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Prepublished online as a Blood First Edition Paper on October 17, 2002; DOI 10.1182/blood-2002-08-2635.
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Blood, 1 March 2003, Vol. 101, No. 5, pp. 1984-1986
NEOPLASIA
Brief report
Leukemic potential of doubly mutant Nf1 and
Wv hematopoietic cells
David A. Ingram,
Mary Jo Wenning,
Kevin Shannon, and
D. Wade Clapp
From the Indiana University School of Medicine, Herman
B. Wells Center for Pediatric Research, Department of
Microbiology/Immunology, Indianapolis, IN; and University of California
at San Francisco, Department of Pediatrics, San Francisco, CA.
 |
Abstract |
The development of molecularly targeted treatments of adult
leukemias warrants investigation of these targets in similar pediatric leukemias. The NF1 tumor suppressor gene, which encodes a
GTPase activating protein for p21ras, is frequently
inactivated in juvenile myelomonocytic leukemia (JMML). Other patients
with JMML acquire activating RAS gene mutations. Recipient
mice reconstituted with Nf1 / fetal
hematopoietic cells develop a myeloproliferative disease (MPD) that
models the human disease. JMML arises from clonal expansion of a
hematopoietic stem cell, and JMML cells and murine
Nf1/ hematopoietic cells are hypersensitive
to granulocyte macrophage-colony stimulating factor and KitL, the
ligand for c-kit. We generated embryos doubly mutant for the
Wv allele of c-kit and Nf1 to ask
if reduction of c-kit activity would delay or prevent the development
of MPD. Despite a reduction in c-kit activity to approximately 10% of
wild-type levels,
Nf1/;Wv/Wv
cells induced MPD in recipient mice.
(Blood. 2003;101:1984-1986)
© 2003 by The American Society of Hematology.
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Introduction |
Mutations in the NF1 tumor suppressor
gene cause neurofibromatosis type I (NF1). NF1
encodes neurofibromin, which negatively regulates p21ras
activity by accelerating the conversion of active
p21ras-GTP to inactive
p21ras-GDP.1,2 Children with NF1 are at
markedly increased risk of developing juvenile myelomonocytic leukemia
(JMML), for which existing treatments are largely
ineffective.3,4 Although Nf1 knockout
mice (Nf1/) die in utero around day
embryonic (E)13.5, recipients of transplanted Nf1/ fetal hematopoietic stem cells
(HSCs) develop myeloproliferative disease (MPD) that
closely models JMML.5,6 This model has been exploited for
preclinical studies of therapeutics that target hyperactive
p21ras.7
Murine Nf1/ fetal liver cells form excessive
numbers of myeloid progenitors in methycellulose cultures containing
low concentrations of kit ligand (KitL),8 and we recently
found that Nf1/ HSCs have a self-renewal
advantage in vivo.9 KitL is a potent survival and
proliferative growth factor for both HSCs and myeloid progenitors
(reviewed in Broudy10). Inasmuch as JMML often arises from
clonal expansion of a HSC and that Nf1/
myeloid progenitors are hypersensitive to KitL, we hypothesized that
genetic inhibition of the KitL/c-kit pathway would alter the
progression of MPD in recipients of Nf1/
fetal liver cells. Evaluating c-kit as a therapeutic target is an
important priority as the tyrosine kinase inhibitor (imatinib mesylate)
induces regression in gastrointestinal stromal tumors (GISTs), a
malignancy characterized by mutations that constitutively activate the
c-kit kinase.11 To assess if impairing c-kit
function would modulate the MPD induced by Nf1 inactivation,
we generated fetal stem cells that were doubly mutant at
Nf1, and the dominant white spotting (W)
locus, which encodes c-kit, performed adoptive transfers and monitored
the recipient mice for development of MPD. We find that the
Wv mutation, which reduces c-kit kinase activity
by approximately 90%, does not suppress the capacity of
Nf1/ fetal liver cells to induce MPD.
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Study design |
Animals and adoptive transfer procedure
Nf1+/ mice in a C57BL/6.129 background
were backcrossed for 13 generations into C57BL/6J mice, which were
purchased from Jackson Laboratories (Bar Harbor, ME).
Wv/Wv mice were purchased from
Jackson Laboratories in a C57BL/6J strain. Studies were conducted with
a protocol approved by the Indiana University Animal Care and Use
Committee. The Nf1 and Wv alleles
were genotyped as previously described.8,12 The crosses used to generate day-E13.5 fetal livers, which contain the experimental groups, are outlined here. Filial (F)0:
Nf1+/; Wv+/+ × Nf1+/+;Wv/Wv.
F1: Nf1+/;Wv/+ × Nf1+/;Wv/+. F2 experimental
groups: Nf1+/+;Wv+/+,
Nf1/;Wv+/+,
Nf1+/+;Wv/Wv,
Nf1/;Wv/Wv.
Syngeneic recipients were given transplants of 3 million
day-E13.5 fetal liver cells generated from the intercrosses following irradiation with 1100 cGy as previously described.9
Animals that underwent transplantation were followed for signs of
MPD,9 and photomicrographs of spleen sections were taken
with an Olympus DP11 (Melville, NY).
Progenitor assays
Colonies derived from myeloid progenitors were enumerated
exactly as described.9 Recombinant granulocyte
macrophage-colony stimulating factor (GM-CSF), interleukin-3
(IL-3), and KitL were purchased from Peprotech (Rocky Hill, NJ), and
recombinant IL-1 and M-CSF were obtained from R&D Systems (Minneapolis,
MN). Cells and recombinant growth factors were added to agar for growth
of low proliferating potential-colony forming cells (LPP-CFCs) and high proliferating potential-colony forming cells (HPP-CFCs), and the
solution was thoroughly admixed before plating. LPP-CFCs and HPP-CFCs
were enumerated in 8% CO2 and 5% O2. LPP-CFCs
and HPP-CFCs were scored by indirect microscopy on days 7 and 14, respectively.
| |
Results and discussion |
Of 300 embryos generated from the Wv × Nf1 intercross, only 3 were doubly mutant
(Nf1/;Wv/Wv) at day
E13.5, and fetal liver cell numbers were markedly reduced. This is
consistent with previous studies using
Wv/Wv mice.12 Despite
these obstacles, one recipient received a transplant successfully from each doubly mutant embryo; 2 of
the recipients developed white blood cell (WBC) counts of
0.045 and 0.050 × 109/L ([45 000 and 50 000
per mm3] compared with an average WBC count of
0.017 × 109/L (17 000 per mm3) in
recipients of wild-type fetal liver cells; n = 10). Each mouse died
at 2 months of age and had gross splenomegaly upon inspection, which is
consistent with MPD previously observed in recipients of
Nf1/-deficient fetal liver
cells.6,9 The third recipient of transplanted Nf1/;Wv/Wv cells
survived to 4 months of age, which allowed a more detailed analysis.
The spleen weight and cellularity were markedly increased compared with
the wild type and Wv/Wv controls and
were comparable to mice that received transplants of
Nf1/ fetal stem cells (Table
1). The MPD in mice that received
transplants of Nf1/ cells is characterized
by a dramatic increase in the numbers of splenic HPP-CFCs and
LPP-CFCs.9 The numbers of HPP-CFCs and LPP-CFCs
were similar in the spleens of recipients injected with singly mutant
Nf1/or
Nf1/;Wv/Wv cells and
were increased 30-fold (HPP-CFCs) and 10-fold (LPP-CFCs) versus mice
that received transplants of wild-type or
Wv/Wv cells (Table 1). Finally, the
spleens of the recipients of transplanted Nf1/;Wv/Wv or
Nf1/ cells demonstrated myeloid cell
infiltration and effacement of splenic architecture, which is
consistent with development of MPD (Figure
1A-D).
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Table 1.
Effect of Wv and Nf1
genotypes on spleen weight, splenic cellularity, WBC
count, and total number of HPP-CFCs and LPP-CFCs per
spleen in primary recipients
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| Figure 1.
Effects of Wv and
Nf1 genotypes on splenic architecture in primary and
secondary recipients.
(A-D) Low-power view (original magnification × 10) of spleen sections
from primary recipients of transplanted fetal liver cells from the 4 F2
experimental groups outlined in "Study design." Recipients of
transplanted Nf1+/+;Wv+/+
and Nf1+/+;Wv/Wv fetal
liver cells show a normal distribution of red pulp and white pulp
(A-B). In contrast, recipients of transplanted
Nf1/;Wv/Wv or
Nf1/;Wv+/+ fetal liver
cells showed expansion of the red pulp with loss of normal splenic
architecture (C-D). (E-H ) A representative low-power view (original
magnification × 10) of spleen sections from secondary recipients of
transplanted low-density mononuclear cells harvested from the bone
marrow of primary recipients of transplanted fetal liver cells from the
4 F2 experimental groups. Secondary recipients of transplanted
Nf1+/+;Wv+/+ and
Nf1+/+;Wv/Wv LDMNCs show
a normal distribution of red pulp and white pulp (n = 7 for each
genotype) (E-F). In contrast, secondary recipients of transplanted
Nf1/;Wv/Wv or
Nf1/;Wv+/+ LDMNCs showed
expansion of the red pulp with loss of normal splenic architecture
(n = 7 for each genotype) (G-H).
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Studies in which bone marrow cells from primary recipients were
transferred into secondary hosts showed that Nf1
inactivation is associated with enhanced proliferative potential in
repopulating stem cells.9 To examine whether the reduction
in c-kit activity altered this previously defined gain of function
potential, we isolated low-density marrow mononuclear cells from each
mouse of the 4 F2 experimental groups and transplanted these cells into secondary recipients. Mice were killed at 6 months of age, and spleens
were examined for evidence of MPD. All recipients of
transplanted Nf1/;Wv/Wv or
Nf1/ cells (n = 8) developed a dramatic
myeloid cell infiltration of the spleen consistent with MPD as
previously observed in the primary recipients and in prior studies. A
representative spleen section from each experimental group is shown in
Figure 1E-H.
In vitro hypersensitivity to GM-CSF in methycellulose cultures is a
hallmark of human JMML blood and marrow cells, a characteristic that is
also observed in Nf1/ fetal liver
cells.5,6 Experiments in which Nf1 and
Gmcsf mutant mice were mated to generate cells for adoptive
transfer demonstrated a central role of this aberrant response to
GM-CSF in the pathogenesis of MPD.13
Nf1/ hematopoietic cells are also
hypersensitive to KitL in colony-forming assays, and KitL and GM-CSF
act synergistically to enhance myeloid progenitor growth from wild-type
and Nf1/ cells.8 These data,
the availability of imatinib mesylate for clinical trials, and the poor
prognosis for patients with JMML led us to ask if reducing c-kit
activity might modulate the course of MPD in recipients of
Nf1/ fetal liver cells. We pursued a genetic
approach to this question because imatinib inhibits c-Abl and  chain
of the platelet-derived growth factor receptor in addition to
c-kit.11 Our data showing that
Nf1/;Wv/Wv fetal
liver cells induced MPD suggest that this disorder is not dependent on
c-kit signaling. Alternatively, 10% of normal c-kit activity may be
sufficient for leukemogenesis, whereas complete inhibition might
interfere with the growth of JMML cells in vivo. However, this
possibility is highly unlikely since
W41/W41;Nf1/
hematopoietic cells (W41 mutants have a 70%
reduction in c-kit activity vs 90% for Wv
strains) are not hypersensitive to KitL in vivo as determined in both
myeloid colony-forming assays and competitive repopulation assays,
which measure HSC function (data not shown). It also possible that
c-kit inhibitors will be ineffective as single agents in JMML, but will
show synergistic activity with drugs that target GM-CSF signaling such
as the peptidomimetic E12R.14 These hypotheses can be
tested by treating mice that received transplants of
Nf1/ cells with imatinib mesylate or with
other pharmacologic inhibitors of c-kit. Recently, a heterozygous
environment was shown to contribute to the development of neurofibromas
in mice in which Nf1 was selectively ablated in Schwann
cells.15 Heterozygous Nf1 mutant cells
infiltrate these lesions, and these cells demonstrate deregulated
p21ras signaling in response to KitL.16,17
Thus, although our data do not support a role for c-kit inhibitors in
JMML, drugs that interfere with this pathway might prove useful for
treating other complications of NF1.
| |
Acknowledgments |
We are indebted to Dr Tyler Jacks for providing
Nf1 mutant mice, and thank Marsha Hippensteel for
exceptional administrative support.
| |
Footnotes |
Submitted August 28, 2002; accepted October 4, 2002.
Prepublished
online as Blood First Edition Paper, October 17, 2002; DOI
10.1182/blood-2002-08-2635.
Supported by National Institutes of Health (NIH) grants 1 KO8
CA096579-01 and K12-HD00850 (D.A.I.), 2 R01 CA74177-06 (D.W.C.), NIDDK
P30 DK49218 (D.W.C.), American Cancer Society (ACS) grant RSG-96-104-06
LIB (D.W.C., K.S.), Department of Defense (DOD) grant
DAMD17-01-1-0711 (D.A.I., D.W.C.), and NIH grant R01 CA762714 (K.S.).
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: David A. Ingram, Indiana University School
of Medicine, Herman B. Wells Center for Pediatric Research, 1044 W
Walnut St, R4/470, Indianapolis, IN 46202; e-mail:
dingram{at}iupui.edu.
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Abstract
Introduction