The therapeutic effect of DNA-hypomethylating agents (HMAs) in AML/MDS is discussed to be via its effects on aberrant gene silencing by reactivation (e.g. through promoter demethylation). While this has been broadly studied in cell line models, only very few studies have addressed the global effects of HMAs in primary blasts serially isolated from AML patients (pts) undergoing HMA treatment (Claus et al., Leuk. Res. 2013, Klco et al., Blood 2013, Welch et al., N. Engl. J. Med. 2016). We therefore conducted prospective serial methylome and transcriptome analyses on AML blasts from pts of the DECIDER trial (NCT00867672), hypothesizing that both random and non-random effects of the HMA may be observed in vivo.
Overall design
Total RNA of serially sorted blasts from 23 patients (at day 0 and 8) was isolated by TRIZOL (Thermo Fisher Scientific, Waltham, MA, USA) extraction and RNA phase was purified with the Qiagen RNeasy MinElute Cleanup Kit. Total RNA concentration was measured with the Qubit Fluorometer (Thermo Fisher Scientific) and integrity was determined by fragment analysis. RNA samples with RQN > 8.5 were further processed with the Affymetrix WT Plus kit and hybridized to GeneChip Human Gene 2.0 ST expression arrays (Affymetrix, Santa Clara, CA, USA) as described by the manufacturer. To avoid a possible batch effect, samples were pipetted in triplicates and randomized across and within plates.
Less...
More...