IDH1-mutated Acute Myeloid Leukemia Research Articles

Isocitrate Dehydrogenase Mutations in Human Cancers: Physiopathologic Mechanisms and Therapeutic Targeting

Isocitrate dehydrogenase (IDH) is a metabolic enzyme responsible for the enzymatic conversion of isocitrate to α-ketoglutarate (α-KG). Mutations in the IDH gene result in a novel gain-of-function, with development of neomorphic enzymatic activity determining the pathological reduction of α-KG to (R)-2-hydroxyglutarate. The accumulation of this pathological metabolite (onco-metabolite) in cancer cells is, to a large extent, responsible for the development of several cancers, including acute myeloid leukemia (AML), low-grade gliomas (LGGs) or chondrocytic tumors. Furthermore, various experimental studies have shown that IDH mutations represent an early, driver event, conserved during tumor progression in neoplasias such AML and LGG. Given all these observations, potent and selective IDH inhibitors have been developed and are currently under investigation in phase I/II clinical studies. In particular, AG-221, a first-in-class inhibitor of mutant IDH2, was tested in hematological patients with refractory/relapsing AML or myelodysplasia and showed an overall response rate of 59/159 (37%), as well as a good safety profile. Similarly, AG-120, an inhibitor of mutant IDH1, was tested in 66 relapsing/refractory AML patients and showed an overall response rate of 36%, with a complete response rate of 16%. A new IDH inhibitor, AG-811 displayed the capacity to inhibit both mutants IDH1/2 and to penetrate the blood: brain barrier, a property that would be suitable for treatment of glioma patients. On the other hand, additional observations have suggested that IDH-mutant AMLs are sensitive to treatment with BCL-2 inhibitors and to the differentiative induction with all-trans retinoic acid. In conclusion, the collective studies carried out in recent years on the characterization of IDH-mutant tumors highlight an admirable paradigm of the virtuosic transfer from basic research (with improvements in our understanding of the physio-pathological role played by IDH mutations in the development of some tumors) to clinical studies (with the development of selective, potent and clinically-active IDH inhibitors).

The oncometabolite R-2-hydroxyglutarate activates NF-κB-dependent tumor-promoting stromal niche for acute myeloid leukemia cells.

Mutations of isocitrate dehydrogenase 1 (IDH1) and IDH2 in acute myeloid leukemia (AML) cells produce the oncometabolite R-2-hydroxyglutarate (R-2HG) to induce epigenetic alteration and block hematopoietic differentiation. However, the effect of R-2HG released by IDH-mutated AML cells on the bone marrow microenvironment is unclear. Here, we report that R-2HG induces IκB kinase-independent activation of NF-κB in bone marrow stromal cells. R-2HG acts via a reactive oxygen species/extracellular signal-regulated kinase (ERK)-dependent pathway to phosphorylate NF-κB on the Thr254 residue. This phosphorylation enhances the interaction of NF-κB and the peptidyl-prolyl cis-trans isomerase PIN1 and increases the protein stability and transcriptional activity of NF-κB. As a consequence, R-2HG enhances NF-κB-dependent expression of cytokines including IL-6, IL-8 and complement 5a to stimulate proliferation of AML cells. In addition, R-2HG also upregulates vascular endothelial adhesion molecule 1 and CXCR4 in stromal cells to enhance the contact between AML and stromal cells and attenuates chemotherapy-induced apoptosis. More importantly, we validated the R-2HG-activated gene signature in the primary bone marrow stromal cells isolated from IDH-mutated AML patients. Collectively, our results suggest that AML cell-derived R-2HG may be helpful for the establishment of a supportive bone marrow stromal niche to promote AML progression via paracrine stimulation.

Mutant IDH1 Downregulates ATM and Alters DNA Repair and Sensitivity to DNA Damage Independent of TET2

Mutations in the isocitrate dehydrogenase-1 gene (IDH1) are common drivers of acute myeloid leukemia (AML) but their mechanism is not fully understood. It is thought that IDH1 mutants act by inhibiting TET2 to alter DNA methylation, but there are significant unexplained clinical differences between IDH1- and TET2-mutant diseases. We have discovered that mice expressing endogenous mutant IDH1 have reduced numbers of hematopoietic stem cells (HSCs), in contrast to Tet2 knockout (TET2-KO) mice. Mutant IDH1 downregulates the DNA damage (DD) sensor ATM by altering histone methylation, leading to impaired DNA repair, increased sensitivity to DD, and reduced HSC self-renewal, independent of TET2. ATM expression is also decreased in human IDH1-mutated AML. These findings may have implications for treatment of IDH-mutant leukemia.

Molecular Pathways: Isocitrate Dehydrogenase Mutations in Cancer.

IDH1 and IDH2 are homodimeric enzymes that catalyze the conversion of isocitrate to α-ketoglutarate (α-KG) and concomitantly produce reduced NADPH from NADP(+) Mutations in the genes encoding IDH1 and IDH2 have recently been found in a variety of human cancers, most commonly glioma, acute myeloid leukemia (AML), chondrosarcoma, and intrahepatic cholangiocarcinoma. The mutant protein loses its normal enzymatic activity and gains a new ability to produce the "oncometabolite" R(-)-2-hydroxyglutarate (R-2-HG). R-2-HG competitively inhibits α-KG-dependent enzymes which play crucial roles in gene regulation and tissue homeostasis. Expression of mutant IDH impairs cellular differentiation in various cell lineages and promotes tumor development in cooperation with other cancer genes. First-generation inhibitors of mutant IDH have entered clinical trials, and have shown encouraging results in patients with IDH-mutant AML. This article summarizes recent progress in our understanding of the role of mutant IDH in tumorigenesis.Clin Cancer Res; 22(8); 1837-42. ©2016 AACR.

Pretreatment d-2-hydroxyglutarate serum levels negatively impact on outcome in IDH1-mutated acute myeloid leukemia

Mutations in isocitrate dehydrogenases (IDHs) 1 and 2 frequently occur in acute myeloid leukemia (AML) and result in the production of the oncometabolite d-2-hydroxyglutarate (D2HG). D2HG has been shown to promote leukemogenesis even in the absence of mutated IDH, but the prognostic significance of pretreatment serum D2HG levels in patients with IDH-mutated AML is unclear. We measured D2HG serum levels in 84 patients with IDH-mutated AML treated in the prospective, randomized multicenter AML2003 trial of the German Study Alliance Leukemia. Multivariate Cox regression showed D2HG levels to negatively impact on event-free survival (EFS) as a continuous variable in the entire IDH(mut) cohort (P=0.04), with no effect on overall survival (OS). In a subgroup analysis, the negative impact of D2HG on EFS was found to be restricted to patients with mutations in IDH1 (P=0.003), adjusted for age, leukocyte count, serum lactate dehydrogenase and European LeukemiaNet risk score. We thus conclude that pretreatment D2HG serum levels may yield prognostic information in patients with IDH1-mutated, but not in IDH2-mutated AML, possibly due to different subcellular localizations of IDH1 and IDH2.

Abstract A2-66: Integrative analysis of gene expression and metabolism in isocitrate dehydrogenase 1 mutant acute myeloid leukemia and myeloid knock in mouse model

Abstract Isocitrate dehydrogenase 1 (IDH1) mutations are common drivers of acute myeloid leukemia (AML). While mutant IDH1 is known to disrupt DNA methylation, gene expression, and metabolism in cell lines, it is not known how mutant IDH1 alters global metabolism in vivo. The purpose of this study is to find differential enzymes and metabolites in IDH1 wild type versus mutant AML and knock in mice by integrating differential genes and metabolites in a metabolic network. Differential genes were found by comparing gene expression and DNA methylation in IDH1 mutant (n = 12) and wild type (n = 89) cytogenetically normal AML patients from The Cancer Genome Atlas. Differential metabolites were found by untargeted liquid chromatography-mass spectrometry of urine, plasma, and bone marrow hematopoietic stem and progenitor cells from unpaired 1-2 year old mixed-sex IDH1 myeloid knock in (n = 13) and control mice (n = 14). Integrative analysis was performed using the MetScape 2 Cytoscape plugin. 71 differential genes and 52 differential metabolites were mapped to the global metabolic network. The set of differential genes and metabolites formed 10 connected subnetworks containing reactions in sphingolipid, phospholipid, and bile acid metabolism. Two enzymes in the subnetworks were differentially expressed in an independent IDH1 mutant versus wild type AML dataset: acyl-CoA thioesterase 4 (ACOT4) and galactosylceramidase (GALC) (P < 0.05, false discovery rate ≤ 0.3). ACOT4 and GALC were significantly upregulated (P < 0.02) in acute promyelocytic leukemia cells treated with all-trans retinoic acid. In conclusion, we have found novel differential genes and metabolites in IDH1 mutant systems which may be relevant to leukemia differentiation treatment. Integrative analysis of gene expression and metabolite levels could be useful for finding dysregulated metabolic subnetworks in cancer. Citation Format: Alan Tseng, Wanda Li, Kim-Chung Lee, Karrie Yung, Ching-Wan Lam, Tak Mak. Integrative analysis of gene expression and metabolism in isocitrate dehydrogenase 1 mutant acute myeloid leukemia and myeloid knock in mouse model. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-66.

Abstract B1-18: Integrative analysis of gene expression and metabolism in isocitrate dehydrogenase 1 mutant acute myeloid leukemia and myeloid knock in mouse model

Abstract Isocitrate dehydrogenase 1 (IDH1) mutations are common drivers of acute myeloid leukemia (AML). While mutant IDH1 is known to disrupt DNA methylation, gene expression, and metabolism in cell lines, it is not known how mutant IDH1 alters global metabolism in vivo. The purpose of this study is to find differential enzymes and metabolites in IDH1 wild type versus mutant AML and knock in mice by integrating differential genes and metabolites in a metabolic network. Differential genes were found by comparing gene expression and DNA methylation in IDH1 mutant (n = 12) and wild type (n = 89) cytogenetically normal AML patients from The Cancer Genome Atlas. Differential metabolites were found by untargeted liquid chromatography-mass spectrometry of urine, plasma, and bone marrow hematopoietic stem and progenitor cells from unpaired 1-2 year old mixed-sex IDH1 myeloid knock in (n = 13) and control mice (n = 14). Integrative analysis was performed using the MetScape 2 Cytoscape plugin. 71 differential genes and 52 differential metabolites were mapped to the global metabolic network. The set of differential genes and metabolites formed 10 connected subnetworks containing reactions in sphingolipid, phospholipid, and bile acid metabolism. Two enzymes in the subnetworks were differentially expressed in an independent IDH1 mutant versus wild type AML dataset: acyl-CoA thioesterase 4 (ACOT4) and galactosylceramidase (GALC) (P < 0.05, false discovery rate ≤ 0.3). ACOT4 and GALC were significantly upregulated (P < 0.02) in acute promyelocytic leukemia cells treated with all-trans retinoic acid. In conclusion, we have found novel differential genes and metabolites in IDH1 mutant systems which may be relevant to leukemia differentiation treatment. Integrative analysis of gene expression and metabolite levels could be useful for finding dysregulated metabolic subnetworks in cancer. Citation Format: Alan Tseng, Wanda Li, Kim-Chung Lee, Karrie Yung, Ching-Wan Lam, Tak Mak. Integrative analysis of gene expression and metabolism in isocitrate dehydrogenase 1 mutant acute myeloid leukemia and myeloid knock in mouse model. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr B1-18.

AG-221, an Oral, Selective, First-in-Class, Potent IDH2-R140Q Mutant Inhibitor, Induces Differentiation in a Xenotransplant Model

Somatic point mutations in the active site of IDH (isocitrate dehydrogenase) 1 and IDH2 genes are observed in acute myeloid leukemia (AML). These mutations lead to the production and accumulation of R-2-hydroxyglutarate (2-HG) in the tumor blast cells as well as in the plasma of patients. High levels of 2-HG have been shown to inhibit alpha-ketoglutarate-dependent dioxygenases, including epigenetic regulators (i.e. histone or DNA demethylases), resulting in altered cellular differentiation. Ex vivo experiments in primary cells from AML IDH2 mutant patients have demonstrated that IDH2 mutant inhibitors are able to revert this phenotype. In order to test the biological activity of AG-221, an oral, reversible and selective inhibitor of mutated IDH2 currently in phase I trials, we developed primary human AML xenograft models.Primary blast cells from 3 AML patients with normal karyotype (n=3), NPM1 mutation (n=3) and FLT3ITD (n=1) were injected into immunodeficient NOD SCID IL2R gamma null (NSG) mice by intrafemoral injection after sub-lethal irradiation. Medullar and peripheral tumoral engraftment was monitored by flow cytometry (using species-specific antibodies) and serum 2-HG measurements. Engraftment of IDH2-R140Q human blast cells was not lethal, but these AML cells persisted over time. Twelve months after injection, IDH2-R140Q blast cell-engrafted mice were randomized into two groups, for treatment with AG-221 (30 mpk) or vehicle (0.5% methylcellulose / 0.2% Tween 80, in water) (n=10 mice; 5 mice per condition). Treatments were administered by oral gavage for 38 days twice per day (BID). Peripheral blood was collected at multiple time points for the determination of pharmacokinetics (PK) and pharmacodynamics (PD), and the assessment of the differentiation effects by AG-221 on human tumor cells.PK/PD analyses showed good plasma exposure of AG-221 and reduction of 2-HG. Furthermore, AG-221 administration also induced a burst of proliferation of human blasts followed by myeloid differentiation starting at day 20 in peripheral blood as measured by the expression of CD11b, CD14, CD15, CD24 and cell morphology. No effects on proliferation or differentiation were seen in the absence of AG-221 administration. Histological analyses of hematopoietic organs in treated animals showed a decrease of infiltrating human cells, as well as obvious morphological changes in the human cell population in AG-221-treated animals compared with vehicle-treated animals. Flow cytometry confirmed the differentiation of the human cells in the spleen and bone marrow of the AG-221-treated mice. Furthermore, cells undergoing differentiation retained the R140Q mutation, demonstrating the differentiating effect of the compound.In conclusion, AG-221 reduced serum 2-HG levels and triggered differentiation of leukemic blast cells engrafted in NSG mice. These results are consistent with what has been observed clinically in IDH2 mutant AML patients treated with AG-221 in a phase I dose escalation trial. DisclosuresQuivoron:Institut National de la Santé Et de la Recherche Médicale (INSERM): Grant Other; Association Laurette Fugain: Grant, Grant Other; Institut National du Cancer (INCa): Grant, Grant Other; Association pour la recherche contre le Cancer (ARC): Grant, Grant Other; AGIOS: Grant Other. David:Institut National de la Santé Et de la Recherche Médicale (INSERM): Grant Other; Institut National du Cancer (INCa): Grant, Grant Other; Association pour la Recherche contre le Cancer (ARC): Grant, Grant Other; Association Laurette Fugain: Grant, Grant Other; AGIOS: Grant Other. Straley:Agios Pharmaceuticals: Employment, Stockholder Other. Travins:Agios Pharmaceuticals: Employment, Stockholder Other. Kim:Agios Pharmaceuticals: Employment, Stockholder Other. Chen:Agios Pharmaceuticals: Employment, Stockholder Other. Zhu:Agios Pharmaceuticals: Employment, Stockholder Other. Bawa:AGIOS: Grant Other. Bernard:Institut National de la Santé Et de la Recherche Médicale (INSERM): Grant Other; Association Laurette Fugain: Grant, Grant Other; Institut National du Cancer (INCa): Grant, Grant Other; Ligue Nationale contre le cancer (LNCC): Grant, Grant Other; AGIOS: Grant Other. Yang:Agios Pharmaceuticals: Employment, Stockholder Other. Agresta:Agios Pharmaceuticals: Employment, Stockholder Other. de Botton:AGIOS: Grant Other. Yen:Agios: Employment. Penard-Lacronique:Institut National de la Santé Et de la Recherche Médicale (INSERM): Grant Other; Association Laurette Fugain: Grant, Grant Other; Institut National du Cancer (INCa): Grant, Grant Other; Association pour la recherche contre le Cancer (ARC): Grant, Grant Other; AGIOS: Grant Other.

AG-221, a Small Molecule Mutant IDH2 Inhibitor, Remodels the Epigenetic State of IDH2-Mutant Cells and Induces Alterations in Self-Renewal/Differentiation in IDH2-Mutant AML Model in Vivo

Somatic mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) are observed in patients with acute myeloid leukemia (AML). Leukemia-associated IDH1/2 mutations result in aberrant accumulation of the oncometabolite 2-hydroxyglutarate (2-HG). The observation that IDH1/2 mutations are mutually exclusive with TET2 mutations led to the finding that IDH1/2-mutant production of 2-HG inhibits TET2 function and induces changes in DNA methylation. These data suggested that small molecule inhibition of mutant IDH enzymes might reverse the aberrant epigenetic remodeling of IDH-mutant leukemia cells and restore normal hematopoietic differentiation.We therefore investigated the in vivo efficacy of AG-221, a potent and selective mutant IDH2 inhibitor in early-phase clinical trials, in murine models of IDH2-mutant leukemia. We first assessed the impact of AG-221 on 2-HG production in hematopoietic cells expressing mutant IDH2-R140Q. AG-221 treatment (10mg/kg or 100mg/kg bid) led to a reduction in 2-HG in vivo (96.7% below pre-treatment levels). Moreover, AG-221 treatment restored megakaryocyte-erythroid progenitor (MEP) differentiation that is suppressed by mutant IDH2 expression in vivo (mean MEP% mean, 39% Veh vs 50% AG-221).We next investigated the impact of mutant IDH2 inhibition with AG-221 on DNA methylation in vivo. We used eRRBS, a bisulfite-based next-generation sequencing platform, to assess the effect of AG-221 therapy on DNA methylation. AG-221 or vehicle therapy treated LSK stem cells (lin- Sca+ c-Kit+) were sorted from mice expressing IDH2-R140Q and evaluated by eRRBS. AG-221 therapy reversed the effects of mutant IDH2; we observed a significant reduction in DNA methylation, including 180 genes that had 20 or more hypomethylated differentially methylated cytosines (DMCs) following treatment. 84 of these genes had reduced methylation at 10 or more DMCs in the gene promoter with AG-221 therapy compared to vehicle. Mutant IDH2 inhibition with AG-221 reversed aberrant methylation at many genes with a known role in hematopoietic proliferation and differentiation, including the master transcriptional factor RUNX1.We next assessed in vivo effects of the small-molecule IDH2-R140Q inhibitor in a mouse model of IDH2-mutant leukemia. We generated mice that simultaneously expressed a constitutive Flt3ITD knock-in allele and a conditional mutant IDH2R140Q knock-in allele. As reported recently using retroviral/transgenic models, Mx1-Cre IDH2R140QFlt3ITD developed fully penetrant, transplantable AML with expansion of c-Kit+ positive blasts in the peripheral blood, and widespread leukemic infiltration. AG-221 inhibited the serial replating capacity of IDH2R140QFlt3ITD expressing cells in vitro. We competitively transplanted IDH2R140QFlt3ITD AML cells and normal bone marrow cells into secondary recipients, and then assessed the effect of AG-221 therapy on leukemia in vivo and on disease burden. AG-221 (100mg/kg bid) treatment of mice engrafted with Mx1-Cre IDH2R140QFlt3ITD AML cells markedly reduced 2HG levels consistent with on target inhibition in vivo. AG-221 therapy induced differentiation of leukemic cells, with an increase in the CD11b+ population and a decrease in the c-Kit+ population in the peripheral blood at 2wks. We next assessed the impact of treatment with both AG-221 therapy with AC220, a potent, specific Flt3 inhibitor in late phase clinical trials. Combined IDH2R140Q and Flt3ITD inhibition resulted in a marked decrease in leukemic burden to vehicle-treated mice, with a significant reduction in leukemic cell chimerism in vivo in the setting of combined inhibition at 2 wks, (mean 45.2 fraction 88% veh, 73% AG-221, p<.01).These data demonstrate that AG-221 inhibits mutant IDH2-mediated 2-HG production in vivo and reverses the effects of mutant IDH2 on DNA methylation in mutant stem/progenitor cells. AG-221 induces differentiation and impairs self-renewal of IDH2-mutant leukemia cells, effects that are further enhanced by simultaneous inhibition of Flt3ITD. Clinical trials combining IDH2 inhibitors with other targeted AML therapies are warranted in order to increase therapeutic efficacy. DisclosuresIntlekofer:Foundation Medicine, Inc: Consultancy. Thompson:Agios: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Travins:Agios Pharmaceuticals: Employment, Stockholder Other. Straley:Agios: Employment, Equity Ownership. Gliser:Agios Pharmaceuticals: Employment, Stockholder Other. Yen:Agios: Employment, Equity Ownership. Levine:Agios: Consultancy, Membership on an entity’s Board of Directors or advisory committees.

Efficacy of Novel Glutaminase Inhibitor CB-839 in Acute Myeloid Leukemia

Inhibition of glutaminase (GLS), the principal enzyme in the glutamine utilization pathway that coverts glutamine (Gln) to glutamate (Glu), is an attractive therapeutic approach in many cancers. Gln plays a unique role in the metabolism of proliferating cancer cells, providing building blocks to sustain cell proliferation and regulating redox homeostasis and signal transduction pathways. Recent findings indicate that leukemic cells depend on Gln as a major carbon source for growth and survival [Willems et al., Blood, 2013]. We previously reported that a subset of acute myeloid leukemia (AML) cell lines are sensitive to Gln deprivation as well as inhibition of glutaminase by the small molecule BPTES [Matre et al. ASH 2013 #606]. Here we report the efficacy of CB-839, a novel, potent, orally bioavailable GLS inhibitor currently under clinical investigation, in the Gln-dependent subset of AML.First, expression of GLS gene splice variants glutaminase C (GAC) and kidney glutaminase (KGA) and of the GLS2 gene was determined through analysis of RNA sequencing data from 173 newly diagnosed AML patients in the TCGA dataset. Of the GLS gene splice variants, the expression levels of GAC were much higher than those of KGA; GLS2 was expressed at low levels. Levels of both GAC and KGA mRNA were significantly higher (two-sample Wilcoxon test) in AML patients with complex cytogenetics and monosomal karyotype (n=31) than in those with diploid AML (n=88, p=0.019 and p=0.01); GAC levels were higher in core-binding factor AML (n=14) than in diploid AML (p=0.018). These findings indicate high expression of the GLSGAC splice variant in specific AML subsets.Analysis of a panel of AML cell lines showed that, in a subset of leukemia cells, CB-839 treatment decreased viable cell number and induced apoptosis. In sensitive cell lines (Molm14, OCI-AML3, MV4;11), CB-839 decreased viable cell number with IC50s between 10nM and 100nM and induced significant apoptosis. HL60, MOLM13, KG1α, and OCI-AML2 cells were less sensitive (IC50 100-1000nM) and responded with minor induction of cell death. CB-839 decreased viability by >40% in blasts from 9 of 20 (45%) primary AML samples.GC- or LC-MS metabolic profiling of OCI-AML3 and THP1 cell lines as well as primary patient samples revealed that GLS inhibition by BPTES or CB-839 was accompanied by concomitant decrease in concentration of downstream GLS metabolites such as glutamate, α-ketoglutarate (a-KG), aspartate, fumarate, and malate. Investigation of the effects of CB-839 on mitochondrial OXPHOS by the Seahorse Bioscience XF96 Analyzer showed that CB-839 exposure for 16 h caused a dose-dependent decrease in maximal respiratory capacity in OCI-AML3 cells, indicating reduced availability of the substrates for OXPHOS. Similar results were obtained upon treatment with BPTES and in AML cells stably transduced with GLS shRNA.Gln, through Glu, is a precursor for cellular α-KG, which can undergo further metabolism through the Krebs cycle or be further metabolized to 2-hydroxyglutarate (2-HG) by mutant isocitrate dehydrogenase (IDH). In THP1 cell lines stably transduced with doxycycline-inducible mutant IDH1-R132H or IDH2-R140Q construct, CB-839 exposure for 4 days reduced intracellular 2-HG oncometabolite levels by >50%. This was associated with induction of differentiation marker CD11b and morphological signs of differentiation in CB-839–treated IDH2-R140Q cells [30%±2% increase in CD11b mean fluorescent intensity (p<0.001)] vs untreated cells; but not in IDH2-WT control cells. Further, IDH2-R140Q THP1 cells were significantly more sensitive to 1mM CB-839 than IDH2-WT cells (61% vs 24% reduction in viable cell numbers). In 4 of 6 IDH1- or IDH2-mutated primary samples, reductions in 2-HG (by 24% [p=0.04]; 31% [p=0.016]; 35% [p=0.033], and 43% [p=0.0056]) were observed upon CB-839 exposure. Preliminary data for an IDH2-R140Q-mutated AML primary sample (n=1) indicate that CB-839 promotes CD11b differentiation in primary AML blasts.In summary, these results indicate that GLS is a relevant therapeutic target in AML, warranting future inclusion of GLS inhibitors in the armamentarium of multi-agent therapeutic approaches. In particular, reduction of production of the oncometabolite 2-HG in conjunction with therapeutic blockade of Gln metabolism may serve as a tailored therapeutic strategy in IDH-mutated AML cells. DisclosuresKonopleva:Calithera Biosciences: Research Funding.

BCL-2 Inhibition As a Synthetic Lethal Approach To Target Isocitrate Dehydrogenase Mutations In Acute Myeloid Leukemia Stem Cells

Mutations in isocitrate dehydrogenase (IDH) 1 and 2 occur in about 15% of acute myeloid leukemia (AML) patients. Studies of paired samples at diagnosis and relapse have demonstrated that IDH mutations, in contrast to FLT3 or RAS mutations, are stable during disease evolution. This finding indicates that a small population of cells harboring IDH mutations can persist in remission and eventually contribute to relapse. Therapeutic strategies that eradicate this IDH-mutated population have the potential to result in long-term remission.Small molecule inhibitors specific for the mutant IDH enzymes have recently been developed (Wang et al Science 2013), but it is not known if they are effective in targeting primary AML cells including the leukemic stem cell (LSC) compartment in vivo. We sought an alternative approach to target IDH-mutated cells based on the concept of non-oncogene addiction which refers to the increased dependence on a subset of non-mutated genes for survival in response to activation of a specific oncogene. This dependency can be exploited therapeutically by inhibiting the activity of these non-oncogenes resulting in selective elimination of malignant cells, a phenomenon known as synthetic lethality (SL).In an effort to identity SL targets against IDH mutations, we performed a pooled lentiviral RNA interference (RNAi) screen to search for genes that, when inhibited, led to the selective elimination of mutant IDH1 expressing cells. Our lentiviral short hairpin RNA (shRNA) library consisted of 27,500 unique shRNAs targeting 5,043 human genes of relevance to cancer biology. Each shRNA was tagged with a unique barcode sequence which permitted downstream identification by sequencing. The lentiviral shRNA library was transduced into a human AML cell line engineered to express mutant IDH1 (R132H) under the control of a doxycycline-inducible promoter. Following transduction, the cells were cultured in doxycycline to deplete shRNAs that were synthetic lethal to mutant IDH1. The relative abundance of each shRNA was subsequently determined by high-throughput sequencing of the barcode. Using a stringent algorithm designed to minimize false positive hits, the prosurvival gene BCL-2 was identified as one of the top SL hits. We confirmed that RNAi-mediated knockdown of BCL-2 expression was selectively lethal to mutant IDH expressing AML cell lines and further demonstrated that exposure to a cell-permeable form of (R)-2-hydroxyglutarate, the oncometabolite produced by mutant IDH, was sufficient to induce BCL-2 dependence. Similarly, pharmacologic inhibition of BCL-2 with ABT-199, a novel orally bioavailable and highly specific inhibitor of BCL-2 (Souers et al Nature Medicine 2013), was significantly more toxic to mutant IDH expressing AML cell lines than isogenic cell lines with wildtype (WT) IDH.We next investigated the impact of IDH mutation status on ABT-199 sensitivity of primary AML cells and found that FACS-purified blasts with IDH mutations were 10-fold more sensitive to ABT-199 than blasts with WT IDH in ex vivo culture conditions. Normal cord blood hematopoietic stem and progenitor cells were highly resistant to ABT-199 treatment ex vivo suggestive of a wide therapeutic index. To demonstrate in vivo activity, we treated immunodeficient NOD/SCID/IL2Rγ-null (NSG) mice engrafted with primary human IDH-mutated leukemic cells with either ABT-199 at a dose of 100 mg/kg/day or vehicle control for 7 consecutive days by oral administration. Bone marrow engraftment analysis before and after treatment showed a 10 to 20-fold reduction in leukemic burden in ABT-199 treated mice, whereas no difference was seen in vehicle-treated mice. Importantly, bone marrow cells collected from ABT-199 treated mice failed to engraft in secondary transplant recipients indicative of a loss of LSC activity. In separate experiments, lentiviral transduction of BCL-2 shRNA vectors into IDH-mutated primary AML cells to knockdown BCL-2 expression impaired their engraftment in NSG mice, further validating the detrimental effect of BCL-2 inhibition on IDH-mutated LSCs.In summary, our results indicate that IDH mutations increase BCL-2 dependence in leukemic cells including LSCs and identify a subgroup of patients that is likely to respond to pharmacologic BCL-2 inhibition. Our data provide the preclinical rationale for investigating the use ABT-199 in this patient subgroup in clinical trials. Disclosures:No relevant conflicts of interest to declare.

Cancer-associated IDH2 mutants drive an acute myeloid leukemia that is susceptible to Brd4 inhibition

Somatic mutations in the isocitrate dehydrogenase (IDH) genes IDH1 and IDH2 occur frequently in acute myeloid leukemia (AML) and other cancers. These genes encode neomorphic proteins that produce the presumed oncometabolite 2-hydroxyglutarate (2-HG). Despite the prospect of treating AML and other cancers by targeting IDH mutant proteins, it remains unclear how these mutants affect tumor development and maintenance in vivo, and no cancer models exist to study the action of IDH2 mutants in vivo. We show that IDH2 mutants can cooperate with oncogenic Flt3 or Nras alleles to drive leukemia in mice by impairing the differentiation of cells of the myeloid lineage. Pharmacologic or genetic inhibition of IDH2 triggers the differentiation and death of AML cells, albeit only with prolonged IDH2 inhibition. In contrast, inhibition of the bromodomain-containing protein Brd4 triggers rapid differentiation and death of IDH2 mutant AML. Our results establish a critical role for mutant IDH2 in leukemogenesis and tumor maintenance and identify an IDH-independent strategy to target these cancers therapeutically.

Glutaminase Inhibition Selectively Slows the Growth of Primary Acute Myeloid Leukemia (AML) Cells with Isocitrate Dehydrogenase (IDH) Mutations.

AbstractAbstract 2624 Introduction:The incidence of mutations in IDH1 and IDH2 (mIDH) in de novo AML is 10–15%. These mutations are enriched in normal karyotype AML, and their presence carries an unfavorable prognostic factor, according to some studies. Furthermore, mutations in IDH1/2 genes have been identified in approximately 5% of myelodysplastic syndromes and 10% of myeloproliferative neoplasms. Although wild-type IDH in cytosol and mitochondria catalyze the conversion of isocitrate to α-ketoglutarate (α-KG) with the production of NADPH, altered amino acids in mIDH1 and mIDH2 reside in the catalytic pocket and result in a neoenzymatic activity, converting α-KG to 2-hydroxyglutarate with the consumption of NADPH. The primary source for α-KG for these cells is glutamine, which is first converted to glutamate by glutaminase and subsequently to α-KG. Because glutamine is the primary source for α-KG, we hypothesized that cells with mIDH are in essence addicted to glutamine via glutaminase activity, such that depletion of glutamine or interruption of its metabolism would be deleterious to cellular metabolism and survival. The aim of this study was to investigate whether inhibition of glutaminase by a small molecule, BPTES (bis-2-[5-(phenylacetamido)-1,3,4-thiadiazol-2-yl]ethyl sulfide), could selectively kill primary AML cells with mIDH1, but not IDH-wild type AML cells. We and others have previously demonstrated that BPTES inhibits glutaminase effectively. Method:Two independent sets of experiments were performed by two separate research groups. One group was blinded to mutant versus wild type IDH status. The other group was blinded to drug identity including solvent versus BPTES and to various BPTES concentrations. Primary AML cells from patients were cultured in RPMI-1640 medium with 20% fetal bovine serum, 20% 5637 cell-conditioned medium and 1% antibiotics with no drug, DMSO control (0.1% concentration) and 20 or 40 microM BPTES. Cells were counted manually on days 2, 4 and 6. Growth curves were generated for viable cells as assessed by trypan blue exclusion. Experiments were performed in triplicates. Results:Growth curves of primary AML cells (with mutation status indicated) with no drug and with DMSO or BPTES (20 or 40 microM) are shown in Figure 1. Cells #2, #3, #5 and #10 carried IDH1 mutations. Cells #4 and #9 were wild type. On day 4, there was approximately a two-fold decrease in the growth of all IDH-mutant AML cells exposed to 20 microM BPTES compared to DMSO. No significant difference in activity was observed between 20 and 40 microM of BPTES. There was no difference in cell growth between exposure to no drug and to DMSO. The growth of wild type AML cells was not significantly affected by the glutaminase inhibitor. Results were consistent between the two research groups. Conclusions:Although IDH mutations are frequently found in AML, a therapeutic strategy targeted at these mutations has not been reported. To the best of our knowledge, this is the first report of a targeted approach to the treatment of IDH-mutant AML. We found that inhibition of glutaminase by a small molecule, BPTES, preferentially slows the growth of primary AML cells with mutant IDH1 versus those AML cells with wild type IDH. Further investigation in xenograft models and pharmacologic studies are ongoing. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Prospective serial evaluation of 2-hydroxyglutarate, during treatment of newly diagnosed acute myeloid leukemia, to assess disease activity and therapeutic response

Mutations of genes encoding isocitrate dehydrogenase (IDH1 and IDH2) have been recently described in acute myeloid leukemia (AML). Serum and myeloblast samples from patients with IDH-mutant AML contain high levels of the metabolite 2-hydroxyglutarate (2-HG), a product of the altered IDH protein. In this prospective study, we sought to determine whether 2-HG can potentially serve as a noninvasive biomarker of disease burden through serial measurements in patients receiving conventional therapy for newly diagnosed AML. Our data demonstrate that serum, urine, marrow aspirate, and myeloblast 2-HG levels are significantly higher in IDH-mutant patients, with a correlation between baseline serum and urine 2-HG levels. Serum and urine 2-HG, along with IDH1/2-mutant allele burden in marrow, decreased with response to treatment. 2-HG decrease was more rapid with induction chemotherapy compared with DNA-methyltransferase inhibitor therapy. Our data suggest that serum or urine 2-HG may serve as noninvasive biomarkers of disease activity for IDH-mutant AML.

Prospective evaluation of serial 2-hydroxyglutarate in acute myeloid leukemia (AML) to determine response to therapy and predict relapse.

6606 Background: Mutations in isocitrate dehydrogenase (IDH1 and IDH2) occur in 10-20% of AML patients and result in production of 2-hydroxyglutarate (2-HG). We hypothesize that serial 2-HG quantification may be used to monitor response and predict relapse in patients with AML. Methods: We are conducting a prospective study at MGH and DFCI to (1) assess changes in serum and urine 2-HG levels during treatment in patients with newly diagnosed AML, (2) compare 2-HG levels in serum, urine, and bone marrow and, (3) serially compare 2-HG levels with IDH1/2mutant allele burden. In those with elevated serum 2-HG (≥1000ng/ml), 2-HG is monitored serially or at relapse. Results: To date, 20 patients have been enrolled, 5 (25%) of whom had elevated baseline serum 2-HG. All 5 were found to have IDH1/2 mutations. The serum 2-HG for these patients was significantly higher than for those who were IDH-wt (median 1933 vs 87ng/mL; p&lt;0.001). Urine 2-HG was also higher in IDH-mutant patients (median 30500 vs 4230ng/mL; p&lt;0.021), as was bone marrow 2-HG (median 9870 vs 309ng/mL; p&lt;0.005). Serum 2-HG levels strongly correlated with that in urine (R2 0.987). Serum 2-HG decreased in all IDH mutant patients on therapy, but more rapidly in those receiving induction chemotherapy (Table), all of whom achieved remission, than those receiving hypomethylatingagents. Conclusions: Patients with IDH-mutant AML have markedly elevated serum and urine 2-HG. 2-HG levels decreased with therapy and there is a strong correlation between serum and urine 2-HG. Data on the sensitivity of serial 2-HG monitoring as well as comparison with mutant IDH1/2allele burden will be presented. This data suggests that serial 2-HG quantification may be a valuable non-invasive biomarker in this genetically defined subset of AML. [Table: see text]

AML patients with IDH1 or IDH2 mutations treated with hypomethylating agents: A case series.

6576 Background: Isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations play a significant role in acute myeloid leukemia (AML), yet optimal treatment for AML patients with IDH mutations remains unclear. IDH mutations have been associated with a globally hypermethylated genomic state, as the result of increased levels of 2-HG, a regulator of histone methylation. We sought to retrospectively determine responses of patients with IDH-mutated AML to treatment with hypomethylating agents. Methods: We reviewed clinical data for AML patients treated at The University of Texas M. D. Anderson Cancer Center from June 2001 to December 2011 whose stored leukemia samples from bone marrow aspirateshad been tested retrospectively for IDH1 and IDH2 mutations. We searched three databases that contained records for 407 AML patients. We selected patients who had been treated with the hypomethylating agents 5-azacitidine or decitabine, alone or in conjunction with other therapeutics. We retrospectively reviewed the patients’ medical records to obtain demographic, laboratory, treatment, and clinical data. We evaluated response measured by remission status, reduction in bone marrow blasts and peripheral blood blasts, and time to relapse. Results: We identified 104 patients (25.6%) who had either an IDH1 or IDH2 mutation, and of these, 8 patients had also been treated with hypomethylating agents. A ninth AML patient, who is IDH2-mutant, is currently undergoing treatment with decitabine alone. Of these 9 patients, three had a durable complete remission without recovery of blood counts (CRp), three had a partial response (PR), with or without blood count recovery, and three had no response. All 9 patients did have an initial decrease in the percentage of bone marrow blasts, peripheral blood blasts, or both. Conclusions: This series of patients demonstrates the possibility that AML patients with IDH1 or IDH2 mutations benefit from the use of hypomethylating agents, and suggests it is worthwhile to prospectively investigate treatment of patients with IDH-mutated AML using hypomethylating agents.

Some novel features of IDH1-mutated acute myeloid leukemia revealed in Chinese patients

Mutations of isocitrate dehydrogenase 1 ( IDH1) have recently been reported in acute myeloid leukemia (AML). However, the characteristics of IDH1-mutated AML are still not known clearly. We analyzed 416 Chinese AML patients and found 28 patients (6.7%) carried this mutation. One homozygous IDH1 mutant in AML was found. The IDH1 mutations were associated with NPM1 mutations ( P = 0.043) and could coexist with recurrent transcription factor aberrations including AML1-ETO (6/50), PML-RARα (3/77) and CBFβ-MYH11 (1/15). For AML with AML1-ETO fusion gene, IDH1 mut patients may have worse disease-free survival (DFS) than IDH1 wild-type patients.