Abstract
MDS are a clinically heterogeneous group of clonal disorders, which share a high frequency of progression to secondary AML (MDS-AML). In contrast to de novo AML, MDS and MDS-AML are uniformly resistant to conventional chemotherapy. Among the few active drugs in MDS are the nucleoside analog DNA methyltransferase inhibitors (MTIs) 5-azacytidine and decitabine. Since tumor suppressor genes such as CDKN2B can be silenced by DNA methylation in MDS, it is believed that reversal of DNA methylation by MTIs might contribute to their anti-tumor effects. However, it is not clear whether methylation-dependent silencing of specific genes are predictive of response or even correlate with response to MTIs. Like MDS, AML also presents epigenetic silencing of CDKN2B and other genes; yet appear to not be as sensitive to MTIs. Given the particular sensitivity of MDS to MTIs and its resistance to standard AML chemotherapy, we hypothesized that MDS is a biologically distinct disease from AML due largely to extensive epigenetic deregulation, which is missed by single locus studies. In order to test this we studied DNA methylation levels at 24,000 gene promoters in 13 MDS pts. and 16 de novo normal karyotype AML cases, and compared and contrasted these to CD34+ bone marrow cells from 8 healthy donors. For this we used the HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR) assay, a robust method for detection of whole-genome DNA methylation, and using MassArray quantitative methylation for single locus validation. Remarkably, MDS was found to have a far greater number of hypermethylated genes than AML or normal CD34+ cells, while AML and CD34+ cells had similar number of methylated genes (MDS vs. AML: 6303 vs. 4177 promoters, p=0.026; MDS vs. normal CD34+ cells: 6303 vs. 4296 promoters, p=0.056). Using a moderated T test, an aberrant methylation signature of 736 genes (p<0.0000001) was identified in MDS vs. normal CD34+ cells, reflecting extensive epigenetic deregulation in this disease, including p16, CEBPZ, MSH2, CHES1, AKT1, Caspase2, BMP3, DAP and MYOD1. A comparison between MDS and de novo AML identified 498 genes (p<0.00005) differentially methylated, with a clear predominance of hypermethylated promoters in MDS vs. de novo AML. These genes included RUNX2 and 3, GFI1, DAPK2, MDM2, TGFA, CEBPZ and SHARP. Finally, a comparison between normal CD34+ cells and de novo AML demonstrated an aberrant pattern of methylation in 341 genes (p<0.00001), including CXCL1 and 5, PPARD, Caspase 2 HOXA4 and HOXA10, GFI1 and the MLL translocation partner Septin11. 7 of the 13 MDS cases were also examined for gene expression using the Affymetrix Hgu133plus2 array. A significant proportion of the genes that had been found to be methylated were also found to be underexpressed, including p16, DAP, BMP3, HOXA2 and HOXB8. Taken together, our data show that MDS is a unique and distinct biological entity than de novo AML featuring profound and functionally significant genome wide epigenetic deregulation. While the de novo AML methylation profile was clearly different from normal CD34+ cells, it was not as severely altered as MDS. These data also suggest that MTIs are most likely uniquely active in MDS due to their DNA methyltransferase activity.
Targets and genomic constraints of ectopic Dnmt3b expression
