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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 96 No. 3 (August 1), 2000:
pp. 925-932
HEMATOPOIESIS
Inhibition of c-kit receptor tyrosine kinase activity by STI
571, a selective tyrosine kinase inhibitor
Michael C. Heinrich,
Diana J. Griffith,
Brian J. Druker,
Cecily L. Wait,
Kristen A. Ott, and
Amy J. Zigler
From the Division of Hematology and Medical Oncology, Department of
Medicine, Oregon Health Sciences University, and Portland Veterans
Affairs Medical Center, Portland, OR.
 |
Abstract |
STI 571 (formerly known as CGP 57148B) is a known inhibitor of the
c-abl, bcr-abl, and platelet-derived growth-factor receptor (PDGFR)
tyrosine kinases. This compound is being evaluated in clinical trials
for the treatment of chronic myelogenous leukemia. We sought to extend
the activity profile of STI 571 by testing its ability to inhibit the
tyrosine kinase activity of c-kit, a receptor structurally similar to
PDGFR. We treated a c-kit expressing a human myeloid leukemia cell
line, M-07e, with STI 571 before stimulation with Steel factor (SLF).
STI 571 inhibited c-kit autophosphorylation, activation of
mitogen-activated protein (MAP) kinase, and activation of Akt without
altering total protein levels of c-kit, MAP kinase, or Akt. The
concentration that produced 50% inhibition for these effects was
approximately 100 nmol/L. STI 571 also significantly decreased
SLF-dependent growth of M-07e cells in a dose-dependent manner and
blocked the antiapoptotic activity of SLF. In contrast, the compound
had no effect on MAP kinase activation or cellular proliferation in
response to granulocyte-macrophage colony-stimulating factor. We also
tested the activity of STI 571 in a human mast cell leukemia cell line
(HMC-1), which has an activated mutant form of c-kit. STI 571 had a
more potent inhibitory effect on the kinase activity of this mutant
receptor than it did on ligand-dependent activation of the wild-type
receptor. These findings show that STI 571 selectively inhibits c-kit
tyrosine kinase activity and downstream activation of target proteins
involved in cellular proliferation and survival. This compound may be
useful in treating cancers associated with increased c-kit kinase activity.
(Blood. 2000;96:925-932)
© 2000 by The American Society of Hematology.
| |
Introduction |
c-kit, a 145-kd transmembrane glycoprotein, is the
normal cellular homologue of the viral oncogene v-kit and a member of
the receptor tyrosine kinase subclass III family that includes
receptors for platelet-derived growth factor (PDGF), macrophage
colony-stimulating factor, and flt3 ligand.1-3 The c-kit
gene product is expressed by hematopoietic progenitor cells, mast
cells, germ cells, interstitial cells of Cajal (ICC), and some human
tumors.4-8 Studies of mice with inactivating mutations of
c-kit or its ligand, Steel factor (SLF), demonstrated that normal
functional activity of the c-kit gene product is absolutely essential
for maintenance of normal hematopoiesis,9,10
melanogenesis,5 gametogenesis,11 and growth and
differentiation of mast cells and ICC.12-14 We and others showed that SLF is produced by human and murine hematopoietic stromal
cells, including endothelial cells, fibroblasts, and bone marrow-derived stromal cells.15,16 The biologic features
of SLF and c-kit were reviewed by Broudy17 and Lyman and
Jacobsen.18
In addition to its importance in normal cellular physiologic
activities, c-kit plays a role in the biologic aspects of certain human
cancers, including germ cell tumors, mast cell tumors, gastrointestinal stromal tumors (GIST), small-cell lung cancer, melanoma, breast cancer,
acute myelogenous leukemia (AML), and neuroblastoma.19-27 Proliferation of tumor cell growth mediated by c-kit occurs either by a
specific mutation of the c-kit polypeptide that results in ligand-independent activation or by autocrine stimulation of the receptor.21,23 In some types of tumors, inhibition of c-kit activity reduces cellular proliferation, suggesting a role for use of
pharmacologic inhibitors of c-kit in the treatment of c-kit-dependent malignancies.20,21,28,29
STI 571 (formerly known as CGP 57148B), a 2-phenylaminopyrimidine
derivative, is a known inhibitor of the c-abl, bcr-abl, and PDGF
receptor (PDGFR) tyrosine kinases.30,31 This compound is
currently being evaluated in clinical trials for the treatment of
chronic myelogenous leukemia (CML).32,33 There is a close homology between the kinase domains of PDGFR and c-kit. Previous reports on other PDGFR kinase inhibitors noted that some of these compounds can inhibit the kinase activity of c-kit.34,35
Therefore, we speculated that STI 571 would also potently inhibit the
kinase activity of c-kit. Using biochemical and cell-based assays of receptor activation, signal transduction, proliferation, and apoptosis, we found that STI 571 inhibited the SLF-dependent activation of wild-type c-kit kinase activity. The concentration that produced 50%
inhibition (IC50) for these effects was approximately 100 nmol/L, which is similar to the concentration required for inhibition of bcr-abl and PDGFR. We also performed similar studies using a mast
cell leukemia cell line, HMC-1, that expresses a constitutively activated c-kit polypeptide.36,37 STI 571 had a more potent inhibitory effect on the kinase activity of this mutant receptor than
it did on ligand-dependent activation of the wild-type receptor. We
conclude that STI 571 is a potent inhibitor of c-kit kinase activity
and may be useful in treating tumors that are partly or completely
dependent on c-kit for proliferation or survival.
| |
Materials and methods |
Cell culture
M-07e is a human myeloid leukemia cell line that is dependent on the
growth factors interleukin 3, granulocyte-macrophage colony-stimulating
factor (GM-CSF), or SLF (Sigma, St Louis, MO) for
proliferation.38 These cells, a generous gift of Dr Hal Broxmeyer (Indiana University School of Medicine), were cultured in
RPMI 1640 (Gibco-BRL, Rockville, MD) supplemented with 10% fetal-calf
serum (FCS) and 200 U/mL recombinant human GM-CSF (Immunex, Seattle,
WA). HMC-1, a factor-independent cell line derived from a patient with
mast cell leukemia, expresses a juxtamembrane mutant c-kit polypeptide
that has constitutive kinase activity.37,39,40 The HMC-1
cells were generously provided by Dr Joseph Butterfield (Mayo Clinic,
Rochester, MN) and maintained in Dulbecco modified Eagle medium
(Gibco-BRL) supplemented with 10% FCS.
Reagents
Dr Elizabeth Buchdunger (Novartis Pharma, Basel, Switzerland)
generously provided STI 571 for these experiments. Fresh stock solutions of inhibitor (10 mmol/L) were made before each experiment by
dissolving 5 mg STI 571 in 1 mL phosphate-buffered saline (PBS; Gibco-BRL).
Antibodies
An anti-c-kit monoclonal antibody was used at a dilution of 1:200
(Roche Molecular Biochemicals, Indianapolis, IN). An
antiphosphotyrosine (anti-P-Tyr) antibody (PY20) was used at a dilution
of 1:1000 (Transduction Laboratories, Lexington, KY). An
antiphospho-p44/p42 mitogen-activated protein (MAP) kinase antibody
that identifies MAP kinase phosphorylated at threonine 202 and tyrosine
204 was used at a dilution of 1:1000 (New England Biolabs, Beverly,
MA). Antibodies to total MAP kinase and total Akt (New England Biolabs) were both used at a dilution of 1:1000. An antiphospho-Akt antibody that identifies Akt phosphorylated at serine 473 was used at a dilution
of 1:1000 (New England Biolabs). Peroxidase-conjugated goat-antimouse
antibody was used at a dilution of 1:5000 and goat antirabbit antibody
at a dilution of 1:10 000 (Pierce, Rockford, IL).
Protein lysates
M-07e cells were grown in serum-free RPMI 1640 at 37°C for
approximately 18 hours before they were incubated for 90 minutes in the
presence of various concentrations of STI 571. The cells were then
pelleted and resuspended in 1 mL RPMI 1640. STI 571 was added to each
tube to achieve the same concentration used during the 90 minutes of
preincubation. The cells were then incubated with inhibitor and growth
factor (SLF [Sigma Chemical, St Louis, MO] or GM-CSF) for 15 minutes
at 37°C. Subsequently, the cell pellets were lysed with 100 to 250 µL of protein lysis buffer (50 mmol/L Tris, 150 mmol/L sodium
chloride, 1% NP-40, and 0.25% deoxycholate, with addition of the
inhibitors aprotinin, leupeptin, pepstatin, phenylmethyl sulfonyl
fluoride, and sodium orthovanadate [Sigma]). Western immunoblot
analysis was performed as previously described.41
Experiments with HMC-1 cells were performed in the same way except that
neither SLF nor GM-CSF was added.
Proliferation assays
Cells were added to 96-well plates at a density of 20 000
cells/well for HMC-1 and 50 000 cells/well for M-07e. Experiments with
M-07e were performed with use of GM-CSF or SLF as a growth factor
supplement. Experiments using HMC-1 were performed without growth
factor supplementation. Proliferation at 48 hours was measured with an
XTT-based assay (Roche Molecular Biochemicals).
Apoptosis assays
Apoptosis at 48 or 72 hours was measured by using a flow cytometric
assay that assessed staining of cells with annexin V labeled with
fluorescein isothiocyanate, conjugated (FITC; Pharmingen, San Diego,
CA), as well as uptake of the vital stain 7-amino-actinomycin D (7-AAD;
Sigma).42,43
| |
Results |
STI 571 inhibits kinase activity of wild-type and mutant c-kit
polypeptide
We used the factor-dependent human myeloid leukemia cell line M-07e
to test the ability of STI 571 to inhibit the kinase activity of
wild-type c-kit receptor. Lysates prepared from M-07e cells were probed
with an anti-P-Tyr specific antibody. In these experiments, SLF-dependent activation was measured by receptor
autophosphorylation.44,45 No significant c-kit
autophosphorylation was observed in the absence of SLF (Figure
1). Inhibition of SLF-induced c-kit
autophosphorylation by STI 571 was dose dependent, with complete
inhibition observed at both 10 and 1.0 µmol/L. Inhibition was also
apparent at a dose of 0.5 µmol/L, although limited c-kit
autophosphorylation still occurred. At an STI 571 dose of 0.1 µmol/L,
c-kit activation was approximately 50% that in SLF-stimulated cells
not treated with inhibitor. Thus, we found that SLF activates c-kit and
that this activation was effectively inhibited by doses of STI 571 in
the range of 0.1 to 10 µmol/L.
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| Fig 1.
STI 571 inhibits Steel factor (SLF)-dependent
phosphorylation of c-kit.
Serum was withheld from M-07e cells overnight before they were
pretreated with various concentrations of STI 571 for 90 minutes. Cells
were then treated with vehicle (phosphate-buffered saline) or SLF
(final concentration, 200 ng/mL) for 15 minutes. Whole cell lysates
were immunoblotted by using an antiphosphotyrosine (anti-P-Tyr)
antibody. The membrane was stripped and reprobed with a monoclonal
antibody specific for human c-kit. The arrow indicates the position of
the 145-kd isoform of c-kit. Representative results from 1 of 8 independent experiments are shown.
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To determine whether STI 571 modulated expression of c-kit protein, the
membrane was stripped and reprobed with an anti-c-kit primary antibody.
The total amount of expressed c-kit protein did not change after
exposure to STI 571 (Figure 1). Thus, STI 571 was found to inhibit
SLF-dependent c-kit autophosphorylation but not expression of c-kit protein.
Activating mutations of c-kit have been found in several types of human
malignant disease, including mastocytosis and mast cell
leukemia, AML, GIST, and seminoma and dysgerminoma
tumors.22-24,46-49 These mutations occur in 2 distinct
regions of the cytoplasmic portion of the c-kit polypeptide the
juxtamembrane domain and the kinase domain.22,46-48,50 The
mutations result in ligand-independent constitutive kinase
activity.51-53 STI 571 functions as a competitive inhibitor
of adenosine triphosphate (ATP) binding to the kinase domain;
therefore, mutations that alter receptor structure or function might
abrogate the inhibitory effects of STI 571 on c-kit kinase activity.
To test the efficacy of STI 571 in inhibiting the activity of mutant
forms of c-kit, we used a human mast cell leukemia cell line (HMC-1)
that expresses a constitutively activated c-kit
polypeptide.37 Lysates prepared from HMC-1 cells were
probed with an anti-P-Tyr antibody, and receptor activation was
assessed by measuring autophosphorylation. As reported
previously,37 c-kit autophosphorylation was observed in the
absence of SLF (Figure 2). Inhibition of
c-kit autophosphorylation by STI 571 was dose dependent, with complete
inhibition observed at doses of 10 and 1.0 µmol/L. Nearly complete
inhibition occurred at a dose of 0.1 µmol/L. Limited
autophosphorylation of c-kit was observed when STI 571 doses of 0.001 to 0.01 µmol/L were used. Thus, our study showed that STI 571 not
only inhibits the autophosphorylation of the mutated c-kit receptor in
HMC-1 cells but is also a more potent inhibitor of this mutated
receptor than it is of the wild-type c-kit receptor. To determine
whether STI 571 modulated expression of c-kit protein, the membrane was
stripped and reprobed with an anti-c-kit antibody. There was no change
in the expression of c-kit protein in cells treated with STI 571 (Figure 2). Therefore, we found that STI 571 decreases
autophosphorylation of mutant c-kit polypeptide by inhibiting c-kit
kinase activity rather than by down-regulating expression of c-kit
protein.
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| Fig 2.
STI 571 inhibits autophosphorylation of an activated
mutant form of c-kit.
HMC-1 cells were treated with various concentrations of STI 571 for 90 minutes. Whole cell lysates were prepared and immunoblotted with an
anti-P-Tyr monoclonal antibody. The membrane was stripped and reprobed
with a monoclonal antibody specific for human c-kit. The arrow
indicates the position of the 145-kd isoform of c-kit. Representative
results from 1 of 8 independent experiments are shown.
|
|
STI 571 inhibits c-kit-dependent but not GM-CSF receptor-dependent
activation of MAP kinase
Treatment of hematopoietic cells with SLF or GM-CSF results in
activation of MAP kinase.54-56 We sought to test the
specificity of STI 571 by examining its effects on SLF-dependent and
GM-CSF-dependent activation of MAP kinase in M-07e cells. Complete
inhibition of SLF-dependent activation of MAP kinase occurred at 10-, 1.0-, and 0.5-µmol/L concentrations of STI 571 (Figure
3). At a dose of 0.1 µmol/L STI 571, MAP
kinase-activation levels were significantly lower than those in
control SLF-stimulated cells. Total MAP kinase expression was not
altered by treatment with STI 571 (Figure 3).
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| Fig 3.
STI 571 inhibits SLF-dependent activation of
mitogen-activated protein (MAP) kinase.
Whole cell lysates of M-07e cells were prepared and immunoblotted with
a polyclonal antibody specific for the doubly phosphorylated forms of
p44 and p42 MAP kinase (Erk1 and Erk2). The membrane was stripped and
reprobed with an antibody for total p44 and p42 MAP kinase. The arrows
indicate the locations of p44 and p42 MAP kinase. Representative
results from 1 of 8 independent experiments are shown.
|
|
To confirm the specificity of STI 571, we examined the compound's
effect on GM-CSF-dependent activation of MAP kinase. At concentrations
of 0.1 to 10 µmol/L, STI 571 did not affect GM-CSF-induced MAP
kinase activation in M-07e cells (Figure
4). Moreover, treatment with STI 571 did
not alter total expression of MAP kinase. Thus, we found that STI 571 inhibits SLF-dependent but not GM-CSF-dependent activation of MAP
kinase.
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| Fig 4.
STI 571 does not inhibit granulocyte-macrophage
colony-stimulating factor (GM-CSF)-dependent activation of MAP kinase.
Serum was withheld from M-07e cells overnight. The cells were then
pretreated with various concentrations of STI 571 for 90 minutes before
the addition of vehicle or GM-CSF (final concentration, 200 U/mL) for
15 minutes. Whole cell lysates were immunoblotted with an antibody
specific for phospho-MAP kinase. The membrane was stripped and reprobed
with an antibody for total p44 and p42 MAP kinase. The arrows indicate
the locations of p44 and p42 MAP kinase. Representative results from 1 of 2 independent experiments are shown.
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To confirm that STI 571 could inhibit signal-transduction events
downstream of an activated mutant form of c-kit, we treated HMC-1 cells
with various concentrations of inhibitor. Complete inhibition of MAP
kinase activation occurred at 10- and 1.0-µmol/L concentrations of
STI 571 (Figure 5). Partial inhibition was
observed at a dose of 0.1 µmol/L, and no inhibition occurred at a
dose of 0.01 µmol/L. Total MAP kinase expression was not altered by treatment with STI 571 (Figure 5). Therefore, we found that STI 571 inhibits the kinase activity of this mutant form of c-kit and blocks
c-kit-dependent activation of MAP kinase.
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| Fig 5.
STI 571 inhibits c-kit-dependent activation of MAP
kinase in HMC-1 cells.
HMC-1 cells were treated with various concentrations of STI 571 for 90 minutes. Whole cell lysates were immunoblotted with an antibody
specific for phospho-MAP kinase. The membrane was stripped and reprobed
with an antibody for total p44 and p42 MAP kinase. The arrows indicate
the locations of p44 and p42 MAP kinase. Representative results from 1 of 8 independent experiments are shown.
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STI 571 inhibits c-kit-dependent activation of Akt
SLF-dependent activation of the c-kit receptor results in activation
of phosphatidylinositol 3 kinase (PI3K).55,57 One of the
downstream events of the PI3K signal-transduction cascade is
phosphorylation and resultant activation of the proto-oncogene Akt.58 To assess the effect of STI 571 on this pathway, we
probed lysates from M-07e cells treated with SLF (with and without STI 571) with an antibody specific for the activated (phosphorylated) form
of Akt. At doses ranging from 1.0 to 10 µmol/L, STI 571 markedly inhibited SLF-dependent Akt phosphorylation (Figure
6). Partial inhibition of Akt activation
occurred at a dose of 0.1 µmol/L STI 571. The kinase inhibitor had no
effect on the expression of total Akt protein (Figure 6). Thus, STI 571 inhibited SLF-dependent activation of Akt but not expression of total
Akt protein.
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| Fig 6.
STI 571 inhibits SLF-dependent activation of Akt.
Whole cell lysates of M-07e cells were prepared and immunoblotted with
a polyclonal antibody specific for Akt protein that had been activated
by phosphorylation of serine 473. The membrane was stripped and
reprobed with an antibody to total Akt. Representative results from 1 of 8 independent experiments are shown.
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To test whether STI 571 could inhibit mutant c-kit receptor activation
of the signal-transduction pathways leading to Akt, we performed
similar studies using HMC-1 cells. STI 571 in doses ranging from 0.1 to
10 µmol/L strongly inhibited Akt phosphorylation (Figure
7). In contrast, no inhibition occurred at
a dose of 0.01 µmol/L. STI 571 had no effect on the expression of
total Akt protein (Figure 7). Therefore, the observed decrease in
phosphorylated Akt protein was found to be a result of inhibition of
Akt activation and not due simply to an effect on total cellular Akt
expression. Thus, we conclude that STI 571 inhibition of mutant c-kit
kinase activity blocks both MAP kinase activation and Akt activation.
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| Fig 7.
STI 571 inhibits c-kit-dependent activation of Akt in
HMC-1 cells.
Whole cell lysates of M-07e cells were prepared and immunoblotted with
a polyclonal antibody specific for Akt protein that had been activated
by phosphorylation of serine 473. The membrane was stripped and
reprobed with an antibody to total Akt. Representative results from 1 of 8 independent experiments are shown.
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STI 571 inhibits c-kit-dependent proliferation but not GM-CSF
receptor-dependent proliferation
Proliferation assays were conducted to study the effect of STI 571 on both SLF-dependent and GM-CSF-dependent proliferation of M-07e
cells. At inhibitor concentrations of 10 and 1.0 µmol/L, SLF-induced
proliferation was significantly decreased, by 97.6% ± 6.0% and 94.7% ± 5.3%, respectively, compared with that of cells treated with SLF but not STI 571 (Figure
8). With 0.1 µmol/L STI 571, cellular
proliferation was reduced by 59.9% ± 7.0%, and with 0.01 µmol/L, proliferation was decreased by 11.5% ± 4.8%. The decrease in proliferation in the presence of 0.1 to 10 µmol/L inhibitor was significant (P  .001).
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| Fig 8.
STI 571 inhibits SLF-dependent proliferation of M-07e
cells.
Serum was withheld from M-07e cells overnight before they were used in
proliferation assays. Cells were plated in a 96-well plate at a
concentration of 50 000 cells/well and grown in RPMI-1640 supplemented
with 10% fetal-calf serum (FCS) with and without 200 ng/mL SLF. STI
571 was added at the same time as SLF. Proliferation at 48 hours was
measured with an XTT-based assay system. Results are expressed as the
percentage of maximal proliferation (SLF only) (± SD).
Representative results from 1 of 6 independent experiments are shown.
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To test the specificity of STI 571 further, we measured the
proliferation of GM-CSF-stimulated M-07e cells in the presence and
absence of STI 571. Proliferation was not significantly affected by
the addition of STI 571, even at a dose of 10 µmol/L (Figure 9).Thus, we demonstrated that STI 571 selectively inhibits SLF-dependent but not GM-CSF-dependent
proliferation of M-07e cells.
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| Fig 9.
STI 571 has no effect on GM-CSF-dependent proliferation
of M-07e cells.
Serum was withheld from M-07e cells overnight before they were used in
proliferation assays. Cells were plated in a 96-well plate at a
concentration of 50 000 cells/well and grown in RPMI-1640 supplemented
with 10% FCS with and without 200 U/mL GM-CSF. STI 571 was added at
the same time as GM-CSF. Proliferation at 48 hours was measured with an
XTT-based assay system. Results are expressed as the percentage of
maximal proliferation (GM-CSF only) (± SD). Representative results
from 1 of 2 independent experiments are shown.
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To test the biologic effect of inhibiting the kinase activity of a
mutant c-kit receptor, we cultured HMC-1 cells for 48 hours in the
presence of various concentrations of STI 571. At inhibitor concentrations of 0.1 to 10 µmol/L, proliferation was decreased by
90% to 95% compared with results in cells treated with medium only
(Figure 10). Partial inhibition of
proliferation occurred at a dose of 0.01 µmol/L STI 571, and no
inhibition was observed at a dose of 0.001 µmol/L (data not shown).
The decrease in proliferation with doses of 0.01 to 10 µmol/L
inhibitor was significant (P < .001). Therefore, these
experiments showed that STI 571 inhibits proliferation of HMC-1 cells
with the same dose-response range observed for inhibition of receptor
autophosphorylation, MAP kinase activation, and Akt activation.
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| Fig 10.
STI 571 inhibits proliferation of HMC-1 cells.
HMC-1 cells were plated in 96-well plates at a concentration of 20 000
cells/well and cultured in RPMI-1640 supplemented with 10% FCS with
and without various concentrations of STI 571. Cellular proliferation
at 48 hours was measured with an XTT-based assay system. Results are
expressed as the percentage of maximal proliferation (cells only; no
STI 571) (± SD). Representative results from 1 of 6 independent
experiments are shown.
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STI 571 abrogates the antiapoptotic effect of c-kit activation
The effects of SLF on hematopoietic progenitors are both mitogenic
and antiapoptotic.59,60 The mechanism by which c-kit activation prevents apoptosis is unknown but may involve PI3K-dependent activation of Akt.61,62 In our proliferation assays, we
observed not only a decrease in proliferation but also morphologic
features consistent with cellular death (data not shown). Because of
these morphologic observations and our earlier finding that STI 571 inhibited SLF-dependent activation of Akt, we hypothesized that STI 571 would block the antiapoptotic effects of SLF.
To test our hypothesis, we withheld serum and growth factor from M-07e
cells for 24 hours before treating the cells with either complete
medium (10% FCS and GM-CSF), RPMI and 1% bovine serum albumin (BSA)
with and without SLF (200 ng/mL), or RPMI, 1% BSA, and SLF, and
various doses of STI 571. Apoptosis was assessed 24 and 48 hours later
by using a flow cytometric assay to measure cellular binding of an
annexin V-FITC conjugate, as well as uptake of the vital stain
7-AAD.42 Consistent with findings of previous studies,38 the M-07e cells underwent apoptosis after
removal of specific hematopoietic growth factors (Table
1). Apoptosis could be prevented by adding
GM-CSF or SLF. However, simultaneous addition of SLF and STI 571 at
doses of 10 or 1 µmol/L resulted in abrogation of the antiapoptotic
effect of SLF. The addition of STI 571 at a dose of 0.1 µmol/L had no
effect on the ability of SLF to prevent apoptosis.
HMC-1 cells treated with STI 571 underwent morphologic changes
consistent with cellular death. To test whether STI 571 was inducing
programmed cell death in these cells, we treated the HMC-1 cells for 72 hours with various doses of inhibitor. Apoptosis was assessed with a
flow cytometric assay as described above. In doses of 1 to 10 µmol/L,
STI 571 potently induced apoptosis in HMC-1 cells. A less potent effect
was observed at a dose of 0.1 µmol/L (Table
2), and there was no effect on apoptosis at a dose of 0.01 µmol/L. We conclude that the survival and
proliferation of HMC-1 cells depends on the kinase activity of the
mutant c-kit polypeptide.
| |
Discussion |
STI 571 was identified previously as a potent inhibitor of
the c-abl protein kinase and shown to have similar activity against v-abl and both the p210 and p190 forms of bcr-abl.31,63,64 Additionally, STI 571 was found to inhibit the kinase activity of the
 and  chains of PDGFR.30,31,64 Experiments using cell-based assay systems revealed that the IC50 for
inhibition of these enzymes was 0.2 to 0.3 µmol/L. The selectivity of
STI 571 was confirmed by testing its activity against a panel of
protein kinases. For example, the IC50 was above 100 µmol/L for the kinase activity of epidermal growth factor receptor,
fibroblast growth factor receptor, insulin-like growth factor-1
receptor, v-src, c-fgr, c-lyn, v-fms, protein kinase A, various protein
kinase C isoforms, casein kinases 1 and 2, and cdc2.30,31
In murine xenograft models of human CML, STI 571 was confirmed to have
significant antitumor activity, with minimal toxicity.31
Currently, this compound is being evaluated in clinical
trials for the treatment of CML.32,33
In our studies of cells expressing wild-type c-kit protein, we found
that STI 571 potently inhibited the activity of c-kit kinase activity,
resulting in inhibition of autophosphorylation. Inhibition of receptor
kinase activity markedly decreased activation of MAP kinase and a
PI3K-dependent enzyme (Akt). STI 571 had no effect on total cellular
expression of c-kit, Erk-1, Erk-2, or Akt. Treatment of M-07e cells
with STI 571 inhibited SLF-dependent but not GM-CSF-dependent
proliferation and abrogated the antiapoptotic activity of SLF (but not
GM-CSF). The IC50 for these effects was about 100 nmol/L,
which is similar to the IC50 of 100 to 300 nmol/L observed
with use of bcr-abl or PDGFR as a target.
Because of its structure, c-kit is classified as a type III receptor
tyrosine kinase. All members of this family have an extracellular domain containing 5 immunoglobulin-like domains, a single transmembrane domain, and a cytoplasmic domain with a split kinase domain and a
hydrophilic kinase insert sequence.17 The juxtamembrane and kinase domains of these receptors are strongly conserved.35 Interestingly, STI 571 can inhibit the kinase activity of c-kit, PDGFR, and PDGFR but has no effect on the kinase activity of the
related receptor tyrosine kinases c-fms and flk2/flt3 (data not
shown).31,64,65
c-kit has been implicated in the pathophysiologic mechanisms of a
variety of human tumors, including mastocytosis and mast cell leukemia,
testicular cancer, small-cell lung cancer, GIST, AML, neuroblastoma,
melanoma, and breast cancer. Two general mechanisms of c-kit activation
in malignant cells have been described: (1) autocrine or paracrine
stimulation of the receptor by SLF, and (2) acquisition of activating
mutations.8,21-23,26-28,66-69
Autocrine or paracrine stimulation of c-kit has been observed in some
human cancers, including neuroblastoma and small-cell lung
cancer.20,21,28 Experimental approaches to demonstrating the role of the SLF-c-kit axis in cancer cell biology have been hampered by the lack of a specific way to inhibit c-kit signal transduction. Previous studies used reagents such as the tyrphostins AG1295 and AG1296 to inhibit c-kit kinase activity. These studies, however, were limited by the poor solubility of these compounds in cell
culture media and by variable specificity.29,34 Other approaches for inhibiting c-kit experimentally included use of antisense oligonucleotides, transfection of dominant negative c-kit
constructs, and antibodies that neutralize the c-kit ligand-binding site. The first 2 techniques are limited by delivery efficiency and the
inability to totally inhibit c-kit expression or
activity.20,70,71 Use of neutralizing antibodies is
constrained by the binding affinity of the antibody. Additionally,
autocrine stimulation of receptor by its cognate ligand can, at least
in some cases, occur intracellularly and thus cannot be inhibited by
extracellular antibody.72 In this study, we found that STI
571 specifically inhibits c-kit, in addition to its previously reported
effects on PDGFR, c-abl, v-abl, and bcr-abl. Thus, this compound may be
useful in vitro for defining the role of the SLF-c-kit axis in tumor
cell proliferation.
Activating mutations of c-kit have been described in cases of human
mast cell disorders, seminoma, and GIST.22,23,46,47,73 For
mast cell disorders and GIST, the presence of the c-kit mutation, the
type of mutation, or both, has clinical prognostic
importance.24,46,73,74 Interestingly, these tumors arise in
tissue types (mast cells, germ cells, and ICC) whose development
depends on the activity of the SLF-c-kit axis. Indeed, these cells are
absent in mice with inactivating mutations of either SLF or
c-kit.5,13,14,75 The development of tumors in these tissues
may require either mutation of c-kit or an alternative way to activate
the downstream effectors of c-kit signal transduction.
Pharmacologic inhibition of c-kit is a potential novel approach to
treatment of malignancies that are partly or completely dependent on
the activity of an activated c-kit receptor. Although current medical
treatments for seminoma are curative for most patients, there are no
effective medical treatments for patients with advanced mast cell
disease or recurrent or metastatic GIST.76,77 Indeed, a
1999 retrospective review of chemotherapy for GIST found a response
rate of less than 10%.78
We used the HMC-1 cell line to test the ability of STI 571 to inhibit
the kinase activity of a mutant form of c-kit. This factor-independent
cell line was originally derived from a patient with mast cell leukemia
and expresses a constitutively activated c-kit
protein.37,40 In our studies, STI 571 potently inhibited receptor autophosphorylation, MAP kinase activation, and Akt activation without affecting levels of c-kit, Erk-1, Erk-2, or Akt. Additionally, doses of 0.01 to 10 µmol/L STI 571 inhibited HMC-1 cellular
proliferation. HMC-1 cells appear to be strongly dependent on the
activity of the mutant receptor to prevent apoptosis, since 85% to
95% of cells exposed to 1 to 10 µmol/L STI 571 for 48 to 72 hours
underwent programmed cell death. The IC50 for induction of
apoptosis was approximately 0.1 µmol/L. Thus, STI 571 can potently
inhibit the kinase activity of the mutant c-kit polypeptide expressed
by HMC-1 cells. The effect of STI 571 on receptor activation was even
more potent than that observed when the wild-type c-kit receptor was used as a target. It is unknown whether this particular c-kit mutation
actually increases the sensitivity of the kinase domain to inhibition
by the compound or whether the observed differences in kinase
sensitivity resulted from unrelated factors, such as differential
cellular uptake of the compound.
On the basis of these studies, we propose that STI 571 may be useful
for treatment of malignancies that are completely or partly dependent
on the activity of wild-type or mutant c-kit for proliferation or
survival. In phase I and II clinical trials of treatment of patients
with CML, STI 571 was effective and well tolerated.32,33 In
these trials, trough levels of 1 µmol/L were readily obtained in
patients, and this level of STI 571 is an order of magnitude greater
than the IC50 for inhibition of c-kit (B. Druker,
unpublished data, March 2000). Further studies are warranted to
determine the potential efficacy of inhibiting c-kit kinase activity as
a novel strategy for treatment of human cancers.
| |
Acknowledgment |
We thank Michael Moody for his help in preparing figures.
| |
Footnotes |
Submitted January 13, 2000; accepted March 30, 2000.
Supported by a Merit Review Grant from the Department of Veterans
Affairs (M.C.H.).
Reprints: Michael C. Heinrich, R&D-19, 3710 SW US Veterans
Hospital Road, Portland, OR 97207; e-mail: heinrich{at}ohsu.edu.
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.
| |
References |
1.
Yarden Y, Kuang WJ, Yang-Feng T, et al.
Human proto-oncogene c-kit: a new cell surface receptor tyrosine kinase for an unidentified ligand.
EMBO J.
1987;11:3341-3351.
2.
Besmer P, Murphy JE, George PC, et al.
A new acute transforming feline retrovirus and relationship of its oncogene v-kit with the protein kinase gene family.
Nature.
1986;320:415-419[Medline]
[Order article via Infotrieve].
3.
Small D, Levenstein M, Kim E, et al.
STK-1, the human homolog of Flk-2/Flt-3, is selectively expressed in CD34+ human bone marrow cells and is involved in the proliferation of early progenitor/stem cells.
Proc Natl Acad Sci U S A.
1994;91:459-463[Abstract/Free Full Text].
4.
Ishikawa K, Komuro T, Hirota S, Kitamura Y.
Ultrastructural identification of the c-kit-expressing interstitial cells in the rat stomach: a comparison of control and Ws/Ws mutant rats.
Cell Tissue Res.
1997;289:137-143[Medline]
[Order article via Infotrieve].
5.
Russell ES.
Hereditary anemias of the mouse: a review for geneticists.
Adv Genet.
1979;20:357-359[Medline]
[Order article via Infotrieve].
6.
Nocka K, Majumder S, Chabot B, et al.
Expression of c-kit gene products in known cellular targets of W mutations in normal and W mutant miceevidence for an impaired c-kit kinase in mutant mice.
Genes Dev.
1989;3:816-826[Abstract/Free Full Text].
7.
Natali PG, Nicotra MR, Sures I, Santoro E, Bigotti A, Ullrich A.
Expression of c-kit receptor in normal and transformed human nonlymphoid tissues.
Cancer Res.
1992;52:6139-6143[Abstract/Free Full Text].
8.
Turner AM, Zsebo KM, Martin F, Jacobsen FW, Bennett LG, Broudy VC.
Nonhematopoietic tumor cell lines express stem cell factor and display c-kit receptors.
Blood.
1992;80:374-381[Abstract/Free Full Text].
9.
Lowry PA, Zsebo KM, Deacon DH, Eichman CE, Quesenberry PJ.
Effects of rrSCF on multiple cytokine responsive HPP CFC generated from SCA+Lin murine hematopoietic progenitors.
Exp Hematol.
1991;19:994-996[Medline]
[Order article via Infotrieve].
10.
Migliaccio G, Migliaccio AR, Valinsky J, et al.
Stem cell factor induces proliferation and differentiation of highly enriched murine hematopoietic cells.
Proc Natl Acad Sci U S A.
1991;88:7420-7424[Abstract/Free Full Text].
11.
Dolci S, Williams DE, Ernst MK, et al.
Requirement for mast cell growth factor for primordial germ cell survival in culture.
Nature.
1991;352:809-811[Medline]
[Order article via Infotrieve].
12.
Tsai M, Takeishi T, Thompson H, et al.
Induction of mast cell proliferation, maturation, and heparin synthesis by the rat c-kit ligand, stem cell factor.
Proc Natl Acad Sci U S A.
1991;88:6382-6386[Abstract/Free Full Text].
13.
Ward SM, Burns AJ, Torihashi S, Harney SC, Sanders KM.
Impaired development of interstitial cells and intestinal electrical rhythmicity in steel mutants.
Am J Physiol.
1995;269:C1577-C1585[Abstract/Free Full Text].
14.
Huizinga JD, Thuneberg L, Kluppel M, Malysz J, Mikkelsen HB, Bernstein A.
W/kit gene required for interstitial cells of Cajal and for intestinal pacemaker activity.
Nature.
1995;373:347-349[Medline]
[Order article via Infotrieve].
15.
Heinrich MC, Dooley DC, Freed AC, et al.
Constitutive expression of steel factor gene by human stromal cells.
Blood.
1993;82:771-783[Abstract/Free Full Text].
16.
Aye MT, Hashemi S, Leclair B, et al.
Expression of stem cell factor and c-kit mRNA in cultured endothelial cells, monocytes and cloned human bone marrow stromal cells (CFU-RF).
Exp Hematol.
1992;20:523-527[Medline]
[Order article via Infotrieve].
17.
Broudy VC.
Stem cell factor and hematopoiesis.
Blood.
1997;90:1345-1364[Free Full Text].
18.
Lyman SD, Jacobsen SEW.
c-kit ligand and flt3 ligand: stem/progenitor cell factors with overlapping yet distinct activities.
Blood.
1998;91:1101-1134[Free Full Text].
19.
Hibi K, Takahashi T, Sekido Y, et al.
Coexpression of the stem cell factor and the c-kit genes in small-cell lung cancer.
Oncogene.
1991;6:2291-2296[Medline]
[Order article via Infotrieve].
20.
Krystal GW, Hines SJ, Organ CP.
Autocrine growth of small cell lung cancer mediated by coexpression of c-kit and stem cell factor.
Cancer Res.
1996;56:370-376[Abstract/Free Full Text].
21.
DiPaola RS, Kuczynski WI, Onodera K, et al.
Evidence for a functional kit receptor in melanoma, breast, and lung carcinoma cells.
Cancer Gene Ther.
1997;4:176-182[Medline]
[Order article via Infotrieve].
22.
Tian Q, Frierson HFJ, Krystal GW, Moskaluk CA.
Activating c-kit gene mutations in human germ cell tumors.
Am J Pathol.
1999;154:1643-1647[Abstract/Free Full Text].
23.
Hirota S, Isozaki K, Moriyama Y, et al.
Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors.
Science.
1998;279:577-580[Abstract/Free Full Text].
24.
Worobec AS, Semere T, Nagata H, Metcalfe DD.
Clinical correlates of the presence of the Asp816Val c-kit mutation in the peripheral blood mononuclear cells of patients with mastocytosis.
Cancer.
1998;83:2120-2129[Medline]
[Order article via Infotrieve].
25.
Beck D, Gross N, Brognara CB, Perruisseau G.
Expression of stem cell factor and its receptor by human neuroblastoma cells and tumors.
Blood.
1995;86:3132-3138[Abstract/Free Full Text].
26.
Ning ZQ, Li J, Arceci RJ.
Activating mutations of c-kit at Asp816 are associated with constitutive activation of Stat signal transduction pathways in acute myeloid leukemia [abstract].
Blood.
1999;94:264a.
27.
Beghini A, Cairoli R, Morra E, Larizza L.
In vivo differentiation of mast cells from acute myeloid leukemia blasts carrying a novel activating ligand-independent C-kit mutation.
Blood Cells Mol Dis.
1998;24:262-270[Medline]
[Order article via Infotrieve].
28.
Timeus F, Crescenzio N, Valle P, et al.
Stem cell factor suppresses apoptosis in neuroblastoma cell lines.
Exp Hematol.
1997;25:1253-1260[Medline]
[Order article via Infotrieve].
29.
Krystal GW, Carlson P, Litz J.
Induction of apoptosis and inhibition of small cell lung cancer growth by the quinoxaline tyrphostins.
Cancer Res.
1997;57:2203-2208[Abstract/Free Full Text].
30.
Buchdunger E, Zimmermann J, Mett H, et al.
Inhibition of the Abl protein-tyrosine kinase in vitro and in vivo by a 2-phenylaminopyrimidine derivative.
Cancer Res.
1996;56:100-104[Abstract/Free Full Text].
31.
Druker BJ, Tamura S, Buchdunger E, et al.
Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells.
Nat Med.
1996;2:561-566[Medline]
[Order article via Infotrieve].
32.
Druker BJ, Talpaz M, Resta D, et al.
Clinical efficacy and safety of an Abl specific tyrosine kinase inhibitor as targeted therapy for chronic myelogenous leukemia [abstract].
Blood.
1999;94:368a.
33.
Druker BJ, Sawyers CL, Talpaz M, Resta D, Peng B, Ford J.
Phase I trial of a specific ABL tyrosine kinase inhibitor, CGP 57148, in interferon refractory chronic myelogenous leukemia patients [abstract].
Proc Am Soc Clin Oncol.
1999;18:7a.
34.
Kovalenko M, Gazit A, Bohmer A, et al.
Selective platelet-derived growth factor receptor kinase blockers reverse sis-transformation.
Cancer Res.
1994;54:6106-6114[Abstract/Free Full Text].
35.
Rousset D, Agnes F, Lachaume P, Andre C, Galibert F.
Molecular evolution of the genes encoding receptor tyro |
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Abstract
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