Characterization of the Proteins HPIP and VENTX2 as Novel Regulatory Proteins of Human Hematopoiesis

The hallmark of hematopoietic stem cells (HSC) is their ability of
self-renewal and differentiation into multiple hematopoietic cell
lineages. Although the molecular network controlling stem cell fate
decisions is largely unknown, multiple studies have attributed a
key role to transcription factors in this developmental process. In
this context the family of homeobox genes was characterized as
‘master genes’ of this early hematopoietic development. The
identification of new genes involved in normal and leukemic
hematopoiesis and the development of therapies against deregulated
processes in hematopoiesis are the major goals in experimental and
clinical hematology. . The identification of new genes involved in
normal and leukemic hematopoiesis and the development of therapies
against deregulated processes in hematopoiesis are the major goals
in experimental and clinical hematology. Therefore, the focus of
this thesis was the characterization of two novel putative
regulatory proteins of early human hematopoiesis, the hematopoietic
PBX-interacting protein (HPIP) and the human Vent-like Homeobox
gene (VENTX2) and to investigate to the activity of the FLT3
protein kinase inhibitor SU5614 on leukemic blast from AML patient
samples. Using complex in vitro assays we analyzed the impact of
constitutive expression of HPIP and VENTX2 on stem cell and early
human hematopoietic development. To detect clonal progenitor cells
primary and secondary colony-forming-unit (CFC) assays were
performed. In addition the in vitro equivalent of HSC long-term
culture initiating cells were detected with the (LTC-IC) assay. We
were able to show that the constitutive expression of HPIP can
rapidly lead to increased numbers of cells detected on the level of
committed clonogenic progenitor cells and LTC-ICs. In addition, the
production of CFC per LTC-IC is markedly enhanced when cord blood
(CB) cells are transduced with HPIP as compared to the control.
Notably, besides its effect on maintenance of primitive
hematopoietic progenitor cells, constitutive expression of HPIP did
not block terminal hematopoietic differentiation. Additional we
could show that the constitutive expression of HPIP leads to an
increase of myeloid cells in transplanted NOD/SCID mice. These data
characterize HPIP as a novel regulator of the early human
hematopoietic stem cell, demonstrating that its constitutive
expression has a notable impact on self renewal and differentiation
of human hematopoietic stem cells. In vitro and in vivo analyses
shed light on the understanding to the function of the homeobox
gene VENTX2. On the level of the most primitive hematopoietic
progenitors we could not observe a significant increase in the
frequency of HSCs. Furthermore, the number of colonies generated
per LTC-IC did not significantly differ between the VENTX2 arm and
the control arm. A strong effect was obtained on the level of
clonogenic progenitor cells. VENTX2 increased the production of
myeloid cells 1.7-fold in comparison to the control. Secondary
replating assay confirmed the amplificatory effect of VENTX2
transduced cells in the number of secondary G-CFU indicating that
VENTX2 promote myeloid lineage differentiation. Interestingly, on
the level of clonogenic progenitors VENTX2 expression resulted in a
significantly decreased growth of erythroid colonies by 4.2-fold
compared to the control suggesting that constitutive expression of
VENTX2 may inhibit early erythroid differentiation. This inhibition
did not occur on the level of primitive hematopoietic cells
detected by Limiting Dilution LTC-IC assay where VENTX2 increased
within a 2.2 fold compared to the control. The observation that
VENTX2 overexpression drives CD34+ to differentiate into myeloid
lineage was additional proved by in vivo experiments. In NOD/SCID
mice VENTX2 induced a 3-fold increase in the proportion of CD15+
mature myeloid cells within the GFP-positive compartment compared
to the control. A 7-fold increase was observed in the total of
CD38+ GFP+ cells in comparison to the MIG mice control (p