Cancer Drivers Affected by Aberrant DNA Methylation in MDS and AML

Abstract 1716

Cytosine methylation is an epigenetic mark affecting accessibility of DNA to transcription. Cancer is associated with hypermethylation in CpG islands (dense clusters of CpG sites frequently present around gene transcription starts) and hypomethylation of sparse CpG sites outside CpG islands. Complex changes of DNA methylation in leukemia permanently disturb epigenetic regulation and participate in leukemogenesis. To characterize epigenetic aberrations in myeloid neoplasms, we analyzed DNA methylation in 16 patients with myelodysplastic syndrome (MDS), 7 patients with acute myeloid leukemia (AML) and 5 healthy controls. Using Digital Restriction Enzyme Analysis of Methylation, we quantified DNA methylation at CpG dinucleotides within approximately 40,000 CCCGGG restriction sites across the genome. We analyzed methylation differences between healthy controls and patients with MDS and AML. CpG sites within CpG islands (CGI sites) are typically not methylated in normal tissues. We found 18,738 CGI sites with methylation <5% in normal controls. MDS and AML patients showed heterogeneous hypermethylation >20% in these sites, ranging from 5 to 2720 (median 186) hypermethylated sites in individual patients. The median number of hypermethylated CGI sites was 146 in MDS and 1234 in AML patients. Altogether, we found 5069 CGI sites corresponding to 2183 genes differentially hypermethylated in MDS or AML. GpG sites outside CpG islands (NCGI sites) are generally methylated. We found only 3262 NCGI sites unmethylated (<5% methylation) in normal controls. Hypermethylation pattern of these NCGI sites in individual MDS and AML patients was similar to that of CGI (r=0.85), with 5–388 (median 38) sites hypermethylated over 20%. Altogether, we found 848 NCGI sites corresponding to 629 genes hypermethylated. Hypermethylation affecting both CGI and NCGI sites was found in 273 genes. In order to identify potential drivers in the plethora of methylation changes, we compared the hypermethylated genes with the Sanger Institute “Cancer Consensus” listing 457 genes. The list of 2539 hypermethylated genes contained 74 genes (3%) from the cancer list (51 in CGI, 10 in NCGI and 13 in both CGI and NCGI). Next we analyzed hypomethylation events in MDS and AML. We found 10,509 CpG sites (1210 CGI, 9299 NCGI) with methylation level >80% in normal controls. Methylation levels <30% in MDS and AML patients were observed at 1–439 (median 23) sites. Hypomethylation affected mostly NCGI sites and the numbers of sites hypomethylated in individual patients positively correlated with hypermethylation at CGI sites (r=0.39). The total of 1153 hypomethylated sites corresponded to 777 genes. Twenty-two genes (3%) were present on the cancer list. Six genes (CBFA2T3, FGFR3, FLI1, MLLT1, PHOX2B and PRDM16) showed both hyper and hypomethylation in different parts of the gene when compared to normal controls. Interestingly, translocations involving 5 of these genes have been reported in blood malignancies. The number of ‘cancer’ genes affected by epigenetic events in individual patients was 1–29 (median 8) in MDS and 2–44 (median 20) in AML. In summary, we have detected tens to thousands of CpG sites with aberrant methylation in MDS and AML patients. Our data suggest that approximately 3% of DNA hypermethylation and hypomethylation events are potential drivers in the leukemogenic process in MDS and AML. DNA methylation changes were detected in 90 genes (13%) of the 457 cancer gene list. Our findings thus support the importance of epigenetics in leukemia.