Involvement of TET2 in Regulation of Epigenetic Silencing in MDS and Related Myeloid Malignancies.

Abstract 2908

Poster Board II-884

Genomic (chromosomal gain losses and UPD) and genetic damage (mutations) are hallmarks of MDS and related myeloid malignancies. In addition, aberrant hypermethylation of CpG islands, leading to epigenetic silencing of tumor suppressor and differentiation genes, appears to play an important pathogenic role in evolution to AML. While various mechanisms leading to chromosomal instability have been identified, the pathogenesis of epigenetic instability leading to aberrant methylation remains unexplored. The recent discovery of mutations in TET2 gene in MDS/MPN, sAML and especially CMML provided an intriguing possibility that dysfunction of this bona fide tumor suppressor gene may result in increased promoter methylation. This hypothesis is based on the description of the function of the highly related TET1 gene: the corresponding protein mediates hydroxylation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which prevents the DNMT1-catalyzed maintenance methylation during cell division. Consequently, TET2 mutations could lead to epigenetic instability and accumulation of aberrantly hypermethylated CpG sites.

Both homozygous and heterozygous TET2 mutations were found suggesting that the presence of WT allele or residual activity is not protective. A large proportion of these mutations results in introduction of frame shifts or stop codons resulting in loss of function whilst missense mutations may lead to decreased function. To clarify the function of TET2 and consequences of the mutations we transduced M5 THP-1 AML cell line with lentiviral vectors, containing partial TET2 cDNA (TET2+) and shRNA (TET2-), to achieve overexpression or knockdown of TET2 gene. Following selection, TET2+ cell line showed 3-fold TET2 mRNA overexpression while TET2- cells demonstrated 85% knockdown of TET2 gene as compared to control cells. Based on the putative function of TET2, we performed thin layer chromatography (TLC) comparing 5hmC DNA content in THP1, TET2+, TET2- and appropriate control cells. Parental THP1 cells revealed abundant level of 5hmC. Whilst a 1.5-fold increase of 5hmC content was found in TET2+ cells, it remained unchanged in TET2- cells. However, early results showed a 20% decrease in 5hmC in bone marrow of patients with myeloid malignancies and homozygous TET2 mutations. Illumina 27k methylation arrays were used to assess the effects of altered TET2 expression on methylation patterns. Although global methylation levels, as expressed by averaged β-values, did not significantly differ, the number of hypermethylated CpG sites, defined by individual β-values >0.9, was decreased by 25% (3756 vs. 2839) in TET2+ cells. No difference was seen in TET2- cells vs. controls. However, a number of promoter CpG in TET2- displayed >2-fold change in methylation level compared to TET2+, including MGMT, PCGF2, PTPRO and NOX1. Among differentially silenced promoter CpG we selected CDKN2B (p15) promoter as a marker for methylation activity. When p15 promoter controlling a luciferase reporter was transfected into TET2+ and control cells, a 2.4-fold increase of luciferase activity occurred, consistent with increased demethylating activity in cells expressing higher levels of TET2. Additionally, qPCR showed a 2.5-fold increase in p15 expression in TET2+ cells. Based on this result we also studied methylation status of p15 promoter in patients with WT (N=20) and mutant TET2 (N=27); patients with MDS and MDS/MPN who carried TET2 mutations presented consistent methylation of various promoters including p15 in 2/3 mutant cases vs. 1/3 WT cases.

Whereas proliferation kinetics was not significantly altered by changes in TET2 levels, 72h treatment with decitabine (0.5 μM) resulted in a differential effect on TET2+ and TET2- cells. TET2+ cells were not significantly affected however survival of TET2- cells decreased by 30%. In addition, TET2+ cells showed a relative resistance to HDAC inhibitor Entinostat (1μM) with 26% inhibition of proliferation vs. 60% seen in TET2- cells measured after 72h by cell proliferation assay (MTS).

In sum, our results suggest that TET2 gene may be involved in epigenetic regulation of promoter CpG that are relevant to proliferation/differentiation of myeloid cells and changes in the expression/activity of this gene may result in altered methylation and thereby epigenetic silencing patterns.