In Vitro Decitabine Treatment Demonstrates Heterogeneous Changes in Methylation and Gene Expression in Primary AML Samples.

Abstract 2527

Acute myeloid leukemia (AML) is a hematopoietic neoplasm with high mortality that is typically treated with daunorubicin/cytarabine induction chemotherapy. Alternative therapies with cytosine analogs such as decitabine are also used in some cases with a variable clinical response that some have estimated to be as high as 25%. The mechanism of these agents is unclear, but at low doses they produce passive DNA hypomethylation by inhibiting DNMT1. Although the impact of these drugs on cell growth and DNA methylation in AML cell lines has been evaluated¹ , studies using primary cells are limited; importantly, most have involved extended drug treatments that may be confounded by the differentiation of the treated cells² . In addition, some evidence suggests that decitabine has a differential effect on methylation in patients who respond to treatment² , but the utility of this phenotype as an in vitro biomarker for decitabine responsiveness is unknown.

In this study, we used a novel in vitro culture system for primary leukemia cells to explore the initial genomic effects of short-term low dose decitabine on primary samples from 22 AML patients. Primary bone marrow or blood samples from these patients were cultured on HS27 stromal cells in DMEM supplemented with beta-mercaptoethanol and 15% FBS along with hSCF, hIL3, hIL-6, hTPO and hFLT3L for an initial 4-day period prior to daily treatment for 3 days with either 100 nM decitabine, 100 nM cytarabine, or vehicle controls. Cells were then evaluated for growth, cell cycle effects, and differentiation (by flow cytometry and morphologic evaluation). DNA was prepared from all samples for 5-methylcytosine content measurements by mass spectrometry, and 8 samples were selected for genome-wide methylation and gene expression profiling with the Illumina Human Methylation 450 and Affymetrix Human Exon 1.0ST array platforms. Mass spectrometry revealed a mean decrease in 5-mdC of 29% (range: 13% to 62%) in the decitabine-treated samples; in comparison, cytarabine treatment resulted in a mean increase in 5-mdC of 5% (range: −10% to 37%). Methylation arrays also showed a modest shift toward lower methylation values, but unsupervised hierarchical clustering demonstrated that methylation patterns were driven by sample-specific differences and not drug treatment. Analysis of methylation changes showed the most pronounced hypomethylation at CpGs with high baseline methylation levels, irrespective of CpG island and gene-based annotation, suggesting that the initial methylation status of each CpG is responsible for preferential effects of decitabine, rather than its genomic context. Methylation at promoter-associated CpGs showed a small but statistically significant negative correlation with change in gene expression, but expression changes at individual genes were not consistent across the samples, including genes previously shown to be regulated by methylation-dependent mechanisms (eg. CDKN2B and CDx H1).

In addition to these findings, we observed that a sample from a long-term decitabine responder had an exaggerated in vitro response to decitabine (58% decrease in 5-mdC after 6 days of treatment), compared to a cohort of decitabine non-responders; a sample from a second patient also showed marked hypomethylation by both mass spectrometry and methylation array, although this patient was not treated with decitabine. While more investigation is needed, this observation might suggest that extreme in vitro hypomethylation in response to decitabine could serve as a biomarker for a clinical response.

In summary, our study showed that short-term low dose decitabine treatment has modest but detectable effects on DNA methylation and gene expression, but these changes did not result in activation of any canonical gene expression pathway at this early time point. We found that the baseline methylation status of a CpG appears to be the best predictor of decitabine-induced hypomethylation, with highly methylated CpGs showing the greatest change. We also observed that hypomethylation is highly variable across primary samples and at specific genes, implying that single gene approaches for measuring decitabine effect may be problematic. Finally, extreme in vitro decitabine-induced hypomethylation should be further investigated as a biomarker for decitabine responsiveness.