Abstract

Lef-1 is a nuclear protein of the Lef/Tcf family of transcription factors, known to be a key component of the Wnt/β-catenin signalling pathway. Lef-1 is crucially linked to normal B- and T-cell development. Furthermore, its aberrant expression has been associated with T-cell lymphoma and CLL. However, there are few data about the potential role of this transcription factor in normal hematopoietic stem cell and progenitor development. Aim of this project was to clarify the expression pattern of Lef-1 in early hematopoietic progenitor cells and to test whether constitutive expression of this transcription factor affects early hematopoietic development. Analysis of Lef-1 expression by semi-quantitative RT-PCR and Real Time PCR demonstrated Lef-1 expression in both lymphoid (B220+, CD4+, CD8+) and myeloid (Gr1+, Mac1+) subpopulations, but also in highly purified hematopoietic stem cells (Sca1+/Kit+/Lin). In order to prove functional relevance of Lef-1 expression, constitutive expression of Lef-1 and of a constitutive active Lef-1 mutant (CA-Lef-1; with a Lef-1 activating β-catenin domain) was induced in primary murine bone marrow (BM) cells by retroviral gene transfer, using a MSCV based retroviral construct with an IRES-GFP cassette. At the level of clonogenic progenitor cells, both Lef-1 and CA-Lef-1 increased the colony forming potential of progenitors in vitro by more than 2-fold compared to the empty vector control (n=4, p<0.03). At the level of the short-term repopulating stem cell, Lef-1 remarkably increased the size and the number of spleen colonies resulting in a 8fold increase in the CFU-S frequency compared to the control (median 120 CFU-S/1x105 versus 15 CFU-S/1x105 cells, respectively; p< 0.001; Lef-1 n=5, control n=13). To assess the impact of Lef-1 on long-term repopulating stem cells mice were transplanted with BM cells transduced either with Lef-1 or CA-Lef-1. Both Lef-1 constructs severely perturbed normal hematopoietic development inducing a reduction of lymphoid cells with an inversion of the lymphoid/myeloid ratio (ratio 0.03 vs. 5.8 in the non-transduced compartment) and accumulation of neutrophils in the peripheral blood (97 % Gr1 positive cells versus 15 % in the non-transduced compartment) as well as in the spleen (lymphoid/myeloid ratio 0.2 vs. 6.9 and 87 % versus 12 % Gr1+ cells). 3 mice (1 Lef-1 and 2 CA-Lef-1 mice) succumbed to a lethal myeloproliferative syndrome, one mouse (Lef-1) developed acute leukemia, which was readily transplantable into secondary and tertiary recipients and showed indefinite IL-3 dependent cell growth in vitro. Taken together, these data show that ordered expression of Lef-1 plays a key role in early hematopoietic development and that deregulation of this transcription factor favors the development of myeloid malignancies.

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