Identification of MDS-Specific and Methylation Associated Downregulation of Survivine and WT1 in Highly Purified Hematopoietic Progenitor Cells during In Vitro Lineage Specific Differentiation.

The molecular pathogenesis of myelodysplastic syndromes (MDS) is poorly understood. To detect epigenetic alterations of key regulatory genes during lineage-specific hematopoietic differentiation, CD34+ cells from healthy donors (n=7) and MDS patients with low risk (LR; RA/n=6, RARS/n=3) and high risk (HR; RAEB/n=4, RAEB-T/n=2) MDS according to IPSS were in vitro differentiated with EPO, TPO or GCSF and harvested at days 0, 4, 7 and 11. For each lineage at each time point, promotor methylation analysis of genes involved in cell cycle control (p14, p15, p16, CHK2), DNA-repair (hMLH1), apoptosis (p73, survivine, DAPK) and differentiation (RARb, WT1) was performed by methylation specific PCR (MSP) of bisulfite converted genomic DNA. Corresponding gene expression was analyzed by oligonucleotide microarrays. Remarkably in CD34+/d0 cells p16 was methylated in 0% controls in contrast to 22% in MDS LR- and 50% in MDS HR-patients. Likewise in CD34+/d0 cells WT1 methylation was found in 0% controls but in 62% MDS LR- and 33% MDS HR-patients. During differentiation (d4, d7, d11) survivine promoter methylation was found in all 3 hematopoietic lineages in MDS only (LR/EPO: 22%, LR/TPO: 11%, LR/GCSF: 55%; HR/EPO: 67%, HR/TPO: 33%, HR/GCSF: 67%). WT1 promotor methylation during differentiation was almost absent in controls (EPO: 0%, TPO: 0%, GCSF: 29%) in contrast to MDS (LR/EPO: 22%, LR/TPO: 11%, LR/GCSF: 55%; HR/EPO: 17%, HR/TPO: 17%, HR/GCSF: 67%). CHK2 methylation during differentiation was not found in controls but in MDS patients (LR/EPO: 44%, LR/TPO: 22%, LR/GCSF: 22%; HR/EPO: 33%, HR/TPO: 33%, HR/GCSF: 50%). Interestlingly, mRNA expression of survivine was significantly reduced in methylated samples during erythropoiesis (p=0,04). Unexpectedly p73 RNA-expression was lower during granulopoiesis (p=0,052) in unmethylated samples. Similarly p16 mRNA showed significantly reduced expression during erythropoiesis (p=0,034) and on day 10 (p=0,064) and day 14 (p=0,035) during lineage specific differentiation in general in unmethylated specimen. Furthermore in low risk MDS WT1 RNA-expression was significantly reduced for methylated samples (p=0,01). P16 hypermethylation of CD34+/d0 cells shows a trend to correlate with MDS IPSS, but higher p16 mRNA expression values were found in methylated cases. Similarly a trend for IPSS risk correlation is shown for survivine promotor methylation during lineage specific differentiation, making it a potential marker of MDS progression, which leads to reduced mRNA expression particularly in erythropoiesis. For WT1 and CHK2 methylation patterns specific for MDS were identified, corresponding to reduced expression in WT1 promotor methylated LR-MDS. The detailed exploration of epigenetic alterations of these genes during early hematopoesis in MDS may give new insights into the pathophysiology of MDS. In particular this study identified survivine and WT1 as genes with lower methylation associated expression in MDS hematopoietic precursor cells which may ultimately contribute to the clonal expansion of the malignant cell clone.