Transcriptional Down-Regulation of the Wnt Antagonist SFRP1 Is Associated to Promoter Hypermethylation as Detected by Pyrosequencing in Hematopoietic Cells of Patients with Different Risk-Types of MDS, ALL and AML.

Abstract 1751

Poster Board I-777

SFRP1 is one of five known secreted frizzled related proteins (SFRPs) whose expression has been recently observed to be down-regulated due to hypermethylation in acute and chronic leukaemia, but so far not in myelodysplastic syndrome (MDS). SFRPs are extracellular antagonists of the Wnt-dependent signalling pathway that plays an important role in the pathogenesis of solid tumours and hematopoietic malignancies.

We examined the mRNA expression and the promoter methylation of SFRP1 and the mRNA expression of the activating Wnt membrane receptor frizzled 3 (Fzd3) in bone marrow (BM) mononuclear cells derived from 78 patients with MDS (low risk, n=18; int-1, n=27; int-2, n=19 and high risk, n=14), 23 patients with acute myeloid leukaemia (AML) and 20 patients with acute lymphoblastic leukaemia (ALL) at the time of initial diagnosis as compared to healthy individuals (n=24). We performed real time quantitative RT-PCR to determine mRNA transcription levels of both genes as well as DNA-pyrosequencing to quantify promoter methylation of SFRP1. In addition, we analyzed highly purified CD34+ cells from 45 MDS patients (low risk, n=11; int-1, n=12; int-2, n=11 and high risk, n=11) and 18 healthy controls.

In both unselected BM and CD34+ cells of the MDS patients (BM: 15 (83%) of low risk, 23 (85%) of int-1, 17 (89%) of int-2 and 11 (80%) of high risk MDS; CD34+: 10 (91%) low risk, 10 (83%) int-1, 11 (100%) int-2 and 9 (82%) high risk MDS) a significant decreased SFRP1 expression was detected (BM: 3.4 fold for low risk, 2.9 fold for int-1, 7.9 fold for int-2, 15.4 fold for high risk MDS; CD34+: 3.6 fold for low risk, 2.1 fold for int-1, 8.3 fold for int-2 and 9.2 fold for high risk MDS). In selected MDS-patient samples (12.3%) we observed DNA-hypermethylation of the SFRP1 promoter as compared to normal controls. Furthermore, in AML and ALL BM samples, we could confirm previously reported promoter hypermethylation (AML 50% and ALL 61%) and the association with transcriptional down-regulation of SFRP1 (19.8 fold in 22 (95 %) AML patients, 17.3 fold in 17 (85%) ALL patients). In addition, expression levels of Fzd3 were up-regulated in both acute leukaemia and MDS achieving the level of statistical significance in high-and low risk MDS.

Our data show a significant transcriptional down-regulation of SFRP1 as a common event in AML, ALL and - as demonstrated for the first time - in MDS. The epigenetic inactivation of SFRP1 and the transcriptional up-regulation of the Wnt receptor Fzd3 seem to be associated with an activation of the disease-related affected Wnt signalling pathway in these hematopoietic diseases. Hypermethylation of the SFRP1 promotor may be one of the predominant regulatory mechanism in AML and ALL, but not mainly in MDS.