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S43Poster Presentations/ Experimental Hematology 41 (2013) S23–S75
P1079 - DECLINED PRESENTATION
DECREASED PU.1 AND ENHANCED CITED2 COOPERATE TOMAINTAIN
SELF-RENEWAL
Hein Schepers1,2, Patrick Korthuis1, Gerald de Haan2, Jan Jacob Schuringa1, and
Edo Vellenga1
1Experimental Hematology, University Medical Center Groningen, Groningen,
Groningen, Netherlands; 2Aging Biology and Stem Cells, European Research Institute
for the Biology of Aging (ERIBA), UMCG, Groningen, Groningen, Netherlands
CITED2 has a conserved role in maintaining HSCs. We have shown before that CD34+
cells from a subset of acute myeloid leukemia (AML) patients display enhanced CITED2
expression and interfering with this expression is detrimental for leukemia maintenance.
Ectopic expression of CITED2 in normal CD34+ stem and progenitor cells (HSPCs)
results in increased proliferation and skewed myelo-erythroid differentiation. Microarray
studies indicated that 1648 genes were differentially expressed in HSPCs upon expres-
sion of CITED2. GSEA analysis indicated that CITED2 modulates hypoxia and p53
signaling. This is consistent with an increased percentage of HSCs, which demonstrated
decreased levels of apoptosis. Colony forming cells assays indicated that these cells have
increased CFC forming capacity, but harvesting and replating of these colonies demon-
strated no secondary colony forming activity. This demonstrates that enhanced CITED2
expression cannot maintain the self-renewal characteristics of HSCs, suggesting that
additional genetic changes contribute to self-renewal of leukemic stem cells. We recently
identified PU.1 as a strong negative regulator of CITED2 and enhanced CITED2 expres-
sion in AML samples correlates with low PU.1 expression. Combined lentiviral down-
regulation of PU.1 with overexpression of CITED2 gave a huge proliferative
advantage to transduced CD34+ cells. Also in CFC assays a large increase of progenitor
numbers was observed. To stringently assess whether self-renewal was maintained, cells
were first cultured for 4 weeks on MS5 and subsequently plated into CFC assays, fol-
lowed by secondary and tertiary replating experiments. The only cells that after 10 weeks
of culture still generated colonies were the ones that demonstrated low PU.1 and
increased CITED2 expression, indicating that this combination preserves self-renewal.
Together, these data suggest that in AML with low PU.1, CITED2 not only functions
to increase proliferation and skew myeloid differentiation, but that together, low PU.1
and high CITED2 are necessary to maintain leukemic stem cell self-renewal.
P1080 - MIR-29A MAINTAINS HEMATOPOIETIC STEM CELL SELF-
RENEWAL AND IS REQUIRED FOR LEUKEMIC TRANSFORMATION
Christopher Park1,5, Wenhuo Hu1, James Dooley2, Stephen Chung1,4, Yu Sup Shin1,
Bart De Strooper3, and Adrian Liston2
1Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer
Center, New York, New York, USA; 2Microbiology and Immunology, KU Leuven,
Leuven, Belgium; 3Center for Human Genetics and Leuven Institute for
Neuroscience and Disease, University of Leuven, Leuven, Belgium; 4Medicine,
Memorial Sloan-Kettering Cancer Center, New York, New York, USA; 5Pathology
and Laboratory Medicine, Memorial Sloan-Kettering Cancer Center, New York, New
York, USA
Hematopoietic stem cells (HSCs) are unique among blood cells in their ability to
indefinitely self-renew in addition to their ability to generate all cell types in the
blood system. We previously demonstrated that miR-29a is highly expressed in
mouse and human HSCs, as well as in human acute myeloid leukemia (AML)
stem cells, and that over-expression of miR-29a in immature mouse hematopoietic
cells induces a myeloproliferative disorder that progresses to AML. We have now
extended our studies to investigate the function of miR-29a in HSCs in mice lacking
the miR-29a/b bicistron. Homozygous deletion of miR-29a/b resulted in reduced
bone marrow cellularity and reduction of colony forming capacity in hematopoietic
stem and progenitor cells (HSPCs). The phenotype is mediated specifically by
miR-29a, since miR-29b expression is not significantly altered in HSCs. In addition,
reconstitution oif miR-29a/b null HSPCs with miR-29a, but not miR-29b, rescued
colony formation defects in vitro. Defects in self-renewal were observed in in
miR-29a deficient HSCs in both competitive and non-competitive transplantation
assays, and these deficits were associated with increased cell cycling and apoptosis.
Transcriptomal studies demonstrated widespread gene expression alterations in
HSCs, revealing that miR-29a deficient HSCs were more similar to wild-type
myeloid progenitor cells than to wild-type HSCs. In addition, a number of
miR-29a target genes were up-regulated in miR-29a deficient HSCs including
DNA methylation enzymes (Dnmt3a, -3b) and cell cycle regulators (e.g. Cdk6,
Tcl1, Hbp1, Pten). Knockdown of one of these targets, Dnmt3a, in miR-29a deficient
HSCs resulted in partial restoration of colony formation, providing functional valida-
tion that Dnmt3a mediates part of miR-29a null HSPCs functional defects. miR-29a
loss also abrogated leukemogenesis in the MLL-AF9 retroviral leukemia model.
Together, these studies demonstrate that miR-29a contributes to HSC self-renewal
and myeloid leukemogenesis.
P1081 - LOW C-KIT EXPRESSION IDENTIFIES HEMATOPOIETIC STEM
CELLS WITH ENHANCED SELF-RENEWAL POTENTIAL
Christopher Park1,2, Joseph Shin1, Safak Yalcin1, and Wenhuo Hu1
1Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer
Center, New York, New York, USA; 2Pathology and Laboratory Medicine, Memorial
Sloan-Kettering Cancer Center, New York, New York, USA
A growing body of evidence supports the view that hematopoietic stem cells (HSCs)
are heterogeneous with respect to their self-renewal, lineage, and reconstitution
capacities. However, the molecular mechanisms underlying these differences are
not well-understood. Because gain- and loss-of-function c-Kit mutants exhibit signif-
icant alterations in HSC function, we hypothesized that even small changes in c-Kit
signaling would result in profound effects on HSC function, and thus, we investigated
whether HSCs (Lin-c-Kit+Sca-1+SLAMf150+CD34-) expressing differing levels of
c-Kit may exhibit distinct phenotypes. Our studies demonstrate that HSCs expressing
low levels of c-Kit (lowest 30% of expressors; c-Kitlo) exhibit enhanced self-renewal
and long-term reconstitution potential compared to HSCs expressing high levels of
c-Kit (highest 30% of expressors; c-Kithi) as demonstrated through non-competitive
and competitive transplantation assays. c-Kithi HSCs also exhibit a strong megakar-
yocytic lineage bias, as demonstrated through in vitro culturing and in vivo transplan-
tation studies, and this bias was confirmed by transcriptomal analysis of c-Kithi
HSCs, which exhibited increased expression of megakaryocyte associated genes
such as Gata-1 and reduced expression of self-renewal genes such as HoxB4. c-Kitlo
HSCs readily give rise to c-Kitlo and c-Kithi HSCs both in vitro and in vivo, but
c-Kithi HSCs have little to no capacity to give rise to c-Kitlo HSCs, consistent
with a hierarchical relationship between these two populations. These qualitative
differences between c-Kithi and c-Kitlo HSCs are preserved even in situations of
physiologic stress in which c-Kit expression is altered, notably following 5-FU treat-
ment. Overall, these data indicate that c-Kitlo HSCs are enriched for long-term
reconstituting HSCs. These findings have important implications for investigations
of HSC function as they suggest that most currently used immunophenotypic
definitions of HSCs identify cell populations significantly contaminated by non-
self-renewing and lineage-biased cells.