Tet2 Negatively Regulates Homeostasis and Differentiation of Hematopoietic Stem Cells in Mice

Abstract Abstract 2354 Epigenetic alterations in cancer cells include aberrant DNA methylation and histone modifications. Specifically, cancer cells display global hypomethylation associated with genomic instability as well as promoter hypermethylation associated with inactivation of tumor suppressor, cell cycle or repair-related genes. In the hematopoietic system, whole-genome sequencing and other genetic analyses have led to the discovery of recurrent somatic alterations that contribute to the pathogenesis of a variety of myeloid malignancies by perturbing the epigenetic landscape of cancer cells. Ten-Eleven-Translocation (TET) family enzymes, TET1, TET2, and TET3 modify DNA methylation status by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in a 2-oxoglutarate and Fe2+-dependent manner. TET2 is located in chromosome 4q24, a region undergoing frequent microdeletions and uniparental disomy in patients with a wide spectrum of myeloid malignancies. Somatic mutations in TET2 are some of the most prevalent acquired mutations in myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), MDS/MPN overlap syndromes including chronic myelomonocytic leukemia (CMML), acute myeloid leukemia (AML) and secondary AML (sAML). We previously showed that missense mutations of TET2 in myeloid malignancies are loss-of-function mutations that compromise its dioxygenase activity. TET2 mutations correlate with decreased levels of 5hmC in patients. Tet2-depleted mouse hematopoietic precursor cells are preferentially committed to differentiation towards monocyte/ macrophage lineages in culture. The levels of DNA methylation in patients with high 5hmC versus healthy controls are similar, however, samples from patients with low 5hmC show hypomethylation relative to controls at the majority of differentially methylated CpG sites. Although it is postulated that impaired TET2 activity may potentiate myeloid transformation by influencing hematopoietic stem/progenitor cells (HSPCs), it has yet to be directly tested whether Tet2 mutations or deletions are implicated in abnormal hematopoiesis in vivo. To clarify the function of Tet2 in hematopoietic development, we generated mice with targeted disruption of the Tet2 catalytic domain and found that Tet2 is critical for self-renewal and differentiation of hematopoietic stem cells (HSCs). Ablation of Tet2 specifically repressed Tet2 expression with no effect on the other Tet family members, Tet1 and Tet3. Dot blot analysis showed that Tet2-deficient cells contain significantly diminished levels of genomic 5hmC in several organs examined. Tet2 deficiency augmented the frequency and absolute number of HSPC compartment in a cell-autonomous manner. In competitive transplantation assays, Tet2-deficient HSCs were capable of multi-lineage reconstitution and possessed a competitive advantage over wild type HSCs, resulting in enhanced hematopoiesis into both lymphoid and myeloid lineages. In vitro differentiation assays showed that Tet2 restrains HSCs from undergoing differentiation, as assessed by expression of lineage markers upon differentiation. Despite this antagonizing effect, however, the number of monocyte/ macrophage cells was greater in Tet2−/− cultures compared with controls, and immature Tet2−/− progenitor cells differentiated prematurely into the monocyte/macrophage lineage. These results indicate that Tet2 deficiency alters stem/progenitor cell properties to delay HSC differentiation and induce developmental skewing towards the monocyte/macrophage lineage. Taken together, these studies indicate that Tet2 has a critical role in regulating the expansion and self-renewal of HSCs. Our data suggest that cell fate decisions of HSPC are affected by TET2 mutations that decrease enzymatic activity, and that this phenomenon has a crucial role in the pathogenesis of diverse myeloid malignancies. We are testing whether Tet2 deficiency synergises with other recurrent mutations to promote myeloid malignancies. Because loss-of-function mutations in TET1 or TET3 have not been reported in most TET2-mutated cancer and TET2 loss-of-function seems to facilitate myeloid transformation because of impaired 5hmC production, it might be beneficial from the perspective of cancer therapies to develop strategies to activate the enzymatic activity of other TET proteins. Disclosures: No relevant conflicts of interest to declare.