Long-Term Cell Autonomous Effect of Tet2 Loss in Hematopoietic Cells in Mice.

Abstract 2416

TET2 mutations are frequently observed in myeloid malignancies including myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN) and MDS/MPN. Several groups have already reported that deletion of Tet2 in mice leads to dysregulated hematopoietic stem cell self-renewal and subsequent development of myeloid malignancies. Of note, mice hypomorphic or heterozygous for the Tet2 allele have been reported to show similar phenotypes as those of Tet2-null mice, suggesting that haploinsufficiency of Tet2 plays a role in the development of myeloid malignancies. However, little is known about long-term cell autonomous effects of Tet2 loss in hematopoietic cells: most of the reports were based on relatively short-term observations or did not exclude the influence of Tet2 loss in the niche cells. To study long-term cell autonomous effect of Tet2 loss in hematopoietic cells, we analyzed the hematopoiesis of wild-type recipient mice reconstituted with fetal liver cells from Tet2 hypomorphic mice for a longer period up to 1 year.

Tet2 gene trap mice (Tet2^trap/trap), in which the gene trap vector was inserted into the exon 2 of Tet2 just before the first coding exon, express Tet2 mRNA at the level approximately 20% of that of the wild-type (WT) mice (Shide et al. Leukemia 2012). We transplanted fetal liver cells from E14.5 WT or Tet2^trap/trap mice into lethally irradiated recipient mice. At 4 months after transplantation, the recipient mice reconstituted with Tet2^trap/trap cells showed a significantly increased proportion of monocytes in peripheral blood (PB) compared with those with WT cells (WT=5.59±2.57%, Tet2^trap/trap=12.67±7.45%, p=0.01). While there were no significant differences between the two groups in the bone marrow (BM) compartments including the numbers of Lineage⁻Sca-1⁺c-Kit⁺ (LSK) hematopoietic stem/progenitor cells, extramedullary hematopoiesis in the spleen was markedly enhanced in the recipients with Tet2^trap/trap cells. The proportion of LSK, granulocyte/macrophage progenitors (GMPs) and megakaryocyte/erythroid progenitors (MEPs) in the spleen of recipient mice reconstituted with WT and Tet2^trap/trap cells were 0.002±0.001% vs 0.006±0.001% (p<0.01), 0.007±0.004% vs 0.029±0.01% (p=0.026) and 0.084±0.024% vs 0.25±0.044% (p<0.01), respectively. These findings were compatible with those reported previously and indicated that recipient mice reconstituted with Tet2^trap/trapcells induce chronic myelomonocytic leukemia (CMML)-like disease.

Of note, after a long observation period, particularly after 9 months post-transplantation, mice reconstituted with Tet2^trap/trap cells developed advanced hematological disease and 53.3% (8 of 15) died or were killed because of their moribund condition by 11 months after transplantation. Detailed analysis on moribund as well as surviving mice reconstituted with Tet2^trap/trap cells (n=3 each) revealed that the 2/3 of the mice developed CMML-like disease with monocytosis in PB and evident extramedullary hematopoiesis harboring increased number of LSK and GMPs in spleen, whereas the remaining 1/3 of mice developed MDS/MPN-like disease with severe anemia. The latter mice did not show monocytosis in PB and BM, but displayed dyserythropoiesis accompanied by massive extramedullary erythropoiesis, as we saw increased number of LSK and MEPs, but not GMPs, in spleen. The proportion of Annexin V+ cells in Ter119^highCD71^higherythroblasts in the BM of WT mice, CMML-like disease-carrying mice and MDS/MPN-like disease-carrying mice were 9.46±0.53%, 10.71±0.36%, and 19.04±0%, respectively, suggesting that the enhanced apoptosis led to the severe anemia seen in the MDS/MPN-like disease-carrying mice. Thus, decreased expression of Tet2 is sufficient to promote not only CMML-like disease, but also an MDS/MPN-like disease as well.

Our findings confirmed a long-term cell autonomous effect from insufficient function of Tet2 in the development of hematological diseases. Interested in the molecular mechanism of disease progression, we have identified 1,642 differentially methylated regions (DMRs) in Tet2^trap/trap GMPs compared with WT GMPs by ChIP-sequencing. We are now working to understand how these DMRs are involved in the development of the two distinct diseases associated with hypomorphic Tet2.