The significance of DNA Methyltransferase inhibition and DNA Methylation for therapy response in Acute Myeloid Leukemia

急性髓性白血病（AML）与表观基因组变化相关，包括DNA甲基化的深刻改变。这些变化的起源和相关性并不完全清楚。尽管进行了强化化疗，但大多数患者无法通过当前疗法治愈。DNA甲基转移酶（DNMT）抑制剂，如地西他滨和氮杂胞苷是没有治愈性的，其作用机制还不完全清楚。在第一个资助期内，我们使用还原型亚硫酸氢盐测序（RRBS）来鉴定DNMT 3B活性增加的小鼠白血病和急性早幼粒细胞白血病（APL）患者中DNA甲基化模式的改变。我们发现，高风险和年龄与APL患者的DNA甲基化失调特别相关。在DNMT3B过表达细胞中，白血病的发生和维持受损。具有高DNA甲基化的小鼠白血病原始细胞也显示对氮杂胞苷的敏感性增加。我们已经开始用DNMT3B +/-产生鼠白血病，以分析白血病诱导和DNMT3B活性轻微降低的治疗反应。新资助期的主要目的是识别和修改影响细胞毒性药物和氮杂胞苷治疗反应的DNA甲基化事件。为此，我们将分析相关的表观基因组变化，这些变化决定了小鼠模型中白血病发生和治疗反应的变化。相关基因将通过基因组编辑（TALEN/CRISPR）和/或通过细胞系和原代母细胞中的shRNA转导沉默。此外，通过使用TALE-SssI构建体的靶向表观基因组编辑来调节相关基因组区域。在体外和体内测试对基因表达、细胞功能和治疗反应的功效和作用。作为补充方法，我们将在诊断时、氮杂胞苷暴露后第5天和化疗开始后第15天使用450K甲基化微珠阵列对AML-AZA临床试验进行生物标志物分析。本文的一个主要目的是鉴定与对标准化疗+/-氮杂胞苷的反应增加相关的DNA甲基化模式。将这些结果与具有确定水平的DNMT活性的鼠白血病数据进行比较。此外，AML-AZA生物样本库将为SPP 1463中的几个合作伙伴提供充分表征的标本。综合起来，这些分析将识别对氮杂胞苷和细胞毒性药物的DNA甲基化相关治疗反应。将分析DNA甲基化变化的功能相关性，以确定潜在的机制。

Acute myeloid leukemia (AML) is associated with epigenomic changes including profound alterations in DNA methylation. Origin and relevance of these changes are not entirely clear. Despite intensive chemotherapy, the majority of patients cannot be cured by current therapies. DNA methyltransferase (DNMT) inhibitors such as Decitabine and Azacytidine are not curative and the mechanisms of action are incompletely understood. In the first funding period, we used Reduced Representation Bisulfite Sequencing (RRBS) to identify altered DNA methylation patterns in murine leukemias with increased DNMT3B activity and in Acute Promyelocytic Leukemia (APL) patients. We found out that high risk and age were especially associated with dysregulated DNA methylation in APL patients. In DNMT3B overexpressing cells, leukemia initiation and maintenance were impaired. Murine leukemic blasts with high DNA methylation also showed increased sensitivity towards Azacytidine. We have started to generate murine leukemias with DNMT3B+/- to analyze leukemia induction and therapy response with a slight decrease of DNMT3B activity. The main aim of the new funding period is to identify and modify DNA methylation events that influence therapy response towards cytotoxic drugs and Azacytidine. For this purpose, we are going to analyze the relevant epigenomic changes that determine changes in leukemogenesis and treatment response in the mouse models. Relevant genes will be silenced by genomic editing (TALEN/CRISPR) and/or by shRNA transduction in cell lines and primary blasts. In addition, relevant genomic regions are modulated by targeted epigenomic editing using TALE-SssI constructs. Efficacy and effects onto gene expression, cellular functions and therapy response are tested in vitro and in vivo. As a complementary approach, we are going to perform a biomarker analysis for the AML-AZA clinical trial with 450K methylation bead arrays at the time of diagnosis, after day 5 of Azacytidine exposure and on day 15 after chemotherapy start. One major aim here is to identify DNA methylation patterns that are associated with increased response to standard chemotherapy +/- Azacytidine. These results are compared to the murine leukemia data with defined levels of DNMT activity. In addition, the AML-AZA biobank will provide well characterized specimens to several collaboration partners within the SPP1463.Taken together, these analyses will identify DNA methylation associated therapy response towards Azacytidine and cytotoxic drugs. The functional relevance of the DNA methylation changes will be analyzed to identify the underlying mechanisms.