Limited Effect of TET2 Mutations on Promoter DNA Methylation in Chronic Myelomonocytic Leukemia

Abstract 1365

TET2 enzymatically converts 5-methylcytosine to 5-hydroxymethylcytosine, possibly leading to loss of DNA methylation. TET2 mutations are common in myeloid leukemia and were proposed to contribute to leukemogenesis through DNA methylation. To expand on this concept, we studied chronic myelomonocytic leukemia (CMML) samples. TET2 missense or nonsense mutations were detected in 53% (16/30 patients). By contrast, only 1/30 patients had a mutation in IDH1 or IDH2, and none of them had a mutation in DNMT3A. By bisulfite pyrosequencing, global methylation measured by the LINE-1 assay and DNA methylation levels of 10 promoter CpG islands frequently abnormal in myeloid leukemia were not different between TET2 mutant and wild-type cases. This was also true for 9 out of 11 gene promoters reported by others as differentially methylated by TET2 mutations. We confirmed only two non-CpG island promoters, AIM2 and SP140, as hypermethylated in patients with mutant TET2. These were the only two gene promoters (out of 14 475 genes) previously found to be hypermethylated in TET2 mutant cases. This finding shows that hypermethylation of both AIM2 and SP140 are bona fide markers of TET2 mutant cases in CMML. On the other hand, total 5-methylcytosine levels in TET2 mutant cases were significantly higher than TET2 wild-type cases. Thus, TET2 mutations have a limited impact on promoter DNA methylation in CMML. To confirm this, we performed genome-wide analysis using a next-generation sequencing method for DNA methylation levels in three TET2 mutant cases. TET2 mutant CMMLs had an average of 230 (1.9%) promoter CpG island sites hypermethylated compared to normal blood, which is close to what is generally observed when one compares cancer to normal. By contrast, all three cases had near normal to increased levels of methylation outside CpG islands. The median methylation levels in non-promoter, non-CpG island sites was 88.7% in normal blood compared to 91.7%, 92.1% and 94.6% in the three TET2 mutant cases. Thus, TET2 mutant CMMLs escape the general hypomethylation phenomenon seen in many cancers. All together, our data suggest that TET2 mutant CMML cases may have distinct DNA methylation patterns primarily outside gene promoters.