TET2 Is Essential for Survival in Mice, and Decreased TET2 Expression Enlarges HSC Compartment and Alters Cell Differentiation

Abstract 2471

Loss-of-fuction mutations of Ten-Eleven-Translocation-2 (TET2) was found in a variety of myeloid malignancies. But little is known about the function of TET2 in normal hematopoiesis and in the pathogenesis of myeloid malignancies. To study the function of TET2 in hematopoiesis in vivo, we conducted detailed analyses on hematopoiesis of TET2 gene trap mice (Tang et al. 2008), in which gene trap vector was inserted into exon 2 of TET2.

Homozygous (trap/trap) mice were born at the proportion with Mendelian expectations, but died at a high rate by 2 days after birth. The fraction of trap/trap mice at P0 and P3 was 33% and 12%, respectively. TET2 mRNA expression level in fetal liver cells (FLs) at embryonic day 14.5 (E14.5) was approximately 60% in trap/wt mice and 20% in trap/trap mice compared to wild-type (WT) mice. Homozygous TET2 gene trap mice revealed to be TET2 low expression mice (TET2^low).

In E14.5 TET2^low FLs, cellularity was comparable to WT FLs. The proportion of lineage-marker⁻Mac-1⁺Sca-1⁺ cells containing hematopoietic stem cells (HSCs) and multipotent progenitors was higher in TET2^low FLs compared to WT FLs (0.43±0.05% vs 0.18±0.01%, p<0.01)). In colony forming assay, CFU-GM was significantly increased in number in TET2^low FLs compared to WT FLs (104±18 vs 83±16, p=0.03).

We next transplanted FLs from WT or TET2^low mice into lethally irradiated recipient mice, and the hematological parameters were examined. Twenty weeks after transplantation, there were no significant differences in blood counts between two groups. The difference in peripheral blood (PB) was observed in the proportion of Gr-1⁺/Mac1⁺ cells and monocytes. The proportion of Gr-1⁺/Mac1⁺ cells and monocytes of WBCs in recipient mice transplanted with WT FLs was 20.7±1.6% and 11±1%, respectively, and that in recipient mice transplanted with TET2^low FLs was 29.7±3.4% and 16±6%, respectively (p<0.05). In BM compartments including LSK, there were no significant differences between two groups. TET2^low BM cells presented a functional increase in colony forming progenitors in vitro. The number of CFU-GM in 2 × 10⁴ BM cells from recipient mice transplanted with WT FLs and TET2 FLs was 130±6 and 199±9, respectively (p<0.01).

Recipient mice transplanted with TET2^low FLs showed splenomegaly and extramedullary hematopoiesis. The spleen weights in recipient mice transplanted with WT FLs and TET2^low FLs was 0.076±0.003g and 0.092±0.005g, respectively (p=0.02). The proportion of LSK, CD41⁺, and CD71⁺TER119⁻ cells was higher in recipient mice transplanted with TET2^low FLs compared to those with WT FLs. The number of CFU-GM and CFU-MEP was also higher in TET2^low group.

To determine whether TET2 low expression affects the HSC function in vivo, we performed a competitive repopulation assay by transplanting 1 × 10⁶ CD45.2⁺ FLs from WT or TET2^low mice along with 1 × 10⁶ CD45.1⁺ WT BM cells (BMs), into lethally irradiated CD45.1⁺ recipient mice (1^st recipient mice). After 12 weeks, 1 × 10⁶ BMs from 1st recipient mice were serially transplanted into irradiated recipient mice (2nd recipient mice). The contribution of CD45.2⁺ cells in total and differential lineage of WBC was analyzed. The proportion of CD45.2⁺ in total WBC from 1st recipient mice transplanted with WT and TET2^low FLs was 75±2.6% and 88±1.1%, respectively. In the 2nd recipient mice, the contribution of TET2 low cells was still high as 89±1.1%, although that from WT cells decreased to 54±4.5%. In both myeloid and lymphoid lineages, the contribution of TET2^low cells was higher compared to that of WT cells. The proportion of CD45.2⁺ cells in Gr-1⁺/Mac1⁺ cells, B220⁺ cells, and CD4⁺/CD8⁺ cells in 2nd recipient mice transplanted with WT BMs was 49±6.5%, 72±4.7%, and 41±3.3%, respectively, and that in 2nd recipient mice transplanted with TWT2^low BMs was 92±0.6%, 97±0.7%, and 64±2.7%, respectively.

These data indicate that (1) TET2 is essential for survival in mice, (2) the decreased TET2 expression resulted in the enlargement of HSCs, and cell-autonomous growth and competitive advantage in hematopoietic cells, (3) the decreased TET2 expression altered the cell differentiation skewing toward myeloid/monocytic lineages. TET2 controls the hematopoietic homeostasis.