Combined epigenetic therapy of acute myeloid leukemia: translational studies of in vivo induction of gene expression and DNA hypomethylation

DNA低甲基化氮核苷药物为老年AML/MDS患者提供了积极的、非强化的治疗，但其体内活性的机制尚未完全了解。利用在4组随机II期“Decider”AML试验中接受5-aza-2‘-脱氧胞苷(5-aza-2’-deoxcytidine，5-aza-DC，Decitabine，DAC)与组蛋白脱乙酰酶(HDAC)抑制剂丙戊酸(VPA)和全反式维甲酸(ATRA，一种活跃于急性早幼粒细胞白血病的分化诱导剂)治疗的患者的原始髓系细胞，在特定目的1中，我们将询问表观遗传治疗是否仅在白血病原始细胞中诱导了明显的、早期的DNA甲基化改变，还是在选定的患者中也诱导了正常的“旁观者”T细胞以及正常CD34+造血祖细胞中的DNA甲基化改变。我们还将研究地西他滨和5-氮胞苷在体内引起的差异变化，以及在所研究的原代细胞中去甲基化和重新甲基化是随机还是非随机过程。特定目的2集中在这样一个问题上：治疗引起的DNA甲基化变化与mRNA表达变化相关？在这里，我们还希望通过患者体内持续去压低/去甲基化的基因来产生一个预测血液学反应的“反应信号”。在具体目标3中，我们将解决哪些编码癌症/睾丸抗原的基因被诱导，以及这是否与这些基因的去甲基化有关。我们希望表观基因治疗白血病患者细胞产生的甲基组和转录组图谱将使我们能够确定这种治疗方法的全基因组靶点、恶性血细胞与正常血细胞中地西他滨DNA去甲基化的程度，并有望成为表观治疗临床反应的标志。长期的研究目标是更好地理解表观遗传活性物质在体内的作用机制，包括那些与DNA低甲基化没有直接联系的物质。

DNA hypomethylating azanucleoside drugs provide active, non-intensive treatment of older AML/MDS patients, but the mechanisms governing their in vivo activity is as yet not fully understood. Using primary myeloid blasts from patients treated within the 4-arm randomized phase II "DECIDER" AML trial with 5-aza-2'-deoxycytidine (5-aza-dC, Decitabine, DAC) with or without the histone deacety-lase (HDAC) inhibitor valproic acid (VPA) and all-trans retinoic acid (ATRA, a differentiation-inducing agent active in acute promyelocytic leukemia), in Specific Aim 1 we will ask whether the epigenetic treatment induces distinct, early DNA methylation changes in the leukemic blasts only or also in normal, "bystander" T-cells and, in selected patients, in normal CD34+ hematopoietic precursors. Also we will address differential changes induced by Decitabine and 5-azacytidine in vivo, and whether de- and re-methylation is a random or non-random process in the primary cells studied. Specific Aim 2 focusses on the question: which treatment-induced DNA methylation changes correlate with mRNA expression changes? Here we also hope to generate a "response signature" to predict hematologic response by genes consistently derepressed/demethylated in the patients. In Specific Aim 3 we shall address which genes encoding Cancer/testis antigens are induced, and whether this is associated with demethylation of the genes. We hope that the generated methylome and transcriptome profiles of the cells from the epi-genetically treated leukemia patients will enable us to identify genome-wide targets of this treatment ap-proach, the extent of Decitabine DNA demethylating activity in malignant vs. normal blood cells, and hopefully a signature of clinical response to the epigenetic therapy. The long-term research goal is a bet-ter understanding of the mechanisms of action of epigenetically active agents in vivo, including those not directly linked to DNA hypomethylation.