By
From the Institute of Hematology, Daniel den Hoed Cancer Center and Erasmus University Rotterdam, 3000 DR Rotterdam, The Netherlands
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Abstract |
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In approximately 20% of cases of severe congenital neutropenia (SCN), mutations are found in
the gene encoding the granulocyte colony-stimulating factor receptor (G-CSF-R). These mutations introduce premature stop codons, which result in truncation of 82-98 COOH-terminal
amino acids of the receptor. SCN patients who develop secondary myelodysplastic syndrome
and acute myeloid leukemia almost invariably acquired a GCSFR mutation, suggesting that
this genetic alteration represents a key step in leukemogenesis. Here we show that an equivalent mutation targeted in mice (gcsfr-715) results in the selective expansion of the G-CSF-
responsive progenitor (G-CFC) compartment in the bone marrow. In addition, in vivo treatment of gcsfr-
715 mice with G-CSF results in increased production of neutrophils leading to a
sustained neutrophilia. This hyperproliferative response to G-CSF is accompanied by prolonged activation of signal transducer and activator of transcription (STAT) complexes and extended cell surface expression of mutant receptors due to defective internalization. In view of
the continuous G-CSF treatment of SCN patients, these data provide insight into why progenitor cells expressing truncated receptors clonally expand in vivo, and why these cells may be
targets for additional genetic events leading to leukemia.
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Introduction |
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Severe congenital neutropenia (SCN)1 is a heterogeneous hematopoietic disorder characterized by a profound neutropenia due to a maturation arrest of granulocytic progenitor cells in the bone marrow (BM). As a result, SCN patients suffer from recurrent, often life-threatening, bacterial infections (1). The disease presents with a variable inheritance, and the mechanisms underlying its pathogenesis are largely unknown (2). Granulocyte colony-stimulating factor (G-CSF) is a major regulator of neutrophil production (6, 7). However, although defective production of G-CSF has been suggested as a possible cause of SCN in isolated cases (8, 9), in the majority of SCN patients G-CSF serum levels are normal or elevated (10). In >90% of SCN patients, daily administration of pharmacological doses of G-CSF increases neutrophil levels and significantly reduces infection-related events (1, 13). In contrast, treatment with GM-CSF is rarely effective (13), suggesting that specifically the G-CSF signal transduction pathway is affected in SCN.
G-CSF mediates its effects through activation of its cognate receptor (G-CSF-R), a single-transmembrane protein of the hematopoietin receptor superfamily which forms homooligomeric complexes upon ligand binding (14). Like all members of this group of receptors, G-CSF-R lacks intrinsic tyrosine kinase activity but can activate cytoplasmic tyrosine kinases of the Jak and Src families (17). These kinases tyrosine phosphorylate substrates, including the receptor, to provide docking sites for other proteins, which, in turn, can be phosphorylated as well. Proteins docking to the G-CSF-R complex include members of the signal transducer and activator of transcription (STAT) family. Upon tyrosine phosphorylation, STATs form dimeric complexes and translocate to the nucleus, where they influence gene transcription (24, 25). Of the six members of the STAT family identified in mammalian cells, STAT1, STAT3, and STAT5 have been implicated in G-CSF signaling (19, 26). STAT3 has recently been linked with IL-6-induced macrophage differentiation and G-CSF-induced neutrophilic differentiation (30), whereas STAT5 has been implicated in the control of hemopoietic cell proliferation by some cytokines, including G-CSF (33, 34).
In a subgroup of SCN patients, acquired mutations in the GCSFR gene are found in BM and peripheral blood neutrophils (18, 35). These mutations introduce premature stop codons that result in the truncation of 82-98 COOH-terminal amino acids, a region previously implicated in the control of neutrophilic differentiation (40, 41). In recent studies of a total of 78 SCN patients, 18 cases with point mutations in the critical region in the GCSFR gene were found (38, 42). Among those patients with GCSFR mutations, eight developed acute myeloid leukemia (AML), whereas only one of the patients without a mutation has thus far showed clinical or cytogenetic evidence of leukemic progression (38). This latter patient represents a rare case of autosomal dominant SCN affecting a mother and four siblings. Up to now, no GCSFR mutations have been detected in patients with idiopathic neutropenia, chronic myeloid leukemia (CML), or myelodysplastic syndrome (MDS) without history of neutropenia. However, mutations occur at very low frequency in de novo AML (18, 43). These clinical observations suggest that acquisition of a GCSFR mutation may be a critical step in the leukemic transformation of progenitor cells from SCN patients.
Recently, we generated mice with a mutation in their gcsfr
gene (715) equivalent to one of the nonsense mutations
found in SCN patients (46). Both homozygous and heterozygous
715 mice have reduced numbers of circulating
neutrophils compared with their wt/wt littermates, supporting
the notion that the G-CSF-R COOH terminus has a role in
granulopoiesis in vivo. Strikingly, after injection with G-CSF,
gcsfr-wt/
715 and gcsfr-
715/
715 mice showed increased
peripheral neutrophil counts compared with their wt/wt littermates (46). Analysis of cells labeled with the thymidine analogue 5-bromo-2'-deoxyuridine in vivo indicated that the
rise was due to de novo generation of neutrophils, although specific mechanistic details were not addressed.
In this study, we show that the gcsfr-715 mutation results in an approximately twofold increase in granulocyte
colony-forming cell (G-CFC) content in the BM. In contrast, the numbers of more primitive hematopoietic progenitors, as assessed in a cobblestone area-forming cell
(CAFC) assay (47), are slightly decreased. Sustained G-CSF
treatment results in a continuous neutrophilia in gcsfr-
715 mice, suggesting a persistent increase in G-CSF- induced proliferation of neutrophilic progenitors due to the
G-CSF-R truncation. Finally, we demonstrate altered levels and kinetics of G-CSF-induced activation of STAT
proteins in gcsfr-
715 mice, including a significantly extended activation of STAT proteins that correlated with a
delay in internalization of the truncated G-CSF-R proteins. These altered signaling properties provide potential explanations for the preleukemic nature of GCSFR mutations in SCN.
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Materials and Methods |
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Mice.
gcsfr-Blood and BM Sampling.
Blood samples were collected from the tail vein at fixed time points to avoid variation due to circadian rhythms (48). Blood cell counts were performed on a Sysmex-K1000X automated counter (Toa Medical Electronics Co. Ltd.). To obtain BM cell suspensions, femurs and tibias were crushed in a mortar in HBSS with 5% FCS (HBSS/FCS). BM cells were passed through a 100-µm sieve, spun down, and resuspended. The resulting monocellular suspensions contained 98-99% viable cells, as determined by trypan blue exclusion. For differential countings, blood smears were fixed in methanol, May-Grunwald-Giemsa (MGG) stained. At least 300 blood cells were analyzed in a Axioscope microscope.Isolation of BM Progenitor Cells.
BM cells of two mice were pooled and suspended in HBSS/FCS and subsequently incubated in a 162-cm2 cell culture flask (Costar) in a humidified atmosphere of 5% CO2 in air at 37°C for 1 h. Nonadherent cells were spun down and resuspended in 45% (In Vitro Colony Assays.
BM progenitor cells were plated in triplicate in 35-mm Petri dishes (Falcon; ) containing 1 ml of methylcellulose medium (Methocult M3230; Stem-cell Technologies, Inc.) containing 30% fetal bovine serum, 1% BSA, 0.1 mM 2-ME, 2 mM L-glutamine, without additional growth factor or with 100 ng/ml G-CSF (Amgen, Inc.), 20 U/ml murine GM-CSF (provided by Dr. Steven Neben, Genetic Institute, Cambridge, MA), 100 U/ml murine macrophage (M)-CSF, 4 U/ml human erythropoietin (EPO; a gift of Janssen-Cilag) plus 10 µg/ml murine stem cell factor (SCF) and 0.2 mM hemin (bovine type 1; ). Colonies (50 cells or more) were counted on day 7 or 8 of culture. CAFC assays were performed as described (47). In this limiting dilution assay, phase-dark hematopoietic colonies (cobblestone areas) are formed under a preestablished stromal layer. Early appearing cobblestone areas (week 1-2) represent clones from the relatively mature stem/progenitor cell subset, while the most primitive hemopoietic stem cells, i.e., those with long-term repopulating abilities in vivo, are assessed at week 4-5.Preparation of Nuclear Extracts.
BM cells isolated as described above were washed once with HBSS, resuspended in RPMI/ 10% FCS, and transferred to a tissue culture flask. After 1.5 h, nonadherent cells were transferred to a fresh flask for another 3.5 h before stimulation with murine IL-3 (100 ng/ml) or human G-CSF (100 ng/ml). Stimulation was terminated by adding ice-cold PBS containing 0.1 mM Na3VO4. Cells were lysed in 200 µl hypotonic buffer (20 mM Hepes, 20 mM NaF, 1 mM Na3VO4, 1 mM Na4P2O7, 0.125 µM okadaic acid, 1 mM EDTA, 1 mM EGTA, 0.2% NP-40, 1 mM dithiothreitol, 0.5 µg/ml aprotinin, 0.5 µg/ml leupeptin, 0.5 µg/ml bacitracin, 0.5 µg/ml iodoacetamide, and 1 mM Pefabloc). Nuclei were spun down at 15,000 g for 30 s, and proteins were extracted by rocking for 30 min at 4°C in 20 µl high-salt buffer (hypotonic buffer with 420 mM NaCl, and 20% glycerol). Insoluble materials were removed by centrifugation at 20,000 g at 4°C for 20 min.Electrophoretic Mobility Shift Assays with STAT-binding Oligonucleotides.
Nuclear extracts were incubated with double-stranded [Flow Cytometric Analysis of G-CSF-R Internalization.
G-CSF-R expression on BM cells was measured by flow cytometry. To this end, G-CSF was biotinylated using D-biotinoyl- ![]() |
Results |
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We have previously shown that gcsfr-715 mice are
neutropenic (46). To determine at what stage of myeloid
development the effect of the
715 mutation becomes
phenotypically overt, we performed in vitro colony assays
in the presence of specific growth factors. Depending on
the factor(s) added, these assays provide a quantitative measure of the number of neutrophilic precursors (G-CSF),
granulocyte-macrophage precursors (GM-CSF), macrophage precursors (M-CSF), and erythroid precursors (EPO
plus SCF). Data from a representative experiment are
shown in Fig. 1. The frequency of G-CSF-responsive
CFCs was increased by a factor 2.0 ± 0.6 (mean ± SD of
four independent experiments) in homozygous
715 mice,
and 1.8 ± 0.9 in heterozygous mice compared with wild-type littermates. Although the frequencies of GM-CSF,
M-CSF, and EPO plus SCF responsive progenitors were also
consistently higher in mutant mice, the difference between mutant and wild-type mice was less pronounced than that
in the granulocytic lineage.
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We next evaluated the effect of the mutation on more
primitive hemopoietic cells in the CAFC assay. From the
CAFC numbers per femur of gcsfr-wt/wt, wt/715, and
715/
715 mice (Table I), we calculated the percentage
of CAFCs relative to the CAFC numbers of wild-type
mice (Fig. 2). Although CAFCs at week 1-2 (a measure of
stem cells with short-term repopulating ability) were relatively more abundant in the gcsfr-
715/
715 mice, CAFCs
at week 4-5 (primitive stem cells with long-term repopulating ability) were present in lower numbers than in control mice. Therefore, it appears that the absence of the
COOH-terminal domain of the G-CSF-R leads to a selective expansion of a myeloid progenitor compartment, to some extent at the expense of primitive stem cells.
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Previously, we showed
that gcsfr-wt/715 and gcsfr-
715/
715 mice injected with
G-CSF for 1 wk have elevated blood neutrophil counts on
days 6 and 7 compared with control littermates (46). To establish whether this effect is transient or sustained, gcsfr-wt/wt, gcsfr-wt/
715, and gcsfr-
715/
715 mice were injected
daily with G-CSF (250 µg/kg) for 22 d, and blood neutrophil numbers were monitored. Wild-type mice responded
to G-CSF treatment with a 10-fold increase in their blood
neutrophil levels, mice heterozygous for the
715 mutation with a 65-fold increase, and gcsfr-
715/
715 mice
with a 250-fold increase (Fig. 3). The average blood neutrophil count on day 23 was 45 × 106/ml in
715/
715
mice (n = 4), 27 × 106/ml in wt/
715 mice, and 6 × 106/ml
in wt/wt mice. Thus, it is clear that G-CSF treatment leads to a sustained neutrophilia in gcsfr-
715 mice. Red blood
cell and platelet counts were not influenced by the mutation, suggesting that the gcsfr-
715 mutation selectively enhances the proliferation of myeloid progenitors.
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Because there is increasing evidence
that STAT proteins play an important role in the control of
proliferation and differentiation of hematopoietic cells, we
analyzed both STAT5 and STAT3 activation in BM cells
of gcsfr-wt/wt, wt/715, and
715/
715 mice. At early time points (up to 15 min), G-CSF-induced activation of
STAT5 was largely unaffected while STAT3 activation was
reduced in
715/
715 BM cells (Fig. 4, A and B). However, at later time points of stimulation (60 and 120 min),
when STAT5 activation in wt/wt cells declined, activation
of STAT5 in
715/
715 cells persisted, whereas STAT3 levels became comparable in both. Quantification of the
signals (Fig. 4 C) shows that after 1-3 h of stimulation,
STAT5 activation in BM cells of
715 mice is four- to
fivefold the STAT5 activation in wild-type BM cells,
whereas STAT3 activation after 1-3 h of stimulation is
similar. BM cells from heterozygous mice showed an intermediate response (data not shown). IL-3-induced activation of STAT complexes was equivalent in all genotypes.
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We also studied STAT activation after 10 min of stimulation followed by an extensive wash to remove G-CSF.
Under these conditions, prolonged activation of STAT5,
STAT3, and STAT1 was seen in 715/
715 cells and to a
somewhat lesser extent in wt/
715 cells (Fig. 5, A and B).
These findings suggest that the truncated receptors which
bound ligand during the first 10 min of stimulation were responsible for the prolonged STAT activation, and that
this was not due to stimulation of new receptors recruited
to the cell surface. Because wild-type and truncated receptors show equivalent Kd (41), this suggests that receptor deactivation is altered in truncated receptors.
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The COOH-terminal region of G-CSF-R contains a dileucine motif (STQPLL) that has been implicated
in ligand-mediated receptor endocytosis (52, 53). Due to
the truncation, 715 G-CSF-Rs lack this motif. When exposed to G-CSF, within 30 min, surface expression of
wild-type receptors had decreased to 20% of the initial expression, whereas 80% of the mutant receptors were still present on the cell surface (Fig. 6, A and B). Thus, upon
exposure to G-CSF, truncated G-CSF-Rs reside longer on
the cell surface, which may contribute to their ability to induce sustained STAT activation.
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Discussion |
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Before the therapeutic use of G-CSF for SCN patients, incidental cases terminating in AML were reported (54). After the introduction of G-CSF treatment of SCN patients, mortality due to severe opportunistic bacterial infections diminished, but the incidence of secondary AML gradually increased. The frequency of leukemic progression is currently estimated at 7.5-10% (55). After the discovery of mutations in the gene encoding the G-CSF-R in several SCN patients, a correlation was observed between the acquisition of such mutations and leukemic progression of the disease (37, 42). The consequences of these mutations for the signaling function of G-CSF-R have initially been investigated in myeloid cell lines. These studies revealed that truncated receptors transduce strong proliferative signals, but are defective in maturation signaling (40, 41).
To gain further insight in the contribution of GCSFR
mutations to the pathogenesis of SCN and AML, we recently generated an in vivo model by introducing an SCN-derived nonsense mutation (gcsfr-715) in mice (46). Both
wt/
715 and
715/
715 mice have significantly reduced
numbers of blood neutrophils compared with their wt/wt
littermates, suggesting that the G-CSF-R COOH-terminal domain contributes to neutrophil development in vivo.
This phenotype has remained consistent and independent
of genetic background in the mice generated so far. In contrast, other investigators have reported that blood neutrophil numbers in mice with a similar mutation in the gcsfr
gene were not significantly altered (56). However, a complication of their model is that the expression of the mutant
allele was significantly higher than the wild-type allele.
This might be due to the fact that the PGK-neo cassette used for selection of embryonic stem cells within the gcsfr
gene had not been removed before the generation of animals. This increased expression of truncated G-CSF-R
may compensate for the mutation, and could explain the
absence of neutropenia.
In gcsfr-715 mice, the numbers of CAFCs at week 5 appear slightly reduced in BM, whereas the numbers of
G-CFCs are selectively expanded. As yet, we cannot explain
this relative paucity of primitive stem cells in
715 mice.
One possibility is that expression of truncated G-CSF-Rs
on these cells would lead to an increase in cell cycling and
commitment. However, whether primitive hemopoietic stem cells express G-CSF-R is still controversial (57, 58). The fact that the numbers of IL-3-, M-CSF-, and GM-CSF-responsive CFCs are also increased in gcsfr-
715 mice
suggests that the GM-CFC compartment is already somewhat expanded. Despite the increased G-CFC compartment, the number of circulating neutrophils is significantly reduced in gcsfr-
715 mice (46). This implies that truncated receptors do impair the development to blood neutrophil,
but at what stage between the G-CFC and mature neutrophil this defect becomes manifest remains unclear.
The gcsfr-715 mice showed a sustained neutrophilia in
response to G-CSF administration. The fact that this hyperproliferation persisted after prolonged exposure to G-CSF
is compatible with the notion that the rate of proliferation
of the early myeloid progenitor compartment is increased
in these mice. Increased proliferation due to G-CSF-R
truncation has also been observed in cell lines (40, 41).
However, immortalized cell lines contain multiple genetic
aberrations that influence cell cycle regulation. We have shown here in primary cells that the gcsfr-
715 mutation itself is sufficient for an in vivo hyperproliferative response to
G-CSF. This supports the idea that acquisition of this type
of mutation represents a step in leukemic transformation.
In SCN patients, expansion of progenitor cells with acquired G-CSF-R mutations is observed. Thus, it is conceivable that under continuous G-CSF administration,
such as that received by SCN patients, progenitor cells that
acquire a GCSFR mutation would have a proliferative advantage leading to their in vivo expansion.
STAT proteins are involved in control of multiple cellular processes regulated by cytokines and growth factors,
including cell differentiation, proliferation, and survival.
Since the BM from gcsfr-wt/wt, gcsfr-wt/715, and gcsfr-
715/
715 mice appears similar in composition, also
within the neutrophilic lineage (46), we could directly
compare G-CSF-induced activation of STAT proteins in
the BM of these mice. BM cells from gcsfr-
715 mice displayed normal levels of STAT5, but decreased levels of
STAT3 activation. The latter is most likely due to the absence of specific STAT3-activating mechanism(s) (26, 29).
In view of the putative role of STAT3 in G-CSF-induced
differentiation of neutrophilic progenitors (32), the decreased STAT3 activation could, at least partially, explain the defective maturation to blood neutrophil which leads
to basal neutropenia in gcsfr-wt/
715 and gcsfr-
715/
715
mice. Importantly, truncated G-CSF-Rs induced prolonged
activation of STAT complexes and showed slower internalization rates. However, the relative levels of G-CSF-
induced activation of STAT5 versus STAT3 in gcsfr-
715 BM were consistently higher than in gcsfr-wt BM. This may
shift the proliferation/differentiation balance within the
neutrophilic progenitor cells. There is increasing evidence
that oncogenesis is associated with abnormal STAT signaling (59). For example, constitutive STAT activation has
been reported in cells transformed by Src, Abl, and other
oncoproteins (60). In human tumors, e.g., breast carcinoma cells, lymphomas, and leukemias, STATs are also frequently activated (63, 65). In view of these findings, our
observation that the kinetics of STAT activation by truncated G-CSF-Rs are altered further corroborates the idea
that GCSFR mutations truncating the COOH terminus of
the receptor are a step in leukemogenesis in SCN patients.
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Footnotes |
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Address correspondence to Mirjam H.A. Hermans, Hematology, Erasmus University Rotterdam (EUR), P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. Phone: 31-10-408-79-61; Fax: 31-10-436-23-15; E-mail: hermans{at}hema.fgg.eur.nl
Received for publication 14 October 1998 and in revised form 21 December 1998.
This work was financed by a grant from the Netherlands Organization for Scientific Research NWO (to M.H.A. Hermans, C. Antonissen, and I.P. Touw), a European Molecular Biology Organization Long-term Fellowship (to A.C. Ward), and grants from the Dutch Cancer Society (to I.P. Touw).We are grateful to Karola van Rooyen for graphical assistance and Bernd Groner for antibodies.
Abbreviations used in this paper AML, acute myeloid leukemia; BM, bone marrow; CAFC, cobblestone area-forming cell; CFC, colony-forming cell; EPO, erythropoietin; SCF, stem cell factor; SCN, severe congenital neutropenia; STAT, signal transducer and activator of transcription.
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