HOXB4 confers a constant rate of in vitro proliferation to transduced bone marrow cells

HOXB4 overexpression mediates increased self-renewal of haematopoietic stem cells (HSCs) ex vivo. Since HOXB4-expanded HSCs retain normal differentiation potential and there is no leukaemia development from transduced HSCs, HOXB 4 represents a promising tool for human HSC therapy. However, the increased proliferation capacity of HOXB4 overexpressing fibroblasts resulting from upregulation of JunB, Fra-1 and cyclin D1 protein levels may indicate a potential risk associated with the HOXB4 overexpression approach. This prompted us to investigate the proliferation rate, differentiation and expression of cell cycle regulators directly in bone marrow cultures overexpressing HOXB4. Here we show that in comparison to neo-transduced control bone marrow cultures, HOXB4-overexpressing cultures had a more homogenous morphology and increased numbers of haematopoietic progenitor cells capable to generate primitive colonies in vitro. In contrast, neo-transduced bone marrow cells in long-term cultures showed hallmarks of myeloid differentiation and a reduced secondary colony forming activity. We further show that multilineage repopulating activity in vivo, which was present only in HOXB4 long-term cultures, declined over time. HOXB4 overexpression in vitro did not result in an increase but in a stabilization of the proliferation rate (1.4 - 1.8 cell divisions per day), while the proliferation rate of control neo-transduced bone marrow cultures gradually declined. Correspondingly, increased HOXB4 expression was paralleled by decreased expression levels of cyclins, CDKs and AP-1 family members. These results suggest that the growth rate of HOXB4- compared to neo-transduced bone marrow cells remains constant in long-term cultures along with a suppression of myeloid differentiation. In contrast to HOXB4 overexpression in fibroblasts, bone marrow cells engineered to overexpress HOXB4 do not upregulate AP-1 complex members or cyclins indicating that HOXB4 acts in a cell type-specific way.