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

急性髓系白血病(AML)与表观基因组学改变有关，包括DNA甲基化的深刻变化。这些变化的起源和相关性尚不完全清楚。尽管进行了密集的化疗，但大多数患者无法通过目前的治疗方法治愈。DNA甲基转移酶(DNMT)抑制剂如地西他滨和氮胞苷等不能治愈，其作用机制尚不完全清楚。在第一个资助期，我们使用简化代表性亚硫酸盐测序(RRBS)来鉴定Dnmt3b活性增加的小鼠白血病和急性早幼粒细胞白血病(APL)患者的DNA甲基化模式的改变。我们发现APL患者的高风险和年龄尤其与DNA甲基化异常有关。在Dnmt3b过表达的细胞中，白血病的启动和维持受到损害。DNA甲基化程度高的小鼠白血病细胞对氮胞苷的敏感性也有所增加。我们已经开始产生带有Dnmt3b+/-的小鼠白血病，以分析在Dnmt3b活性略有下降的情况下诱导白血病和治疗反应。新资助期的主要目的是确定和修改影响细胞毒药物和氮胞苷治疗反应的DNA甲基化事件。为此，我们将在小鼠模型中分析决定白血病发生和治疗反应变化的相关表观基因组变化。相关基因将通过基因组编辑(TALEN/CRISPR)和/或通过在细胞系和原代母细胞中的shRNA转导而沉默。此外，相关的基因组区域通过使用TALL-SSSI结构的有针对性的表观基因组编辑来调节。在体外和体内测试对基因表达、细胞功能和治疗反应的疗效和影响。作为补充，我们将在诊断时、氮胞苷暴露后第5天和化疗开始后第15天对AML-AZA临床试验进行生物标记物分析，该试验使用450K甲基化珠阵列阵。这里的一个主要目的是确定与标准化疗+/-氮胞苷反应增强相关的DNA甲基化模式。将这些结果与定义了DNMT活性水平的小鼠白血病数据进行比较。此外，AML-AZA生物库将向SPP1463内的几个合作伙伴提供具有良好特征的标本。这些分析结合在一起，将确定DNA甲基化相关治疗对氮胞苷和细胞毒药物的反应。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.