Comparative characterization of azacytidine-induced RNA and DNA demethylation in myeloid leukemia

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The cytosine derivatives 5-azacytidine and decitabine (5-aza-deoxycytidine) function as inhibitors of DNA methyltransferases and are increasingly used for the treatment of myeloid leukemias. However, clinical drug resistances are frequently observed and the extended mode of action of these drugs remains poorly characterized. During the first funding period, we have used array-based methylation profiling to analyze genomic methylation patterns during myeloid differentiation and during the treatment of myeloid leukemia cells with 5-azacytidine and decitabine and decitabine. We also investigated the function of another 5-azacytidine drug target, the tRNA methyltransferase DNMT2, in considerable detail. Further work focused on the characterization of the factors required for the cellular uptake of 5-azacytidine, which led to the establishment of 5-azacytidine-resistant myeloid leukemia cell lines. These cells were characterized by a number of interesting features, including stable global DNA hypomethylation and a strong suppression of DNMT2 protein expression. We will now identify and characterize cellular factors mediating 5-azacytidine resistance in drug-resistant myeloid leukemia cell lines. We will also further analyze the global hypomethylation observed in 5-azacytidine resistant cells and characterize the role of the RNA methyltransferase DNMT2 in 5-azacytidine resistance. Finally, our findings from preclinical models will be validated in clinical samples through collaborations that were established during the first funding period. Altogether, our results will provide detailed insight into the mechanisms of 5-azacytidine resistance and identify novel candidate biomarkers for patient stratification.