One of the most elusive goals in hematologic research has been to understand how hematopoietic stem cells (HSC) self-renew, since this knowledge could be applied in protocols to expand clinically useful numbers of HSC for bone marrow transplantation and targeted gene therapy for hematologic disorders. A number of studies have suggested an essential role for homeodomain-containing proteins belonging to the Hox gene family in the regulation of adult HSC self-renewal. Experiments where the homeobox gene, Hoxb4, was overexpressed using a retroviral vector in HSC resulted in a 40-fold expansion in long-term repopulating HSC (LT-HSC) numbers in vitro. A similar stimulation of LT-HSC symmetric self-renewal in vitro was observed when Hoxb6 or Hoxa9 were overexpressed in LT-HSC. Loss of Mll, a mammalian trithorax homolog that functions to maintain Hox gene expression, results in the loss of adult LT-HSC in vivo, which further supports the hypothesis that Hox gene activity is essential for HSC self-renewal. Recent experiments where the acute myeloid leukemia oncoprotein NUP98-HOXA10 was expressed in murine hematopoietic progenitor cells using a retroviral vector showed an unprecedented 10,000-fold expansion of LT-HSC over a two-week in vitro culture period. In studies outlined in this proposal, we have observed a similar 10,000-fold in vitro expansion of adult LT-HSC using a related Hox fusion protein, NUP98-HOXA9. The major objective of this proposal will be to delineate the molecular mechanisms responsible for the dramatic enhancement of LT-HSC symmetric self-renewal mediated by NUP98- HOXA9 using genetic and biochemical approaches.
In Aim 1, we will functionally test whether genes commonly up-regulated in LT-HSC by NUP98-HOXA9, Hoxb4, and Hoxa9 contribute to NUP98-HOXA9- mediated symmetric self-renewal.
For Aim 2, we will characterize the extent to which Hoxa5 and Hoxb5 contribute to the potent HSC self-renewal phenotype stimulated by NUP98-HOXA9. Hoxa5 and Hoxb5 are the two most highly up-regulated Hox genes in LT-HSC expressing NUP98-HOXA9 so these paralogous genes may have a unique function in stimulation of LT-HSC expansion. Finally, in Aim 3 we will define the roles of the Hox co-factors, Pbx1 and Pbx3, in LT-HSC self-renewal mediated by Hoxb4 and NUP98- HOXA9 and identify NUP98-HOXA9-interacting proteins in LT-HSC using mass spectrometry. Our ability to expand LT-HSC 10,000-fold in vitro using NUP98-HOXA9 will allow, for the first time, conventional biochemistry to be done using a pure population of LT-HSC. This will facilitate the biochemical characterization of Hox protein complexes that are functioning in LT-HSC to regulate the self-renewal process. Collectively, these studies will provide new insights into the genes and pathways controlling adult LT-HSC symmetric self-renewal that is stimulated by Hox gene activity.
The ability to expand hematopoietic stem cells (HSC) in vitro to clinically useful numbers has tremendous application in the areas of bone marrow transplantation and gene therapy for hematologic disorders. In this proposal, we will explore the underlying molecular basis for the dramatic 10,000-fold ex vivo expansion of adult bone marrow HSC that is stimulated by the Hox fusion protein, NUP98-HOXA9.