Transcriptome profiling by RNA-sequencing of the AML cell line U937.

All-trans retinoic acid (ATRA, RA) has powerful activity in APL; its efficacy in non-APL AML is still unclear, but may be boosted by epigenetic drugs such azanucleoside DNMT inhibitors (Blagitko-Dorfs et al. PLoS ONE 2013). In a randomized phase II study (DECIDER trial, NCT00867672) the addition of RA to decitabine (DAC) in newly diagnosed non-fit older AML patients resulted in a clinically meaningful extension of survival. We hypothesize that in vitro, the add-on of RA to DAC results in cooperative transcriptome changes (possibly associated with demethylation), which may explain at least in part this clinical result. Overall design: The AML cell line U937 was treated with decitabine (DAC) ± ATRA in technical triplicates for 3 consecutive days (with daily change of media and DAC add-on). Cells were harvested and RNA was isolated after 72 hours, Fragment analysis by TapeStation confirmed RQN values =9.7. rRNA was removed and strand-specific libraries of each replicate were sequenced on an Illumina HiSeq 2500 with approximately 40 million reads per sample.

全反式维甲酸（All-trans retinoic acid, ATRA, RA）对急性早幼粒细胞白血病（Acute Promyelocytic Leukemia, APL）具有强效活性；其在非APL型急性髓系白血病（Acute Myeloid Leukemia, AML）中的疗效尚不明确，但可通过氮杂核苷类DNA甲基转移酶抑制剂等表观遗传药物增强（Blagitko-Dorfs等，PLoS ONE，2013）。在一项随机II期临床试验（DECIDER试验，编号NCT00867672）中，对初诊且不适合强化疗的老年AML患者采用地西他滨（Decitabine, DAC）联合RA治疗，可实现具有临床意义的生存延长。本研究提出如下假说：体外实验中，DAC联合RA可引发协同转录组变化（可能与去甲基化相关），该变化至少可部分解释上述临床结果。实验整体设计：以AML细胞系U937为研究对象，采用地西他滨（DAC）±ATRA处理，设置三次技术重复，连续处理3天（每日更换培养基并补加地西他滨）。72小时后收集细胞并提取RNA，通过TapeStation片段分析确认RNA质量编号（Ribosomal RNA Quality Number, RQN）为9.7。去除核糖体RNA后，为每个重复样本构建链特异性文库，随后在Illumina HiSeq 2500平台上进行测序，每个样本约获得4000万条reads。