12th Congress of the European Hematology ... - Haematologica

(e.g. bcr-abl leading to CML). CSC have a fitness advantage r compared
to normal cells with fitness 1. Cells are selected for reproduction proportional
to their fitness. After each reproductive event, one cell chosen at
random from the pool is eliminated. We follow this birth-mutationexport
process for a HSC pool of ~400 cells and perform stochastic simulations
to follow the evolution of the clone and determine time probability
density functions for either extinction or complete invasion by the
mutant stem cells. Results. Stochastic dynamics leads to three possible
scenarios: clonal extinction, latency/stability or complete takeover of
the pool of the HSC pool by CSC. Although stochastic extinction is rare,
this is possible even for mutants with a significant fitness advantage. The
mutant clone can remain latent for many years. The time required for
the mutant clone to reach a threshold compatible with a diagnosis of cancer
varies significantly and has a very broad distribution, especially if the
fitness advantage conferred by the mutation is small. As the fitness
advantage increases, the time to diagnosis decreases and while extinction
is still possible this becomes a rare event. Summary and conclusions.
Stochastic considerations with respect to the active HSC pool are important
and can possibly explain observations such as stability of JAK2
mutant clones in patients with essential thrombocythemia, disappearance
of bcr-abl clones in healthy adults or transient leukemia in children
with Down syndrome. Single mutations have to give a significant fitness
advantage (r>1.7) to the cell for the disease to appear within the observed
timeframes.
0434
A PUTATIVE ROLE FOR VEGFR-1 (FLT-1) IN B CELL COMMITMENT AND DIFFERENTIATION
R. Fragoso, 1 C. Igreja, 1 C. Appleton, 2 A. Henriques, 2 N. Clode, 2 Y. Wu, 3
Z. Zhu, 3 S. Dias1 1 Portuguese institute of Oncology, LISBON, Portugal; 2 Hospital Santa Maria,
LISBON, Portugal; 3 ImClone Systems, NEW YORK, USA
VEGF and its receptors are expressed in the hematopoietic system. A
role for FLT-1 in particular was described in monocyte-macrophage
migration and differentiation, megakaryocytes maturation and dendritic
cell differentiation. Given that the expression of this receptor in the
lymphoid lineage is not known, we studied FLT-1 expression and a its
functions in normal lymphoid progenitors (B cells). To address this question
we induced in vitro CD34 + cord blood cells differentiation into the
B cell lineage using a well established assay (on S17 stromal cells). With
this approach, we observed that FLT-1 is expressed throughout B cell differentiation
increasing along the differentiation process, and reaching
its highest at the small pre-B cell stage. We also neutralized FLT-1 during
B cell differentiation in vitro. Surprisingly, in the presence of the FLT-1
neutralizing antibody (6.12 monoclonal Ab, from ImClone systems), at
the end of the assays (4 different experiments) a significantly higher
number of CD19 + cells were detected. We observed that the expression
of PU.1, Pax5 and E47 was also up-regulated by FLT-1 neutralization. To
understand if VEGF/PlGF signalling through FLT-1 promoted myeloid
differentiation, suppressed B cell differentiation or simply regulated the
quiescent state of hematopoietic stem cells, we studied the in vitro differentiation
of CD34 + /FLT-1 – and CD34 + /FLT-1 + cells (10% of CD34 +
population) using the assay described above. Interestingly, CD34 + /FLT-
1- differentiation in vitro largely promoted B cell differentiation, while
CD34 + /FLT-1+ cells originated mostly myeloid cell differentiation. These
results pointed out for a role of FLT-1 in B cell commitment. In order to
confirm it, we used FLT-1 positive and negative BM precursors to reconstitute
the BM of immunocompremised (sub lethally 350 rad irradiated)
mice. Surprisingly, FLT-1 + precursors lacked the capacity to engraft and/or
reconstitute the BM of irradiated recipients and at the end of 5 days all
mice in this group were dead. On the other hand, FLT-1 – precursors not
only reconstituted the recipient BM but also originated B cells, which
were detected/quantified in both BM and in the spleen. Next, given that
FLT-1 function was mainly associated with cell migration, and since it is
expressed at the BM latest B cell stages, we reasoned that FLT-1 might
have a role in B cells exit from the BM. For this purpose, we used LPS to
induced B cells exit from BM and pre-treated some of the mice with a
FLT-1 neutralizing Ab. FLT-1 neutralization not only prevented LPSinduced
B cells exit from BM but also decreased the number of activated
B cells in the peritoneal cavity. Taken together, our results reveal a role
for FLT-1/VEGF in regulating B cell commitment from precursor cells
and later during B cell differentiation as a chemoattractive signal.
12 th Congress of the European Hematology Association
0435
RXR α DIFFERENTIALLY REGULATES MYELOID SUBLINEAGE DIFFERENTIATION
S. Taschner, 1 C. Kösters, 1 B. Platzer, 2 A. Jörgl, 1 W. Ellmeier, 1 H. Strobl 1
1 2 Medical University Vienna, VIENNA, Austria; Harward Medical School,
BOSTON, USA
Cells of the myeloid lineage including neutrophil granulocytes (G),
monocytes (M) and dendritic cells (DC) are continously regenerated by
bone marrow resident hematopoietic stem cells. Nuclear receptor (NR)
family members are ligand-activated transcription factors that play key
roles in cellular proliferation and differentiation processes including
myelopoiesis. Retinoid X receptor alpha (RXRα) represents the predominant
NR type I and II homo- and heterodimerization partner in myeloid
cells. RXRα partner availability regulated by intracellular RXRα abundance
is thought to determine NR signaling. However its regulation in
primary human neutrophil versus M versus DC differentiation remained
uncharacterized. Here we show that human myeloid progenitors express
RXRα protein at sustained high levels during M-CSF-induced
monopoiesis. In sharp contrast, RXRα is downregulated during G-CSFdependent
late-stage neutrophil differentiation from myeloid progenitors.
Downregulation of RXRα is critically required for neutrophil development
since ectopic RXRα inhibited granulopoiesis by impairing proliferation
and differentiation. Moreover ectopic RXRα was sufficient to
redirect G-CSF-dependent granulocyte differentiation to the monocyte
lineage and to promote M-CSF-induced monopoiesis. Functional genetic
interference with RXRα signaling in hematopoietic progenitor/stem
cells using a dominant-negative RXRα promoted the generation of latestage
granulocytes in human cultures in vitro and in reconstituted mice
in vivo. Therefore, our data suggest that high levels of RXRα?are not
compatible with neutrophil development but redirect common progenitors
towards the M lineage. Furthermore we show that a third sublineage
of myeloid origin - Langerhans-type DC (LC) - are also regulated
by RXRα. RXRα activation inhibited TGFβ1-dependent LC proliferation
and differentiation and arrested LC development at a
CD14loCD11blo/- common LC/intDC/M progenitor stage. Ectopic
expression of the myeloid transcription factor PU.1 - but not RelB - fully
restored LC development from arrested progenitors in human primary
cultures, suggesting that RXRα functionally interferes with PU.1
signaling Taken together we conclude that RXRα abundance and activation
determine the fate of myeloid progenitors towards G, M and DC
in a differentiation stage and cytokine-dependent manner.
haematologica/the hematology journal | 2007; 92(s1) | 161