targeting the molecular characteristics that drive the malignant phenotype, including altered signal transduction and angiogenesis, and more recently, various approaches of immunotherapy.

Integrated genomic characterization of IDH1-mutant glioma malignant progression.

Gliomas represent approximately 30% of all central nervous system tumors and 80% of malignant brain tumors. To understand the molecular mechanisms underlying the malignant progression of low-grade gliomas with mutations in IDH1 (encoding isocitrate dehydrogenase 1), we studied paired tumor samples from 41 patients, comparing higher-grade, progressed samples to their lower-grade counterparts. Integrated genomic analyses, including whole-exome sequencing and copy number, gene expression and DNA methylation profiling, demonstrated nonlinear clonal expansion of the original tumors and identified oncogenic pathways driving progression. These include activation of the MYC and RTK-RAS-PI3K pathways and upregulation of the FOXM1- and E2F2-mediated cell cycle transitions, as well as epigenetic silencing of developmental transcription factor genes bound by Polycomb repressive complex 2 in human embryonic stem cells. Our results not only provide mechanistic insight into the genetic and epigenetic mechanisms driving glioma progression but also identify inhibition of the bromodomain and extraterminal (BET) family as a potential therapeutic approach.

Control of carbon flux to glutamate excretion in Klebsiella pneumoniae: the role of the indigenous plasmid and its encoded isocitrate dehydrogenase.

Klebsiella pneumoniae (NCTC, CL687/80) harbors a large indigenous plasmid (p(C3)), which in addition to encoding for citrate utilization, proline synthesis and glutamate excretion, it uniquely carries the structural gene (icd); encoding isocitrate dehydrogenase (ICDH). Flux analysis revealed that ICDH, despite its role in the generation of NADPH required for glutamate dehydrogenase, is not rate-limiting (controlling) in central metabolism as evidenced by a negative flux control coefficient and an adverse effect of overexpression (14-fold) on glutamate excretion. More significantly, however, this paper presents, for the first time, clear evidence that the accumulation of glutamate and its subsequent excretion is associated with the C3 plasmid-encoded regulatory elements, which trigger a shift-down in the activity of α-ketoglutarate dehydrogenase, both in the K. pneumoniae parental strain as well as in the E. coli exconjugants strains. This finding opens the door for the exploitation of regulatory elements as a tool for manipulating flux in microbial cell factories.

A synthetic lethal approach targeting mutant isocitrate dehydrogenase in acute myeloid leukemia.

Pathogenic mutations of the genes encoding isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) occur in people with acute myeloid leukemia or other tumors. A new study identifies a dependence of IDH-mutated cells on the anti-apoptosis regulator BCL-2 and indicates a 'synthetic lethal' strategy for the treatment of leukemias.

Diagnostic Features and 2-Hydroxyglutarate (2-HG) Levels Among Acute Myeloid Leukemia (AML) Patients with and without Isocitrate Dehydrogenase (IDH) Mutations

Mutations in genes encoding isocitrate dehydrogenase 1/2 (IDH 1/2) enzymes result in increased 2-hydroxyguterate (2-HG) levels, which may provide a non-invasive marker of disease in IDH-mutant AML. The purpose of this study was to characterize patients with IDH-mutant AML by assessing presenting features, concurrent mutations, and 2-HG levels.From 7-2011 through 6-2014, we identified 170 consecutive patients with newly diagnosed AML and measured 2-HG by liquid chromatography-tandem mass spectrometry in serum, urine, marrow aspirate, and marrow pellet samples. All patients had IDH1 R132, IDH2 R140 and R172 testing; 169/170 had hotspot mutational profiling for AKT1, APC, BRAF, CTNNB1, EGFR, ERBB2, KIT, KRAS, MAP2K1,NOTCH1, NRAS, PIK3CA, P53, and PTEN. We assessed FLT3 (168/170), NPM1 (168/170), CEBPA (100/170) mutational status as routine clinical care; 2 patients had BCR/ABL alterations, and 6 were JAK2 positive (of 36 tested). IDH1/2-mutant were compared to wildtype (WT) patients using a Wilcoxon rank sum test, Fisher’s exact test, or Kruskal Wallis test, as appropriate.The group was 54% male; 83% white, 2.4% black, 2.4% Asian, and 6.5% Hispanic. 12 patients had APL. IDH mutations included IDH1 R132C (n=10), IDH2 R172 (n=7), and IDH2 140Q (n=22). Other mutations included NPM1 (20.8%), FLT3-ITD (17.3%), FLT3-TKD (6.6%), NRAS (18.4%), TP53 (3%), KRAS (6.5%), and KIT (1.2%). CEBPA mutations occurred in 13 of 100 patients. IDH mutations (n=39) more frequently co-occurred with normal cytogenetics and NPM1 mutations, vs IDH-WT, consistent with prior reports (Table 1). No patients with favorable cytogenetics harbored an IDH1/2 mutation.Significantly higher 2-HG levels were detected among those with IDH1/2 mutations compared to IDH WT, in the serum (p<0.0001), urine (p<0.0001), marrow aspirate (p<0.0001) and marrow pellet (p<0.0001). Overall, elevated 2-HG levels were present regardless of type of IDH mutation. Serum and marrow pellet 2-HG levels were elevated (> 1000 ng/mL in serum and > 1000ng/2x10^6 cells in marrow pellet) in 30/38 and 24/29 IDH-mutant patients, respectively, compared to 1/129 and 5/117 IDH WT patients. All but 2 IDH-mutant patients displayed either or both an elevated marrow pellet or serum 2-HG. Patients with IDH2 R172 mutations had lower marrow 2HG levels compared to those with R140Q mutations (Figure 1, p=0.0434). The WBC and blast count was lower among IDH2 R172-mutant compared to R140Q- or IDH1 R132C-mutant patients (p= 0.0134 and p=0.0039, respectively); there was no significant difference in serum 2HG levels.All three canonical IDH1/2 mutations have higher 2HG levels compared to IDH WT. IDH2 R172 mutated AML may present with lower WBC counts and peripheral blast percentage compared to IDH1 R132C and IDH2 R140Q mutant AML. Urine and serum 2HG levels effectively identify patients with mutant IDH1/2, of particular relevance given targeted therapies for these mutations.Table 1Characteristics of IDH1/2-mutant and WT AML patients.IDH1/2 MutatedIDH1/2 WTp-valueAge, y (median, range)67 (41-86)65 (20-87)0.4718AML History (n, %)De Novo28 (72%)82 (63%)0.6336Therapy-related4 (10%)15 (11%)MDS2 (5%)17 (13%)Cytogenetic Risk (n, %)Favorable023 (18%)0.0006Intermediate30 (81%)71 (55%)Normal25 (68%)44 (34%)Poor7 (19%)34 (27%)Concurrent Mutations (n, %)NPM115 (38%)20 (16%)0.0033FLT3-ITD3 (8%)26 (20%)0.0907FLT3-TKD2 (5%)9 (7%)1.000CEBPA Heterozygous Homozygous3 (14%) 08 (10%) 2 (3%)0.8118NRAS9 (23%)22 (17%)0.4788KRAS011 (8%)0.0700KIT1 (3%)1 (1%)0.4093P531 (3%)4 (3%)1.000Table 2WBC and 2-HG levels by IDH mutation (median, range). BQL, below quantitative limit.IDH1 R132C N= 10IDH2 R172 N=7IDH2 R140Q N=22IDH WT N=131WBC count10.1 [1.8-333.2]1.6 [0.7-3.2]5.9 [0.9-122.8]6.3 [0.4-315.4]Peripheral blast %33.0 [0-98]1.0 [0-9.8]18.2 [2-91]15 [0-94]Marrow aspirate 2-HG (ng/mL)20,900 [1330-93500] (n=7)4300 [2230-26000] (n=6)30800 [BQL-311000] (n=19)BQL [BQL-22000] (n=116)Serum 2-HG (ng/mL)1807 [101-66207] (n=9)1102 [287-2908] (n=7)1464 [365.1-11696] (n=22)86.9 [BQL-1224] (n=128)Urine 2-HG (ng/mL)33850 [7260-282000] (n=10)7430 [2460-63700] (n=7)11300 [2070-177000] (n=20)3140 [BQL-53200] (n=125)Marrow pellet 2-HG (ng/2*10^6 cells)13680 [193-172,800] (n=7)7480 [1056-79600] (n=5)5560 [564-96400] (n=17)65 [BQL-4040] (n=116) [Display omitted] DisclosuresBrunner:NIH T32 CA 71345-18: Research Funding. Attar:Agios: Employment. Yen:Agios: Employment. Yang:Agios Pharmaceuticals: Employment, Stockholder Other. Straley:Agios: Employment. Agresta:Agios Pharmaceuticals: Employment, Stockholder Other. Stone:Agios: Consultancy. Fathi:Millennium: Research Funding; Seattle Genetics: Advisory Board, Advisory Board Other; Agios: Advisory Board, Advisory Board Other.

SC-19 * METABOLIC ALTERATIONS IN EARLY GLIOMAGENESIS

Gliomas are primary brain tumors with an incidence of over 20,000 new cases in the US annually, a poor prognosis and limited therapeutic options. Despite chemoradiotherapy and surgery, low-grade gliomas (LGGs) transform into high-grade gliomas, leading to neurological deterioration and death. Mutations in genes encoding Isocitrate Dehydrogenase (IDH) isoforms are found in up to 80% of LGGs. Among the three IDH genes, R132H-IDH1 represents the most prevalent mutation in LGGs. The mutation is a gain-of-function change at the catalytic site. Although IDH1 normally functions to convert isocitrate to α-ketoglutarate, the mutation leads to the production of the (R)-2-hydroxyglutarate (2HG) oncometabolite, causing characteristic DNA and histone methylation changes that are thought to contribute to tumorigenesis. To test the hypothesis that mutant IDH1 is a driver of gliomagenesis, we propose a novel approach in which we use human embryonic stem cells (hESCs) to model the tumorigenesis process: we inducibly overexpress the R132H-IDH1 protein in human neural stem cells, neuroblasts, oligodendrocyte progenitors and astrocytes that are derived from hESCs. Using lentiviral constructs and BAC-engineered hESC lines, this approach allows us to investigate the effects of 2HG on differentiation, self-renewal and transformative potential systematically along the human neuroglial axis both in vitro and in vivo. Using hESCs to understand IDH1-mediated oncogenesis is a novel strategy based on modeling a single oncogenic change in a human genetic background, potentially overcoming limitations found in current mouse models and further shedding light on early human gliomagenesis.

Abstract LB-139: IDH1 mutations alter citric acid cycle metabolism and increase dependence on oxidative mitochondrial metabolism

Abstract Mutations in the genes encoding isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in a variety of tumor types, resulting in production of the proposed oncometabolite, 2-hydroxyglutarate (2-HG). How mutant IDH alters central carbon metabolism, though, remains unclear. To address this question, we performed 13C metabolic flux analysis (MFA) on an isogenic cell panel containing heterozygous IDH1/2 mutations. We observe a dramatic and consistent decrease in the ability of IDH1, but not IDH2, mutant cell lines to utilize reductive glutamine metabolism via the carboxylation of α-ketoglutarate to isocitrate. Additionally we find that cells with IDH1 mutations exhibit increased oxidative tricarboxylic acid (TCA) metabolism. Similar metabolic trends were observed in vivo as well, and also in endogenous, non-engineered IDH1/2 mutant cell lines. Interestingly, IDH1-mutant specific inhibitors were unable to reverse the decrease in reductive metabolism, suggesting that this metabolic phenotype is independent of 2-HG. Furthermore, this metabolic reprogramming increases the sensitivity of IDH1 mutant cells to hypoxia or electron transport chain (ETC) inhibition in vitro. IDH1 mutant cells also grow poorly as subcutaneous xenografts within hypoxic in vivo microenvironments. These results suggest that exploiting metabolic defects specific to IDH1 mutant cells could be an interesting avenue to explore therapeutically. Citation Format: Alexandra R. Grassian, Seth Parker, Shawn Davidson, Ajit Divakaruni, Courtney Green, Xiamei Zhang, Kelly Slocum, Minying Pu, Fallon Lin, Chad Vickers, Carol Joud-Caldwell, Franklin Chung, Hong Yin, Erika Handly, Christopher Straub, Joseph D. Growney, Matt Vander Heiden, Anne Murphy, Raymond Pagliarini, Christian Metallo. IDH1 mutations alter citric acid cycle metabolism and increase dependence on oxidative mitochondrial metabolism. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-139. doi:10.1158/1538-7445.AM2014-LB-139

Structural Biology of Mutant IDH2 Inhibition

A number of human cancers harbor somatic point mutations in the genes encoding isocitrate dehydrogenases- 1 and -2 (IDH1, IDH2)[1]. 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). 2HG is a potent inhibitor of DNA methylating enzymes such as TET2[2]. This suggests a connection between cancer related IDH mutations and aberrant epigenetics. As such, IDH represents an important new druggable target in the pursuit of novel cancer therapies. We have 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[3]. While structures of IDH1 and IDH2 were known, this is the first ever structure of an inhibited IDH protein and shows a novel conformation of IDH2. 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 (AML) 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.

Lithium chloride decreases proliferation and migration of C6 glioma cells harboring isocitrate dehydrogenase 2 mutant via GSK-3β

The gene encoding isocitrate dehydrogenase (IDH) is somatically mutated predominantly in secondary glioblastoma multiforme. Mutations of IDH1 and IDH2 lead to simultaneous loss and gain of activities in the production of α-ketoglutarate and 2-hydroxyglutarate, respectively. Lithium chloride was recently proved efficient in inhibiting glioma cell migration. The mechanism of lithium chloride on C6 glioma cells harboring IDH2 mutation has not been studied. Here, we found lithium chloride induced inhibitive effects on cell proliferation of both C6 glioma cells with and without IDH2 mutation, although IDH2 mutation increased the stability of HIF-1α. GSK-3β could be phosphorylated at Ser9 and its activity was inhibited when C6 glioma cells were treated by lithium chloride. The degree of phosphorylation in IDH2(R172G) treatment group was lower than that as compared to the control and IDH2 treatment groups. At the same time, the accumulation of β-catenin in C6 cell nucleus was decreased. Moreover, although the β-catenin and HIF-1α increased the secretion of metalloproteinase-2,-9 in C6 glioma cells harboring IDH2 mutation, the migration potential of lithium chloride-treated C6 glioma cells harboring the IDH2 and its mutant was uniform. These results indicated lithium chloride could decrease the proliferation and migration potential of C6 glioma cells harboring IDH2 mutation.

IDH1/IDH2 but not TP53 mutations predict prognosis in Bulgarian glioblastoma patients.

Mutations in genes encoding isocitrate dehydrogenase isoforms 1 (IDH1) and 2 (IDH2) have been associated with good prognosis for patients with brain neoplasias and have been commonly found together with mutated TP53 gene. To determine the prevalence of IDH1, IDH2, and TP53 mutations and their impact on overall survival 106 glioblastoma patients were analysed. IDH1 mutations were detected in 13 and IDH2 mutation in one patient. Two homozygous samples with R132H mutation in IDH1 gene and a novel aberration K129R in IDH2 gene were found. Sixty-four percent of IDH1/IDH2 mutated tumours harboured also a mutation in TP53 gene. Genetic aberrations in TP53 were present in 37 patients. Statistical analysis of the impact of the studied factors on the overall survival showed that the mutations in IDH1/IDH2, but not the ones in TP53, were associated with longer survival. Also, the impact of age on prognosis was confirmed. This is the first comprehensive study on glioblastomas in Bulgaria. Our results suggest that IDH1/IDH2 but not TP53 mutations together with other prognostic factors such as age might be applied in clinical practice for prediction of outcome in patients with glioblastomas.

Critical Roles Of The IDH2 Mutation In Development and Maintenance Of Acute Myeloid Leukemia

Mutations in genes encoding isocitrate dehydrogenase (IDH) 1 and 2 are frequently observed in numerous cancers including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and angioimmunoblastic t-cell lymphoma (AITL). The roles of mutant IDHs in tumorigenesis remain unclear because of a lack of appropriate cancer models. Here we established a mouse AML model harboring an IDH2 mutation. IDH1/2 mutations in AML frequently occur simultaneously with mutations in other genes such as NPM, DNMT3A, and FLT3. In accordance with these observations, IDH2/R140Q, NPMc, DNMT3A/R882H and FLT3/ITD cooperatively induced AML in the mouse model. When only three out of the four mutant genes were transduced, the onset of AML was delayed in any combinations. These results clearly indicate that all four mutations are necessary for the efficient induction of AML. Gene-expression analysis indicated that IDH2/R140Q and NPMc cooperatively activate Hoxa9/Meis1 and hypoxia pathways to maintain AML cells in vivo. These two pathways are likely to be important for the IDH2/R140Q-mediated engraftment/survival of NPMc+ cells in mice. DNMT3A/R882H further upregulated the expression levels of Meis1. Furthermore, DNMT3A/R882H promoted the maintenance of cells in an undifferentiated state. Previous studies have shown that FLT3/ITD promotes cell growth and survival. Taken together, our results suggest that multiple signaling pathways are activated in this IDH-mediated AML system.The tumor-associated mutant IDHs catalyze the formation of an oncometabolite 2-hydroxyglutarate (2-HG), which dysregulates a set of α-ketoglutarate-dependent dioxygenases that includes epigenetic regulators (TETs and KDM4A), hypoxic signaling molecule (EGLN) and others (collagen prolyl 4-hydroxylases). Because mutant IDHs act via a mechanism that is completely different from those of previously described oncogenes, it has attracted increasing attention as a new therapeutic target. Small molecules that potently and selectively inhibit the mutant IDHs induced differentiation of cancer cells in vitro. However, it remains unclear whether the mutant IDHs are valid targets for cancer therapy in vivo. Here we report that AML harboring IDH2/R140Q can be blocked by conditional deletion of IDH2/R140Q, even after leukemia has developed. Deletion of IDH2/R140Q blocked 2-HG production and the maintenance of Csf-1r+ and c-Kit+ leukemia stem cells, resulting in survival of the AML mice. These results indicate that the IDH2 mutation is critical for the development and maintenance of AML stem cells, and that mutant IDHs are promising targets for anticancer therapy. Disclosures:No relevant conflicts of interest to declare.

Isocitrate dehydrogenase mutations in leukemia

Recent genome-wide discovery studies have identified a spectrum of mutations in different malignancies and have led to the elucidation of novel pathways that contribute to oncogenic transformation. The discovery of mutations in the genes encoding isocitrate dehydrogenase (IDH) has uncovered a critical role for altered metabolism in oncogenesis, and the neomorphic, oncogenic function of IDH mutations affects several epigenetic and gene regulatory pathways. Here we discuss the relevance of IDH mutations to leukemia pathogenesis, therapy, and outcome and how mutations in IDH1 and IDH2 affect the leukemia epigenome, hematopoietic differentiation, and clinical outcome.

Abstract IA20: Epigenetic deregulation of gene expression

Abstract Epigenetic deregulation of gene expression through aberrant DNA methylation or histone modification plays an important role in the malignant transformation of hematopoietic cells. In particular, acute myeloid leukemias (AMLs) can be classified according to epigenetic signatures affecting DNA methylation or histone modifications affecting specific gene sets. Heterozygous somatic mutations in the loci encoding isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in ~20% of AMLs and are accompanied by global DNA hypermethylation and hypermethylation and silencing of a number of specific gene promoters. IDH1/2 mutations are almost completely mutually exclusive with somatic loss of function mutations in TET2, which hydroxylates methylcytosine (mCpG). DNA hydroxymethylation can function as an intermediate step in mCPG demethylation. TET2 mutant de novo AMLs also display global and promoter specific hypermethylation partially overlapping with IDH1/2 mutant cases. Mutations in the IDH1/2 loci result in a neomorphic enzyme that generates the aberrant oncometabolite 2-hydroxyglutarate (2HG) using alpha-ketoglutarate (aKG) as a substrate. 2HG can disrupt the activity of enzymes that use aKG as a cofactor, including TET2 and the Jumonji family of histone demethylases. Expression of mutant IDH isoforms inhibit TET2 hydroxymethylation and Jumonji histone demethylase functions. IDH and TET2 mutant AMLs accordingly exhibit reduced levels of hydroxymethylcytosine and a trend towards reduced histone methylation. Mutant IDH or TET2 loss of function causes differentiation blockade and expansion of hematopoietic stem cells and TET2 knockout results in a myeloproliferative phenotype in mice. Hydroxymethylcytosine is abundance in hematopoietic stem cells, and displays specific distribution patterns, and yet the function of this covalent modification is not fully understood. Recent data link TET2 with the function of cytosine deaminases as a pathway towards DNA demethylation, which has implications as well for B-cell lymphomas and CML lymphoid blast crisis, which are linked with the actions of activation induced cytosine deaminase. Altogether, the available data implicate mutations in IDH1/2 and TET2 in promoting malignant transformation in several tissues, by disrupting epigenomics programming and altering gene expression patterning. Citation Format: Ari Melnick. Epigenetic deregulation of gene expression. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr IA20.

Prognostic significance of IDH mutation in adult low-grade gliomas: a meta-analysis

Mutations in the gene encoding isocitrate dehydrogenase (IDH) have been identified in approximately 65-90 % of low-grade gliomas (LGGs). Various studies examining the relationship between IDH mutation with the clinical outcome in patients with LGGs have yielded inconclusive results. The purpose of the present meta-analysis of literature is to determine this effect. We conducted a meta-analysis of 10 studies (937 patients) that evaluated the correlation between IDH mutation and overall survival (OS). For the quantitative aggregation of the survival results, the IDH mutation effect was measured by hazard ratio (HR). Overall, the pooled HR was 0.585 (95 % CI, 0.376-0.911, p = 0.025, random effect model) for patients with IDH mutation vs patients without IDH mutation. IDH mutation was associated with better overall survival of LGGs. At least this trend was observed in our analysis.