Genomic impact of transient low-dose decitabine treatment on primary AML cells

Abstract

Key Points• Decitabine treatment of in vitro expanded primary AML samples leads to global hypomethylation.• Highly methylated CpGs are most affected by decitabineinduced hypomethylation, with little influence on transcriptional activity.Acute myeloid leukemia (AML) is characterized by dysregulated gene expression and abnormal patterns of DNA methylation; the relationship between these events is unclear. Many AML patients are now being treated with hypomethylating agents, such as decitabine (DAC), although the mechanisms by which it induces remissions remain unknown. The goal of this study was to use a novel stromal coculture assay that can expand primary AML cells to identify the immediate changes induced by DAC with a dose (100nM) that decreases total 5-methylcytosine content and reactivates imprinted genes (without causing myeloid differentiation, which would confound downstream genomic analyses). Using array-based technologies, we found that DAC treatment caused global hypomethylation in all samples (with a preference for regions with higher levels of baseline methylation), yet there was limited correlation between changes in methylation and gene expression. Moreover, the patterns of methylation and gene expression across the samples were primarily determined by the intrinsic properties of the primary cells, rather than DAC treatment. Although DAC induces hypomethylation, we could not identify canonical target genes that are altered by DAC in primary AML cells, suggesting that the mechanism of action of DAC is more complex than previously recognized. (Blood. 2013;121(9):1633-1643) IntroductionAcute myeloid leukemia (AML) is a clonal hematopoietic neoplasm characterized by maturation arrest in the myeloid lineage. Treatment typically consists of induction chemotherapy with an anthracycline and cytarabine with the goal of achieving a complete remission, 1,2 followed by consolidation therapy. Despite these measures, the mortality rate of AML is still very high. Although recent genomic advances have improved our understanding of AML pathogenesis and risk stratification 3,4 the overall outcome is still dismal for most patients, and alternative treatment strategies are needed.One alternative approach for the treatment of AML and myelodysplastic syndromes (MDS) is the use of hypomethylating agents, including the cytosine analogs 5-azacytidine (AZA) and 5-aza-2Ј-deoxycytidine (decitabine; DAC). 5,6 These nucleosides are distinct from other cytosine analogs because they contain a pyrimidine ring modification that results in the covalent trapping of the maintenance DNA methyltransferase (DNMT1). This effect is cell-cycle dependent, because AZA and DAC must first be modified and incorporated into newly synthesized DNA; this leads to passive hypomethylation of DNA as cells divide, because of the depletion and degradation of DNMT1. 7,8 Exposure to these drugs is also associated with cellular differentiation 9,10 and cytotoxicity at higher doses. 11 Although the impact of DAC and AZA on acute myeloid leukemia cell lines ha...

Molecularly targeted drug combinations demonstrate selective effectiveness for myeloid- and lymphoid-derived hematologic malignancies

“…Developments in functional screening technology have produced several assay platforms for the evaluation of responses of tumor cells to exogenous perturbations. Functional screening efforts in hematologic malignancies have often involved the culture of patient cells in conventional 2D tissue culture platforms, sometimes with conventional culture conditions (38)(39)(40) and other times using additives or feeder cell coculture that promote certain phenotypic aspects of the cells, such as preservation of primitive cell differentiation state (41) and cell proliferation (42). A key feature of our ex vivo assay is that it provides drug sensitivity data within 4 d, a time frame that can support and influence clinical decision-making.…”

DNA Hypomethylating Drugs in Cancer Therapy

“…Studies using 27K or 450K methylation arrays (Tsai et al 2012;Klco et al 2013;Pandiyan et al 2013), as well as with whole genome bisulfite sequencing (Lund et al 2014) have shown that DNA demethylating agents cause global hypomethylation throughout the entire genome. However, these studies have also shown that only a small percentage of genes that undergo promoter hypomethylation actually become reactivated (Tsai et al 2012;Klco et al 2013;Pandiyan et al 2013;Lund et al 2014).…”

Pharmacological targeting of -catenin in normal karyotype acute myeloid leukemia blasts

“…13 For all 3 samples, treatment with iCRT3 resulted in a significant decrease in the expansion of viable cells compared to control (P<0.05) (Figure 2A Figure 2B). Interestingly, treatment of this sample with iCRT3 also increased the percentage of CD34 + , CD38…”