The Transcriptional corepressorTle4 Is a Novel Regulator Of Murine Hematopoietic Stem and Progenitor Cells and Their Niche

We previously identified Transducin-like/Enhancer of Split 4 (TLE4) as a potential tumor suppressor gene in acute myeloid leukemia whose loss appears to complement AML1-ETO induced leukemia. In this study we examined the role of Tle4 in normal hematopoiesis. Using Tle4 knockout mice we demonstrated that Tle4 is critical for maintaining a supportive hematopoietic stem cell niche, and also has intrinsic effects on HSC survival and B-cell differentiation.

Tle4 knockout mice exhibit profound defects in bone mineralization

Tle4 knockout mice become runted by 2 weeks of age and generally die by the time of weaning. These mice exhibit a profound defect in mineralization of the bone and bone marrow failure by 3.5 weeks. At birth KO mice showed a significant reduction in the degree of calcification of the skull and long bones, despite no significant differences in overall body size or weight at this early time point. This was confirmed by Von Kossa staining for calcified bone of one-day old tibiae from KO and WT mice. At 21-28 days after birth trabecular bone was almost absent in tibiae and femurs with thin cortices of the long bones. These data indicate Tle4is critical for proper bone mineralization. In further support of this novel role of Tle4 in skeletal development, a mutation in TLE4 has recently been identified in a father and daughter with congenital vertebral malformations including delayed anterior fontanelle closure, shortened vertebral pedicles and kyphoscoliosis.

Tle4 deficient bone marrow stroma is defective in supporting hematopoietic stem and progenitor cells

To determine if the hematopoietic abnormalities in Tle4 KO mice might in part be due to the observed defects in the skeletal or stromal compartment of the BM, we cultured WT LKS cells on WT or KO stromal cells obtained at 2 weeks, prior to the observation of bone marrow hypoplasia. In WT cocultures, 6-15% of recovered cells were positive for both c-kit and Sca-1. In stark contrast, less than 1% of cells were C-kit+Sca-1+, when WT LKS cells were plated in KO stromal cocultures. Long-term coculture experiments in methylcellulose revealed an even more pronounced effect, with cells recovered from WT cocultures generating on average 10-fold more colonies than cells recovered from KO cocultures. Some KO cocultures failed to exhibit any colony forming ability. Therefore, these data suggest that Tle4-/- stromal cells cannot maintain and support HSPC growth as efficiently as WT stromal cells. In addition, Western blots of whole bone lysates from two week old mice showed that the majority of Tle4 KO mice had a reduction in Scf protein levels.

Tle4 deficient hematopoietic stem cells exhibit cell intrinsic defects in B-cell differentiation and survival

Due to the progressive leukopenia in primary Tle4 KO mice, and the extrinsic effects noted above, we sought to determine whether loss of Tle4 intrinsically affected self-renewal and repopulation efficiency of HSC in serial transplantation assays using BM and fetal liver cells.

Whole BM from two-week-old KO and WT animals transplanted into lethally irradiated allogeneic CD45.1 mice showed similar levels in donor chimerism in blood, but significant reductions in the frequency of B cells in KO transplanted mice at 16 weeks. At 32 weeks after transplant, recipient mice of KO BM developed significant leucopenia, mostly due to a decreased frequency of B cells. To exclude any effects of the bone marrow stroma we also performed serial transplantations from fetal livers of embryonic day 13.5 WT and KO fetuses. In this transplant model, both peripheral leukopenia and specific B cell lymphopenia were observed in primary transplant recipients, recapitulating the phenotype observed in recipients of KO BM. Although in the primary transplants no difference in the frequency of HSC between recipients of KO or WT BM was observed, progressively with secondary and tertiary transplants we observed a striking hematopoietic stem cell exhaustion with reduction in LKS cells, CD34-LKS cells, and LKS CD34-CD48-CD150+ long term HSC.

Taken together, these data indicates Tle4 is a critical regulator of hematopoiesis acting by maintaining supportive function of the bone marrow niche as well as intrinsically affecting B-cell differentiation and the survival of hematopoietic stem cells.