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

胞嘧啶衍生物5-氮杂胞苷和地西他滨（5-氮杂脱氧胞苷）作为DNA甲基转移酶的抑制剂，越来越多地用于髓性白血病的治疗。然而，临床耐药性经常被观察到，这些药物的扩展作用模式仍然缺乏特征。在第一个资助期，我们使用基于阵列的甲基化分析来分析髓细胞分化和髓细胞白血病细胞用5-氮胞苷、地西他滨和地西他滨治疗期间的基因组甲基化模式。我们还相当详细地研究了另一个5-氮杂胞苷药物靶点tRNA甲基转移酶DNMT2的功能。进一步的工作集中在细胞摄取5-氮杂胞苷所需因素的表征上，这导致了5-氮杂胞苷耐药骨髓白血病细胞系的建立。这些细胞具有许多有趣的特征，包括稳定的整体DNA低甲基化和DNMT2蛋白表达的强烈抑制。我们现在将鉴定和表征介导耐药骨髓白血病细胞系5-氮杂胞苷耐药的细胞因子。我们还将进一步分析在5-氮杂胞苷耐药细胞中观察到的整体低甲基化，并表征RNA甲基转移酶DNMT2在5-氮杂胞苷耐药中的作用。最后，我们在临床前模型中的发现将通过在第一个资助期建立的合作在临床样本中得到验证。总之，我们的研究结果将为5-氮杂胞苷耐药机制提供详细的见解，并为患者分层确定新的候选生物标志物。

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.