TET2 mutations in Diffuse Large B-Cell Lymphoma: The Role of TET2 in the Regulation of Methylation Patterns at TET2 Target Genes

Cytosine methylation (mC) is a major DNA modification in higher eukaryotic genomes, which is involved in transcriptional silencing. A large amount of data has shown that patterns of DNA methylation are perturbed in hematological cancers including diffuse large B-cell lymphoma (DLBCL). The discovery that the TET hydroxylases convert mC to hydroxymethylcytosine (hmC) is a major break through for our understanding of how DNA methylation is deregulated. Multiple reports describe TET2 (Ten-Eleven Translocation 2) loss-of-function mutations in myeloid malignancies, and a recent study shows that TET2 inactivation perturbs both myeloid and lymphoid development in the mouse, and identifies TET2 mutations in ∼2% of human B-cell lymphoma (Quivoron et al, Cancer Cell 20, 1–14, 2011).

In the present study our aims are to determine the frequency and clinical impact of TET2 mutations in DLBCL, to identify TET2 target genes in CD34+ cells, normal- and malignant B-cells, and evaluate the role of TET2 mutations on the methylation pattern at TET2 targets genes in normal and malignant hematopoiesis.

DNA was isolated from fresh frozen DLBCL (n=110), normal CD34+ cells and B-cells, and a TET2 mutant DLBCL-cell line. Mutation scanning was performed by denaturing gradient gel electrophoresis (DGGE) and automated sequencing. Global methylation profiling was done by Illumina Infinium microarrays, methylation at individual genes by methylation specific melting curve analysis and pyrosequencing. Global mC and hmC patterns were determined by DNA immunoprecipitation and promoter array analysis in cell lines, B-cells and CD34+ cells. TET2 target genes were identified by ChIP followed by deep sequencing. Gene expression by Nimblegen custom made arrays and RT-qPCR.

We identified TET2 mutations in 15% of primary diffuse DLBCL, including missense mutation in the catalytic domain (n=8, 2 of which showed allelic loss), loss-of-function mutations (n=7, one of which showed allelic loss), and missense mutation outside the catalytic domain (n=1 with allelic loss). Somatic origin of these mutations was verified in 11 of the 16 cases where matched normal tissue was available. No difference in overall survival was observed between TET2mut and TET2wt cases (P=0.17). To a large extent, the TET2 targets genes identified by ChIP seq analysis were overlapping in CD34+ cells, normal- and malignant B-cells. Gene ontology analysis showed that TET2 target genes are mainly involved in DNA metabolism and repair, metabolic processes and cell cycle homeostasis. Global methylation in TET2mut and TET2wt cases and gene expression data are being analyzed in DLBCL samples. In addition, the distribution patterns of hmC and mC at TET2 target genes and the relation to gene expression is being analyzed in a TET2 mutant DLBCL cell line, normal B-cells and CD34+ cells.

Here, we show that TET2 mutations are frequent in DLBCL, and identify the TET2 target genes in CD34+ cells, and in normal and malignant B-cells. The role of TET2 mutations for global methylation and for the methylation patterns at TET2 target genes will be presented at the meeting. By investigating the clinical implications of TET2 mutations we aim to identify DLBCL subsets that may benefit from hypomethylating therapy. Furthermore, the identification of hypermethylated TET2 target genes will hopefully contribute to molecular understanding of how TET2 mutations induces malignant transformation.

Christensen:EpiTherapeutics: cofounder of EpiTherapeutics and have shares and warrants in the company. Helin:EpiTherapeutics: cofounder of EpiTherapeutics and have shares and warrants in the company.