DNA METHYLATION PROFILING IN PATIENTS WITH MYELODYSPLASTIC SYNDROME TREATED WITH 5-AZACYTIDINE

Background:

Myelodysplasticsyndromes (MDS) are clonal disorders of hematopoietic stem cells characterized by ineffective hematopoiesis. Aberrant DNA methylation in MDS was documented in several studies. Hypermethylation in the promoter regions may reduce the expression of tumor suppressor genes, whereas hypomethylation may lead to an increased expression of oncogenes. The DNA-hypomethylating drug 5-azacytidine is in clinical use for the treatment of MDS.

Aims:

To date, correlation between DNA methylation and the clinical response to hypomethylating agents has not been found.Therefore, we investigated whether DNA methylation status might predict response to azacytidine treatment.We also analyzed different methylation levels between patients and controls.

Methods:

We assessed methylation status in 31 samples from MDSpatients before 5-azacytidine treatment using the Illumina Infinium HumanMethylation27 BeadChip, which interrogates 27,578 CpG sites, selected predominantly from the promoter regions of 14,000 annotated genes. DNA was isolated from CD34+ cells separated by magnetic bead from bone marrow. DNA was subsequently modified by sodium bisulfite. The nonparametric Mann-Whitney test was used for comparison of β-values between responders and nonresponders and between patients and control group.

Results:

We found significant promoter hypermethylation in CD34+ cells in 10 genes with transcription regulator activity (e.g. GATA4, WT1, EGR3, MAPK15, MYF6 and PAX3) and 10 genes involved in embryonic organ morphogenesis (e.g.HOXA2, HOXA5, HOXB6 and TCF21) (P<0.001) in MDS patients compared to controls. The contrary, we detected promoter hypomethylation in genes participating in regulation of programmed cell death (CASP3, IL3, MAPK, PRAME and TNFSF14) and genes are involved in cellular response to stress (e.g. DDB1, MBD4, MAPK1 and PARP3) (P<0.005). To determine whether DNA methylation could predict response to 5-azacytidine treatment, we compared DNA methylation at baseline with clinical responses in 31 MDSpatients. Fourteen patients of 31 (45 %) achieved complete remission or partial remission, 10 had stable disease (32%), and 7 showed progression (23%). We found significant differences in methylation status in 45 genes (P<0.05) between groups of responders and nonresponders. The affected genes were functionally annotated with the David database. 20 genes (e.g. HMP19, NOS, PDCD, 1LG21, VPS53 and GPR92) from these were integral to membrane.Furthermore, among the genes with the largest methylation difference between responders and nonresponders belonged HSD17B4 (0.29 vs 0.75, p <0.001), HMP19 (0.37 vs 0.77, P<0.01) and DAAM2 (0.51 vs 0.15, P<0.001).

Summary / Conclusion:

Our study shows aberrant promoter methylation in MDS patientscompared to controls and different levels of methylation between groups of patients with unlike response to therapy. We suppose that the success of 5-azacytidine therapy may depend on its transport into cells. Drug must be transported into cells before its incorporation into DNA and subsequently inactivates DNA methyltransferases. Cellular transport across membranes is crucial for uptake of this highly polar hydrophilic molecule. We detected significantly altered levels of methylation in large number of genes for proteins, which are integrated into the membrane in responders vs nonresponders. These changes of methylation level may influence the absorption of the active substance.

This work was supported by grants NT13899, NT13847and by the project for conceptual development of research organization (00023736) from the Ministry of Health of the Czech Republic.

Abstrakta v časopise Haematologica 2013, Suppl1

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