Genomic studies in acute myeloid leukemias (AML) have identified mutations which drive altered DNA methylation, including TET2 and IDH2. Here we show that models of AMLs resulting from TET2 or IDH2 mutations combined with FLT3ITD mutations are sensitive to 5-Azacytidine or to the IDH2 inhibitor AG-221, respectively. 5-Azacytidine and AG-221 treatment induced an attenuation of aberrant DNA methylation and transcriptional output, and resulted in a reduction in leukemic blasts consistent with anti-leukemic activity. These therapeutic benefits were associated with restoration of leukemic cell differentiation, and the normalization of hematopoiesis was derived from mutant cells. By contrast, combining AG-221 or 5-Azacytidine with FLT3 inhibition resulted in a reduction in mutant allele burden, progressive recovery of normal hematopoiesis from non-mutant stem-progenitor cells, and reversal of dysregulated DNA methylation and transcriptional output. Together, our studies suggest combined targeting of signaling and epigenetic pathways can increase therapeutic response in AML.
Point mutations in isocitrate dehydrogenase (IDH) define distinct subsets of acute myelogenous leukemia (AML). IDH is a metabolic enzyme that interconverts isocitrate and α-ketoglutarate (α-KG), but cancer-associated point mutations in IDH1 and IDH2 confer a neomorphic activity that allows reduction of α-KG to the oncometabolite R-2-hydroxyglutarate (2-HG). High levels of 2-HG have been shown to inhibit α-KG-dependent dioxygenases including histone and DNA demethylases, which play a key role in regulating the epigenetic state of cells, but the relationship between 2-HG and oncogenesis is not completely understood. Consistent with 2-HG promoting cancer via an effect on chromatin structure, patients harboring IDH mutations display a CpG island methylator phenotype (CIMP) and several studies have shown that overexpression of IDH mutant enzymes can induce histone and DNA hypermethylation as well as block cellular differentiation. In addition, mice engineered to express IDH1-R132H in hematopoietic tissue have increased early hematopoietic progenitors, splenomegaly, anemia, hypermethylated histones and altered DNA methylation patterns similar to those found in AML patients harboring IDH1/2 mutations.[i] Taken together, these data suggest that cancer-associated IDH mutations may induce a block in cellular differentiation to promote tumorigenesis. To investigate whether selective pharmacological inhibition of the mutant IDH1 enzyme could provide an effective way to lower intracellular 2-HG levels and restore normal differentiation, we treated TF-1 cells or primary human AML patient samples expressing mutant IDH1 with AG-120, an oral, selective, first-in-class, potent IDH1 mutant inhibitor currently in phase I clinical trials. Treatment with AG-120 decreased intracellular 2-HG levels, inhibited growth factor independent proliferation and restored erythropoietin (EPO)-induced differentiation in TF-1 IDH1-R132H cells. Similarly, pharmacological inhibition of mutant IDH1 enzyme with AG-120 in primary human blast cells cultured ex vivo provided an effective way to lower intracellular 2-HG levels and induced myeloid differentiation. Taken together, these data demonstrate that AG-120 is effective at lowering 2-HG levels and restoring cellular differentiation, and support further clinical development of this compound. Figure 1: Diagnosis and karyotypes of primary AML patient samples used in ex vivo studies Figure 1:. Diagnosis and karyotypes of primary AML patient samples used in ex vivo studies PB = peripheral blood, BM = bone marrow Figure 2: Percent 2-HG remaining relative to DMSO control after 6-day treatment with AG-120 in IDH1 R132H or IDH1 R132C patient samples Figure 2:. Percent 2-HG remaining relative to DMSO control after 6-day treatment with AG-120 in IDH1 R132H or IDH1 R132C patient samples or following 6 days of treatment with control (DMSO) or AG-120 (0.5, 1.0, and 5.0 μM) Figure 3: Relative proportion of cell types in human AML bone marrow samples untreated Figure 3:. Relative proportion of cell types in human AML bone marrow samples untreated [i] M. Sasaki et al., IDH1(R132H) mutation increases murine haematopoietic progenitors and alters epigenetics. Nature 488(7413):656-9, 2012. Disclosures Hansen: Agios Pharmaceuticals: Employment, Stockholder Other. Quivoron: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. Straley:Agios Pharmaceuticals: Employment, Stockholder Other. Lemieux:Agios Pharmaceuticals: Employment, Stockholder Other, US20130190249 (pending) Patents & Royalties. Popovici-Muller:Agios Pharmaceuticals: Employment, Stockholder Other. Fathi:Agios Pharmaceuticals: Advisory board participation Other. Gliser:Agios Pharmaceuticals: Employment, Stockholder 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. 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. Dorsch:Agios Pharmaceuticals: Employment, Stockholder Other. Yang:Agios Pharmaceuticals: Employment, Stockholder Other. Su:Agios Pharmaceuticals: Employment, Stockholder Other. Agresta:Agios Pharmaceuticals: Employment, Stockholder Other. de Botton:AGIOS: Grant Other. 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. Yen:Agios Pharmaceuticals: Employment, Stockholder Other.
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. Disclosures Intlekofer: 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.
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. Disclosures Quivoron: 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.
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