Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2

Abstract

TET2 is a close relative of TET1, an enzyme that converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. The gene encoding TET2 resides at chromosome 4q24, in a region showing recurrent microdeletions and copy-neutral loss of heterozygosity (CN-LOH) in patients with diverse myeloid malignancies. Somatic TET2 mutations are frequently observed in myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), MDS/MPN overlap syndromes including chronic myelomonocytic leukaemia (CMML), acute myeloid leukaemias (AML) and secondary AML (sAML). We show here that TET2 mutations associated with myeloid malignancies compromise catalytic activity. Bone marrow samples from patients with TET2 mutations displayed uniformly low levels of 5hmC in genomic DNA compared to bone marrow samples from healthy controls. Moreover, small hairpin RNA (shRNA)-mediated depletion of Tet2 in mouse haematopoietic precursors skewed their differentiation towards monocyte/macrophage lineages in culture. There was no significant difference in DNA methylation between bone marrow samples from patients with high 5hmC versus healthy controls, but samples from patients with low 5hmC showed hypomethylation relative to controls at the majority of differentially methylated CpG sites. Our results demonstrate that Tet2 is important for normal myelopoiesis, and suggest that disruption of TET2 enzymatic activity favours myeloid tumorigenesis. Measurement of 5hmC levels in myeloid malignancies may prove valuable as a diagnostic and prognostic tool, to tailor therapies and assess responses to anticancer drugs.

Figure 1. The catalytic activity of Tet2 is compromised by mutations in predicted catalytic residues

a, Schematic representation of TET2. The catalytic core region contains the cysteine-rich (Cys-rich) and double-stranded beta-helix (DSBH) domains. Three signature motifs conserved among 2OG- and Fe²⁺-dependent dioxygenases are shown,. Substitutions in the HxD signature that impair the catalytic activity of TET1, leukemia-associated mutations in the C-terminal signature motifs, and corresponding substitutions introduced into murine Tet2 are indicated. b, Tet2 expression results in increased 5-hmC by immunocytochemistry. HEK293T cells transfected with Myc-tagged wild type and mutant Tet2 were co-stained with antibody specific for the Myc epitope (red) and antiserum against 5-hmC (green). DAPI (blue) indicates nuclear staining. c, Tet2 expression results in loss of nuclear 5-mC staining. HEK293T cells transfected with wild type and mutant Myc-tagged Tet2 were co-stained with antibody specific for the Myc epitope (green) and antiserum against 5-mC (red). d, Equivalent expression of wild type and mutant Myc-Tet2. CD25⁺ cells were isolated from HEK293T cells transfected with bicistronic Tet2-IRES-human CD25 plasmids, and Tet2 expression in whole cell lysates was detected by immunoblotting with anti-Myc. β-actin serves as a loading control. e, Genomic DNA purified from CD25⁺ HEK293T cells over-expressing wild type or mutant Tet2 was treated with bisulfite to convert 5-hmC to CMS (Suppl. Fig. 5a). CMS was quantified by dot blot assay using anti-CMS and a synthetic bisulphite-treated oligonucleotide containing a known amount of CMS. As positive and negative controls, we included DNA from CD25⁺ HEK293T cells transfected with TET1 catalytic domain (TET1-CD) or TET1-CD with mutations in the HxD motif (TET1-CD-HxDmut).

Figure 2. TET2 mutational status correlates with 5-hmC levels in patients with myeloid malignancies

a, Quantification of 5-hmC by anti-CMS dot blot. Left, A representative dot blot of genomic DNA isolated from bone marrow aspirates of patients with MDS/MPN and TET2 mutational status as indicated. A synthetic oligonucleotide with a known amount of CMS was used as standard. Right, The linear portion of the standard curve was used to estimate the amount of 5-hmC in DNA from patient samples. b, Bar graph of data from Fig. 2a. The three patients with TET2 mutations show lower 5-hmC levels than the three patients with wild type TET2. Error bars indicate standard deviation (n=3). c, Correlation of 5-hmC levels with TET2 mutational status. CMS levels in bone marrow samples from healthy donors and patients with myeloid malignancies (Suppl. Table S1) are shown as the median of triplicate measurements (Suppl. Fig. 7b). In the TET2 mutant group, squares, triangles, diamonds and the star indicate homozygous, hemizygous, heterozygous and biallelic heterozygous mutations, respectively (for detail definition, see online methods). The horizontal bar indicates the median for each group. p-values for group comparisons were calculated by a two-sided Wilcoxon rank sum test. Patients bearing TET2 mutations show uniformly low 5-hmC expression levels.

Figure 3. Tet2 regulates myeloid differentiation

a, b, Tet2 shRNA represses Tet2 mRNA and protein expression. a, c-Kit⁺ stem/progenitor cells from bone marrow of C57BL/6 mice were transduced with retroviruses (Suppl. Fig. 10). After selection with puromycin for 3 days, Tet2 mRNA expression was assessed by quantitative RT-PCR. Error bars show the range of duplicates. b, HEK293T cells were cotransfected with expression plasmids encoding Myc-tagged Tet2 and retroviral shRNAs. 48 h later, Tet2 protein expression was quantified by anti-Myc immunoblotting of whole cell extracts. c, Effect of Tet2 depletion on myeloid differentiation. Lin⁻ cells purified from bone marrow of C57BL/6 mice were transduced with control (scramble) or shTet2 retroviruses, then cultured in the presence of 50 ng/ml stem cell factor (SCF), puromycin (2 µg/ml) and cytokines (10 ng/ml) as indicated (also see Suppl. Fig. 10). After 4 days, flow cytometric analysis of Mac-1 vs. F4/80 (left panel) or CD115 (also known as M-CSFR, right panel) was performed. All cells were GFP⁺ at the day of analysis.

Figure 4. Relation of 5-hmC levels to DNA methylation status

a, Normalised 5-hmC (CMS) levels in DNA from three different groups: healthy controls (black diamonds), patients with mutant TET2 (red symbols) and patients with wild type TET2 (blue circles). Among TET2 mutants, we distinguish homozygous (squares), hemizygous (triangles), heterozygous (small diamonds) and biallelic heterozygous (star) mutations (for definitions see Online Methods). The horizontal bar indicates the median for each group. The number of samples in each group is indicated. b, Histogram of normalised 5-hmC (CMS) levels in DNA from healthy donors (black diamonds), patients with mutant TET2 (red rectangles) and patients with wild type TET2 (blue circles). The frequency was calculated based on a Gaussian kernel estimator. The local minimum between both modes was used as a threshold (vertical dotted line) between low and high 5-hmC values. c, Density of methylation values for healthy controls (black), high 5-hmC samples (blue) and low 5-hmC samples (red) of all sites (top panel), sites outside CpG islands (middle panel) and sites inside CpG islands (lower panel). d, Boxplot for group-specific methylation for the only two hypermethylated sites (SP140, AIM2; top panel) and the top nine hypomethylated sites (lower panels) between healthy controls and low 5-hmC samples (total number of differentially sites was 2512).