Disruption of Tet2 Leads to Enhanced Self-Renewal and Competitive Repopulating Capacity of Fetal Liver Hematopoietic Stem Cells

Abstract 2331

TET2 (ten-eleven-translocation 2) gene has been reported to be frequently mutated in various human myeloid malignancies, including myeloproliferative neoplasms, myelodysplastic syndromes, acute myeloid leukemia, and chronic myelomonocytic leukemia. These observations suggest critical roles of TET2 dysfunction in molecular pathogenesis of myeloid malignancies. Recent studies using conditional knockout mouse model indicated that mouse Tet2 loss leads to clonal dominance of adult hematopoietic stem cells (HSCs) in competitive repopulation assay. However, self-renewal capacity of adult HSCs has never been addressed precisely by serial transplantation assay. In addition, the effect of Tet2 loss on hematopoietic stem/ progenitor cells was examined only in the BM, but not in the fetal livers (FLs). Since FL HSCs and adult HSCs differ in several aspects of their phenotypes and functions, we speculated that Tet2 might be involved differently in the regulation of FL and adult hematopoiesis.

To address this issue, we analyzed E14.5 FL cells from Tet2 gene-trap (Tet2^gt) mice. In these mice, gene trap-cassette was inserted into the second intron, just before the first coding exon. RT-PCR analysis showed that over 99% of Tet2 mRNAs from endogenous promoter were trapped by the gene-trap cassette in Tet2^gt/gt mice, showing that Tet2^gt allele can be considered as a null allele. Initial analysis showed that Tet2^gt/gt embryos developed normally, but most Tet2^gt/gt mice were perinatally lethal. Total numbers of FL cells and the numbers of committed progenitors in FLs as revealed by colony assays were not significantly different between each genotype. Interestingly, Tet2^gt/gt embryos displayed significant increase in lineage (Lin)(-)Sca-1(+)c-Kit(+)(LSK) fraction compared to wild type (WT) (Tet2^+/+) littermate (2.42±0.66% vs. 1.17±0.18%, p=0.02). In addition, common myeloid progenitor (CMP) fraction (IL7Rα(-), Lin(-), Sca-1(-), C-Kit(+), CD34(+), FcgRII/ III(low)) was significantly increased in Tet2^gt/gt FLs compared to WT (9.04±1.09% vs. 6.26±0.53%, p=0.008). In serial transplantation assays, donor cells derived from Tet2^+/gt and Tet2^gt/gt FLs showed significantly higher peripheral blood chimerism in secondary and tertiary recipient mice as compared to that of WT cells, showing that disruption of Tet2 leads to the enhanced self-renewal capacity of FL HSCs. Moreover, donor-derived HSC fraction (CD34⁻LSK cells) was significantly expanded in the recipients of Tet2^gt/gt FL cells, suggesting that increased self-renewal capacity is cell intrinsic to Tet2^gt/gt HSCs. We have also examined differentiation of Tet2-mutant FL cells in the recipients' peripheral blood, and found that Tet2^gt/gt cells displayed impaired differentiation to Gr-1(+)CD11b(+) mature granulocytes (WT vs. Tet2^gt/gt = 5.02±1.35% vs. 11.5±3.09% in the primary recipients) and slight, but significant increase of B cells. Liquid culture of FL cells with cocktails of cytokines in vitro demonstrated that Tet2^gt/gt FL cells retained higher percentage and number of LSK, Lin- and c-Kit⁺ cells after the culture for 7-days compared to WT cells, showing enhanced resistance of Tet2^gt/gt cells to differentiative stimuli in in vitro culture. It is of note that Tet2^+/gt mice showed a significant increase in hematopoietic stem/progenitor fraction (LSK) in the BM compared to wild type littermate (0.48±0.11% vs. 0.32±0.04%, p=0.04). However, they presented no signs of extramedullary hematopoiesis such as splenomegaly and expansion of LSK cells in spleens during an observation up to 35-weeks.

Taken together, we demonstrate that Tet2 critically regulates self-renewal and long-term repopulating capacity of FL HSCs and has pleiotropic functions in myeloid and lymphoid differentiation. These data strongly indicate that Tet2 is an essential regulator of BM and FL hematopoiesis. In addition, enhanced HSC self-renewal, expansion of HPC and myeloid progenitors and perturbed myeloid differentiation induced by TET2 ablation likely to set molecular basis for myeloid transformation, which explains high incidence of loss-of-function mutations of TET2 in myeloid malignancies.