Exploring Experimental Hematology: June 2021 (Volume 98)

DNA methylation therapy joins forces in IDH2-mutant AML

Isocitrate dehydrogenases 1 and 2 (IDH1/2) are frequently mutated in Acute Myeloid Leukemia (AML), with nearly 20% of patients carrying gain-of-function point mutations in these genes (Ley et al., 2013). IDH2 is a metabolic enzyme that catalyzes the conversion of isocitrate to 2-oxoglutarate during the Krebs cycle. Patients carrying IDH2 gain-of-function mutations produce instead high levels of the oncometabolite 2-hydroxyglutarate (2-HG), which inhibits oxoglutarate-dependent enzymes such as the TET family of methylcytosine dioxygenases, responsible for active DNA demethylation (Xu et al., 2011). As a consequence, IDH2 mutations in AML patients induce DNA hypermethylation and inhibit hematopoietic differentiation (Figueroa et al., 2010).

Azacytidine (AZA) and enasidenib (ENA) are commonly used AML therapies which induce DNA hypomethylation, albeit through different mechanisms. AZA is a nucleoside analog that inhibits DNA methyltransferase enzymes (DNMTs) and ENA impairs IDH2 mutant catalytic activity (Wang et al., 2013). In a study recently published in Experimental Hematology (MacBeth et al., 2021), Macbeth and colleagues hypothesized that combining both drugs in IDH2-mutant leukemia will produce a synergistic effect to drive hypomethylation and restore AML differentiation.

To test this hypothesis, the authors first performed combination or single agent treatments on a leukemia cell line model overexpressing IDH2 mutation (TF1 IDH2^R140Q cells) and evaluated dose-dependent erythroid differentiation via a hemoglobinization assay. Compared to single agent treatments, combination treatment with high AZA and ENA doses showed the highest rate of hemoglobinization.

Their findings were further strengthen using primary cells from AML patients carrying IDH2^R172 and IDH2^R140 mutations. Differentiation was assessed by flow cytometry using CD34⁺ (marking stem and progenitor cells) and CD15⁺ (marking granulomonocytic cells). Combination treatment showed a reduction in CD34 and increased CD15 intensity, indicative of myeloid differentiation, an effect that was not detected in IDH2-WT AML samples.

Next, they assessed genome-wide methylation profiles of AZA+ENA treatment on TF1 IDH2^R140Q cells. 5hmC levels were evaluated using hydroxymethylated DNA immunoprecipitation (hMeDIP) sequencing, showing an increase in 5hmC levels in ENA treated and ENA+AZA treated cells, with no measurable effects in AZA-only treated cells. Subsequently, 5mC was measured using EERBS (Enhanced Representation Bisulfite Sequencing), showing a decrease in both single-agent AZA and combination treatment, but no difference in single-agent ENA, concordant with the known mechanism of action of this IDH2 inhibitor. Overall, ENA+AZA combination showed a greater degree of DNA hypomethylation than both single-agent treatments alone.  These findings support a model in which restoration of TET activity cooperates with the inhibition of DNMTs to cooperatively induce DNA hypomethylation in leukemic cells.

Further mechanistic studies would be needed to elucidate the precise molecular mechanisms of AZA+ENA synergistic effect, such the identification of methylation patterns at specific genomic regions and functional regulatory elements responsible for the increased myeloid differentiation. DNA hypomethylation agents are widely used in AML therapy, although only a modest proportion of patients respond. Therefore, resolving the underlying mechanism of action of DNA methylation therapies and designing new rational combinations to enhance response rates continues to be a promising avenue for AML treatment.

 

Figueroa, M. E., Abdel-Wahab, O., Lu, C., Ward, P. S., Patel, J., Shih, A., Li, Y., Bhagwat, N., Vasanthakumar, A., Fernandez, H. F., Tallman, M. S., Sun, Z., Wolniak, K., Peeters, J. K., Liu, W., Choe, S. E., Fantin, V. R., Paietta, E., Löwenberg, B., . . . Melnick, A. (2010). Leukemic IDH1 and IDH2 mutations result in a hypermethylation phenotype, disrupt TET2 function, and impair hematopoietic differentiation. Cancer Cell, 18(6), 553-567. https://doi.org/10.1016/j.ccr.2010.11.015

Ley, T. J., Miller, C., Ding, L., Raphael, B. J., Mungall, A. J., Robertson, A., Hoadley, K., Triche, T. J., Jr., Laird, P. W., Baty, J. D., Fulton, L. L., Fulton, R., Heath, S. E., Kalicki-Veizer, J., Kandoth, C., Klco, J. M., Koboldt, D. C., Kanchi, K. L., Kulkarni, S., . . . Eley, G. (2013). Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. N Engl J Med, 368(22), 2059-2074. https://doi.org/10.1056/NEJMoa1301689

MacBeth, K. J., Chopra, V. S., Tang, L., Zheng, B., Avanzino, B., See, W. L., Schwickart, M., Figueroa, M. E., Quek, L., & DiMartino, J. F. (2021). Combination of azacitidine and enasidenib enhances leukemic cell differentiation and cooperatively hypomethylates DNA. Exp Hematol, 98, 47-52.e46. https://doi.org/10.1016/j.exphem.2021.03.003

Wang, F., Travins, J., DeLaBarre, B., Penard-Lacronique, V., Schalm, S., Hansen, E., Straley, K., Kernytsky, A., Liu, W., Gliser, C., Yang, H., Gross, S., Artin, E., Saada, V., Mylonas, E., Quivoron, C., Popovici-Muller, J., Saunders, J. O., Salituro, F. G., . . . Yen, K. E. (2013). Targeted Inhibition of Mutant IDH2 in Leukemia Cells Induces Cellular Differentiation. Science, 340(6132), 622-626. https://doi.org/doi:10.1126/science.1234769

Xu, W., Yang, H., Liu, Y., Yang, Y., Wang, P., Kim, S. H., Ito, S., Yang, C., Wang, P., Xiao, M. T., Liu, L. X., Jiang, W. Q., Liu, J., Zhang, J. Y., Wang, B., Frye, S., Zhang, Y., Xu, Y. H., Lei, Q. Y., . . . Xiong, Y. (2011). Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of alpha-ketoglutarate-dependent dioxygenases. Cancer Cell, 19(1), 17-30. https://doi.org/10.1016/j.ccr.2010.12.014

Authored by: Alba Rodriguez-Meira, PHD, ISEH Publications Committee and Efosa Enoma, DFCI, Harvard

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