Combination Treatment of Acute Myeloid Leukemia Cells with DNMT and HDAC Inhibitors: Predominant Synergistic Gene Downregulation Associated with Gene Body Demethylation

DNA-hypomethylating agents approved for older AML patients, are clinically tested in combination with histone deacetylase inhibitors (HDACi).The mechanism of action of these drugs is still under debate. In colon cancer cells, 5-aza-2''deoxycytidine (DAC) can downregulate oncogenes and metabolic genes by reversing gene body DNA methylation, thus implicating gene body methylation as a novel drug target. We asked whether DAC-induced gene body demethylation in AML cells is also associated with gene repression, and whether the latter is enhanced by HDACi. Transcriptome analyses revealed that a combined treatment with DAC and HDACi panobinostat or valproic acid effected significantly more transcripts than the sum of the genes regulated by either treatment alone, demonstrating a quantitative synergistic effect on genome-wide expression in U937 cells. This effect was particularly striking for downregulated genes. Integrative analysis of the methylome and transcriptome showed that a massive downregulation of genes, including oncogenes (e.g.c-MYC) and epigenetic modifiers (e.g. KDM2B and SUV39H1) often overexpressed in cancer, was associated predominantly with gene body DNA demethylation, and changes in acH3K9/27. These findings have implications for the mechanism of action of combined epigenetic treatment, and for a better understanding of responses in trials where this approach is clinically tested. Overall design: Examination of H3K9 acetylation and H3K27 acetylation in U937 cells after decitabine +- panobinostat/entinostat treatment获批用于老年急性髓系白血病（AML, Acute Myeloid Leukemia）患者的DNA低甲基化药物，目前正与组蛋白去乙酰化酶抑制剂（HDACi）联合开展临床试验。此类药物的作用机制迄今仍存在争议。 在结肠癌细胞中，5-氮杂-2'-脱氧胞苷（5-aza-2''deoxycytidine, DAC）可通过逆转基因体DNA甲基化下调癌基因与代谢基因的表达，由此将基因体甲基化确立为全新的药物靶点。本研究旨在探究AML细胞中DAC诱导的基因体去甲基化是否同样与基因抑制相关，以及该基因抑制效应是否可被HDACi增强。 转录组分析结果显示，DAC联合HDAC抑制剂帕比司他（panobinostat）或丙戊酸（valproic acid）处理后，所调控的转录本数量显著高于单一疗法分别调控的基因数之和，证实二者在U937细胞中对全基因组表达具有定量协同效应，且该协同效应在下调基因中尤为显著。 甲基化组与转录组的整合分析显示，大量基因的显著下调——包括癌症中常高表达的癌基因（如c-MYC）以及表观遗传调控因子（如KDM2B与SUV39H1）——主要与基因体DNA去甲基化及组蛋白H3K9/27乙酰化（acH3K9/27）的改变相关。本研究结果为联合表观遗传治疗的作用机制提供了新的科学见解，也有助于更深入理解该疗法在临床试验中的应答情况。 实验整体设计：检测地西他滨（decitabine）联合或不联合帕比司他（panobinostat）、恩替诺特（entinostat）处理后，U937细胞中的H3K9乙酰化与H3K27乙酰化水平。