Approximately 15% of AML patients have an IDH2 mutation which leads to production of the oncometabolite 2-hydroxyglutarate (2HG). Accumulation of 2HG inhibits aKG-dependent DNA and histone demethylases, resulting in epigenetic disregulation, which in turn leads to a block in cellular differentiation, promoting AML. AG-221 is a selective inhibitor of IDH2 mutant enzyme and is in development for AML patients carrying IDH2 mutation. Clinical responses to AG-221 therapy have been observed, including decreases in blast percentages and evidence of differentiated functional blood cells; however, preliminary analysis indicated that the mutant allelic burden was not reduced in a majority of subjects treated with AG-221. Azacitidine (AZA) is another epigenetic modifying agent with clinical activity in AML. We hypothesized that combining AG-221 with AZA could synergize in releasing the differentiation block in IDH2-mutant AML cells and enhance cell killing. TF-1:IDH2R140Q are human erythroleukemia cells engineered to express R140Q-mutant IDH2 and model the differentiation block conferred by 2HG accumulation. TF-1:IDH2R140Q cells were treated with AG-221, AZA or the combination of AG-221 + AZA, and measures of cell differentiation (hemoglobinization, KLF1 and HBG qRT-PCR, CD34/CD38 flow cytometry) and death (IncuCyte Zoom caspase 3/7) were evaluated. Measures of cell differentiation were evaluated in TF-1:IDH2R140Q cells, using an in vitro erythropoietin differentiation assay. Single agent AG-221 and AZA increased heme production in a dose-dependent manner, as evidenced by increased red color of cells. With AZA + AG-221 combination, hemoglobinization was greater than with single agents. Dose-dependent increases in RNA expression of differentiation markers KLF1 and HBG were observed with single agents, and the combination resulted in additive, or greater than additive, increases. Quantification of hematopoietic stem /progenitor cell populations demonstrated that as single agents, both AG-221 and AZA reduced CD34+/CD38+ and CD34+/CD38- cell populations, and the combination resulted in additive or greater than additive decreases. Real-time quantification of cell death showed that single agent AG-221 had no effect, while single agent AZA increased apoptosis. Concurrent combination of AZA + AG-221 increased cell death beyond that of single agents. Whole genome expression data (RNA-seq) showed enrichment of differentiation and cell death gene signatures with the combination when compared to single agents alone. We have demonstrated the beneficial effects of combining AG-221 + AZA in the TF-1:IDH2R140Q AML cell line, including greater than additive increases in hemoglobinization and expression of differentiation markers, reduced stem /progenitor cell populations, and potentiation of death. Further exploration of the molecular mechanism of the combination is ongoing.

Citation Format: Vivek S. Chopra, Brian Avanzino, Konstantinos Mavrommatis, Adam Olshen, Jorge DiMartino, Kyle J. MacBeth. Functional characterization of combining epigenetic modifiers azacitidine and AG-221 in the TF-1:IDH2R140Q AML model. [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 2280.