Investigating the Biological and Molecular Mechanisms Underpinning the Engraftment/Selection Advantage Conferred to Hematopoietic Stem Cells by HOXB4.

Abstract

We have recently demonstrated that co-expression of HOXB4 enables the enhanced delivery of HSC harbouring a second therapeutic trans-gene. Nonetheless, it is of great importance to elaborate the current knowledge about the mechanism of HOXB4 action in order to both evaluate the safety implications of its use in a clinical strategy, and to gain greater insight into the regulation of HSC self-renewal/expansion. To these ends we have performed an extensive in vitro analysis of the consequences of HOXB4 overexpression in primary murine BMC and in a murine multipotent myeloid progenitor cell line (FDCP-mix). We demonstrate for the first time in murine cells, that ectopic HOXB4 reduces the responsiveness of murine hematopoietic cells to differentiation stimuli. Furthermore, by performing a detailed investigation into the kinetics of FDCP-mix differentiation, we reveal that HOXB4 overexpression results in a specific differentiation delay as opposed to an outright block. We propose that an analogous delay is in operation in repopulating cells in order that the shift to increased assymetrical self-renewal, a requirement for stem cell expansion, is achieved. Notwithstanding this, it is clear that any perturbation in differentiation constitutes an increased risk of cellular transformation if this technology were transferred to a clinical setting. In order to further define the repercussions of ectopic HOXB4 delivery, we have developed a retroviral vector which encodes an activatable version of HOXB4. We have shown that this vector is able to mediate an in vitro differentiation delay in primary murine BMC and FDCP-mix as well as enable enhanced engraftment of BMC in vivo, both dependent upon the addition of the estrogen analogue; tamoxifen. Using this system, we are currently examining the effect of ectopic HOXB4 on the transcriptome of FDCP-mix cells, in addition to performing an in depth study into the biological mechanisms affected by HOXB4 overexpression in BMC in vivo. We envisage that these model systems will be particularly amenable to the manipulation required for target gene identification/validation.