Epigenetic Silencing of a Novel Candidate Tumor Suppressor “miR” Predicts Poor Prognosis In High-Risk MDS and AML.

A tumor suppressor has long been sought for in the commonly deleted region on chromosome 5q in myelodysplastic syndrome and acute myeloid leukaemia (MDS/AML). MicroRNAs (miRs) may have tumor suppressor function since they may repress the expression of proto-oncogenes through post-transcriptional regulation by binding to the 3′UTR of mRNA. It has recently been shown that several types of haematological malignancies have globally altered expression of miRs. DNA methylation cause targeted down regulation of individual tumor suppressor genes that may be directly involved in the pathogenesis of MDS/AML and we speculated whether epigenetic downregulation of miRs also plays a role in MDS/AML pathogenesis.

Cell lines were treated with the hypomethylating agent 5-azacytidine. Upregulated miRs were identified by microarray (Exiqon) analysis and confirmed by RT qPCR analysis. Transcription start site was determined by 5′RACE and promoter methylation by methylation specific melting curve analysis. MiR expression and processing were analysed by qPCR, Northern blotting, and si-RNA mediated knock down of Drosha. Clinical data were collected from patient files.

We focused on one upregulated miR that locates to the commonly deleted region of AML/MDS. We found that the processing of this “miR” was independent of Drosha, and northern blotting showed that this was in fact a different type of non-coding RNA (ncRNA). The gene that encodes this ncRNA has a CpG island at the transcription start site. This CpG island is unmethylated in normal CD34+ cells, normal peripheral blood lymphocytes and methylated in three different myeloid cells lines (HL60, NB4, F-36P). Normal CD34+ cells and normal peripheral blood lymphocytes expressed the ncRNA, while no expression was seen in the cell lines. However, treatment by 5-azacytidine derepressed expression of this ncRNA. Bone marrow mononuclear cells from 101 high-risk MDS and AML patients, isolated at the time of diagnosis, were examined for methylation of the ncRNA promoter. Methylation was detected in 48% of the patients. Patients with no methylation have a significant better survival as compared to patients with methylation of the promotor.

This is the first example of targeted methylation of this type of RNA that has not previously been implicated in cancer. The preliminary results indicate that methylation of this ncRNA may be used as a prognostic marker for survival in high-risk MDS- and AML patients. We are currently investigating the function of this ncRNA by performing stable transfections into MDS/AML cell lines.

Jones:Lilly: Consultancy; Millipore: Consultancy.