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

12 th Congress of the European Hematology Association
Stem cell biology and microenvironment
0431
INVERSE REGULATION OF HEMATOPOIETIC PROGENITOR CELL EGRESS BY MT1-MMP
AND RECK
A. Avigdor, 1 Y. Vagima, 2 P. Goichberg, 2 S. Shivtiel, 2 M. Tesio, 2 A. Dar, 2
A. Kalinkovich, 2 I. Petit ,2 O. Perl, 1 E. Rosenthal, 1 I. Resnick, 3 I. Hardan, 1
A. Nagler, 1 T. Lapidot2 1 The Chaim Sheba Medical Center, TEL-HASHOMER; 2 The Weizmann Institute
of Science, REHOVOT; 3 Hadassah Medical Center, JERUSALEM, Israel
Background. hematopoietic stem and progenitor cells continuously exit
from the bone marrow (BM) reservoir to the blood circulation as part of
homeostasis and host defense. Clinical recruitment of progenitors to
the peripheral blood (PB) is achieved by chemotherapy and repeated G-
CSF stimulations, which mimic physiological egress of stem and progenitor
cells during injury and inflammation. Yet, mechanisms governing
progenitor cell trafficking during steady state homeostasis and clinical
mobilization are not fully understood. Membrane type-1 metalloproteinase
(MT1-MMP) and its endogenous inhibitor, reversion-inducingcysteine-rich
protein with Kazal motifs (RECK), are established key regulators
of tumor and endothelial cell motility. Methods and Results. In our
study we found that egress of human hematopoietic CD34 + progenitors
requires cell autonomous changes in their motile properties inversely
mediated by MT1-MMP and its endogenous inhibitor, RECK. By flow
cytometry analysis we detected higher MT1-MMP and lower RECK
expression on circulating human CD34 + cells and maturing leukocytes
as compared to immature BM cells. MT1-MMP expression was even
more prominent on CD34 + cells obtained from PB of G-CSF-treated
healthy donors whereas RECK labeling was barely detected. In addition,
five daily injections of G-CSF to NOD/SCID mice, previously engrafted
with human cells, increased MT1-MMP and decreased RECK expression
on human CD45 + leukocytes, human immature CD34 + and primitive
CD34 + /CD38 – /low cells, in a PI3K/Akt1-dependent manner, resulting
in elevated MT1-MMP activity. Inverse regulation of MT1-MMP
and its inhibitor RECK by G-CSF induced mobilization was confirmed
by in situ immuno-labeling of BM sections, as well as by human MT1-
MMP and RECK mRNA expression analysis of leukocytes repopulating
the BM of chimeric mice. Blocking MT1-MMP function halted mobilization,
while RECK neutralization promoted egress of human CD34+ progenitors
in the functional model of NOD/SCID chimeric mice. Treatment
with MT1-MMP neutralizing Ab or with tissue inhibitor of metalloproteinase-2
(TIMP-2) reduced the in vitro chemotactic response to
SDF-1 of human G-CSF-mobilized CD34 + cells via matrigel coated filters.
In contrast, blocking RECK function by neutralizing Ab, thus abrogating
RECK-mediated inhibition of MT1-MMP, facilitated SDF-1induced
migration of steady state human BM CD34 + cells. Following G-
CSF-induced mobilization, we also observed a reduction in CD44
expression on human leukocytes and, specifically, on immature CD34 +
progenitor cells in the BM of chimeric mice. This was accompanied by
accumulation of CD44 cleaved products of molecular weights, expected
for MT1-MMP activity, in the BM supernatants. In chimeric mice coinjected
with MT1-MMP-neutralizing Ab, less cleavage of CD44 was
detected upon G-CSF mobilization, whereas in the absence of a mobilizing
signal, increasing MT1-MMP activity by anti RECK Ab injection
facilitated CD44 proteolysis on the BM cells. Finally, MT1-MMP expression
correlated with the number of CD34 + cells, collected on the first
apheresis day in 29 consecutive patients with lymphoid malignancies
and in 21 healthy donors treated with G-CSF. Conclusions. our results
indicate that MT1-MMP and RECK oppositely control G-CSF inducedhematopoietic
progenitor cell mobilization, by regulating CD44 surface
expression and ultimately, cell adhesion and motility. These molecules
might serve as targets for new approaches to improve clinical stem cell
mobilization and repopulation.
160 | haematologica/the hematology journal | 2007; 92(s1)
0432
SNX5, A NOVEL GENE LINKED TO FANCONI ANAEMIA CAUSES HAEMATOPOIETIC
FAILURE WHEN KNOCKED DOWN IN ZEBRAFISH
C. Flynn, 1 E.M. Mendenhall, 2 C.M. Verfaillie1 1 Stem Cell Institute Leuven (SCIL), LEUVEN, Belgium; 2 Dept. of Medicine &
Stem Cell Institute, MINNEAPOLIS, USA
Background. Sorting nexin 5 (SNX 5) is a member of the sorting nexin
family, a diverse group of cellular trafficking proteins unified by the
presence of a phospholipid binding domain (PHOX) which is involved
in protein-protein interactions. While the function of SNX5 is unknown,
there is evidence from yeast two hybrid studies that SNX5 binds to
FANCA, and SNX5 is one of the 20 genes located on a region on mouse
chromosome 2 associated with accelerated stem cell aging. More recently
SNX5 has been shown to be a partner of Mindbomb- an E3 ubiqitin
ligase essential for notch signal activation. Aims. We have shown that
SNX5 is significantly higher expressed in CD34 + CD33 – CD38 – Rho + (Rhohi)
cells from umbilical cord blood (UCB) and bone marrow (BM),
depleted of SCID repopulating cells (SRCs) than in CD34 + CD33 –
CD38 – cKIT + Rho- cells (Rholo), containing all SRCs. We are investigating
the role of SNX5 in Fanconi anaemia and developmental
haematopoiesis. Results. A large scale high throughput functional analysis
was carried out in zebrafish and genes differentially expressed
between Rholo and Rhohi cells were knocked down using morpholino
(MO) antisense oligonucleotides. Morpholinos are useful tools which
can specifically inhibit the translation of target mRNA. 16 out of 70
genes knocked down, including snx5, demonstrated decreased circulating
blood in the zebrafish injected. Further analysis has shown that MO
knock down of snx5 in zebrafish (n=152) results in haematopoietic failure
with normal vasculature, with normal expression of scl and gata-1
by in situ hybridization, indicating a defect at or beyond the haematopoietic
stem cell (HSC) stage. Quantitative RT-PCR was used to further
confirm the phenotype. Levels of haemoglobin, l-plastin and myeloperoxidase
mRNA in snx5 morphants compared to uninjected controls,
were significantly lower, consistent with a multi-lineage haematopoietic
differentiation defect. The decrease in circulating blood could be partially
rescued by overexpressing human SNX5 cDNA, confirming specificity
of the morphant phenotype. To determine whether SNX5 might
be linked to Fanconi anaemia (FA), we measured levels of SNX5 mRNA
and protein in EBV transformed cell lines from patients with Fanconicomplementation
group A (FANCA), -complementation group C
(FANCC) or unknown complementation group (FANC-NX). We have
found no significant difference between SNX5 mRNA and protein
expression in 11 Fanconi cell lines tested compared to Raji controls. The
association of SNX5 and Fanconi anaemia in mammalian cell lines has
been inconclusive. Studies examining SNX5 over expression and knockdown
in human UCB using lentivirus, the impact on haematopoietic
engraftment and differentiation in a NOD SCID murine model are in
progress and this data will be presented. Conclusions. Knockdown of
snx5 in zebrafish results in decreased blood, with an effect at or beyond
the definitive HSC. There is no quantitative defect of SNX5 in Fanconi
anaemia cell lines and mammalian studies are ongoing to determine its
association with the HSC.
0433
IMPACT OF STOCHASTIC DYNAMICS ON THE ACTIVE HEMATOPOIETIC STEM CELL POOL
D. Dingli, 1 A. Traulsen, 2 J.M. Pacheco3 1 Mayo Clinic, ROCHESTER, USA; 2 Harvard University, CAMBRIDGEUSA;
3University of Lisbon, LISBON, Portugal
Background. Hematopoiesis is maintained by a small group of
hematopoietic stem cells (HSC) that may contribute to blood formation
for many years, if not for the lifetime of the individual. HSC replicate
approximately 1/year and this slow replication rate is one mechanism
to minimize the risk of acquiring mutations in this important pool of
cells. However, HSC do acquire mutations that can lead to either neoplastic
proliferation (e.g. chronic myeloid leukemia, CML) or marrow
failure (e.g. paroxysmal nocturnal hemoglobinuria, PNH). Aims. Given
that the size of the HSC is small, we aim to determine the potential role
of stochastic effects on the evolution of mutant clones that originate in
this pool of cells by mathematical modeling. Methods. We develop a stochastic
model of HSC dynamics based on the Moran process where the
cell population is maintained strictly constant. Cells are chosen for reproduction
based on their fitness and each time a cell is chosen for reproduction,
it has a probability to mutate into a cancer stem cell (CSC). In
this model we consider that one mutation is enough to lead to cancer