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Prepublished online as a Blood First Edition Paper on April 17, 2002; DOI 10.1182/blood-2001-12-0350.
NEOPLASIA
From the Department of Surgical and Radiological
Sciences, School of Veterinary Medicine, University of California at
Davis, and from Sugen, Inc, South San Francisco, CA.
Mutations in the proto-oncogene c-kit, including point
mutations, deletions, or duplications in the negative regulatory
juxtamembrane (JM) domain or point mutations in the catalytic domain,
have been observed in human and canine cancers and often result in
constitutive activation of Kit in the absence of ligand binding. To
identify a receptor tyrosine kinase (RTK) inhibitor capable of blocking the function of mutant Kit, we evaluated 3 indolinones (SU11652, SU11654, and SU11655) that act as competitive inhibitors of adenosine triphosphate binding to several members of the split kinase family of
RTKs, including VEGFR, FGFR, PDGFR, and Kit. Mast cell lines expressing
either wild-type (WT) Kit, a point mutation in the JM domain, a tandem
duplication in the JM domain, or a point mutation in the catalytic
domain were used for these studies. All 3 indolinones inhibited
phosphorylation of WT Kit in the presence of stem cell factor at
concentrations as low as 0.01 µM. Autophosphorylation of both JM
mutants was inhibited at 0.01 to 0.1 µM, resulting in cell cycle
arrest within 24 hours, whereas autophosphorylation of the catalytic
domain mutant was inhibited at 0.25 to 0.5 µM, resulting in cell
death within 24 hours. poly(ADP-ribose) polymerase (PARP) cleavage was
noted in all Kit mutant lines after indolinone treatment. In
summary, SU11652, SU11654, and SU11655 are effective RTK inhibitors
capable of disrupting the function of all forms of mutant Kit. Because
the concentrations of drug necessary for receptor inhibition are
readily achievable and nontoxic in vivo, these compounds
may be useful in the treatment of spontaneous cancers expressing Kit mutations.
(Blood. 2002;100:585-593) The proto-oncogene c-kit was first
identified as the oncogenic component of the acutely transforming
Hardy-Zuckerman 4-feline sarcoma virus.1 It encodes Kit, a
type 3 receptor tyrosine kinase (RTK) that binds the ligand stem cell
factor (SCF).2 Structurally, Kit is related to the
receptors for platelet-derived growth factor, vascular endothelial
growth factor, fibroblast growth factor, and FLT-3 ligand (reviewed in
Ashman3). All contain an extracellular ligand-binding
domain composed of 3 to 7 immunoglobulinlike regions, a transmembrane
domain, a negative regulatory juxtamembrane (JM) domain, and 2 kinase
domains in which resides a kinase insert. SCF-Kit interactions promote
the development of mast cells from hematopoietic progenitors; mice deficient in either SCF or Kit (Sl or W mutant
mice) exhibit a near-complete absence of mast cells, anemia, and
thrombocytopenia.2,4-6 These mice are also sterile and
lack skin pigmentation because of the absence of melanocytes. With
regard to mast cells, SCF-Kit interactions are intimately involved in
several critical processes. Most important, Kit signaling is required
for the differentiation and maturation of mast cells in
vivo.2,7-10 Other activities attributable to SCF-Kit
binding include chemotaxis and haptotaxis of mast cells and the
promotion of cell survival and
proliferation.2,7,8,11,12
Several years ago, investigators began to study the potential role of
Kit dysfunction in malignant mast cells. Point mutations in
c-kit that lead to constitutive activation of Kit in the
absence of ligand binding were identified in 3 malignant mast cell
lines (human HMC-1, rat RBL, and mouse P815), providing an indication that dysregulation of Kit may promote uncontrolled growth or survival of mast cells.13-15 Following this discovery, similarly
activating mutations were identified in the CD34+
hematopoietic precursors from patients with mastocytosis and in cells
from patients with urticaria pigmentosa, thus providing the first
evidence that c-kit may play a role in spontaneous mast cell
disease.16,17 Recent studies have consistently
demonstrated the presence of activating point mutations in the
catalytic domain of c-kit in human patients with aggressive
mastocytosis, mast cell leukemia, and hematologic disorders with
associated mastocytosis.17-23
In contrast to the disease in humans, mast cell neoplasms are one of
the most common malignant tumors of the dog, representing between 7%
and 21% of all canine tumors.24,25 Given the link between
mast cell disorders and dysfunctional Kit, we investigated whether
similar mutations in c-kit were present in canine mast cell
tumors (MCTs). Although c-kit derived from the canine MCTs did not contain the previously described activating mutations, 30% to
50% of the tumors examined had novel mutations consisting of tandem
duplications in exons 11 and 12 of the gene.26 As is the
case with tandem duplications in exons 11 and 12 of Flt-3, these
mutations also result in constitutive phosphorylation of Kit in the
absence of ligand binding.26-29
In humans and dogs, malignant mast cell disease often suggests an
extremely poor prognosis. To date, no reliable effective chemotherapeutic agents have been identified.25,30-34
Systemic mast cell disorders in humans have been treated with
interferon- The purpose of this study was to identify an RTK inhibitor
capable of blocking the function of multiple forms of mutant Kit. This
is of particular importance because STI571 does not appear to be an
effective inhibitor of the catalytic domain Kit mutant (most commonly
found in human mastocytosis) and because resistance to STI571 has been
noted in treated patients, making the development and application of
additional inhibitors necessary.52-57
The earliest small molecule with an indolinone chemical structure to
enter clinical testing (SU5416; semaxanib; Sugen Inc, South San
Francisco, CA) was targeted to inhibit a single RTK, VEGFR2, based on
the hypothesis that broadly targeting multiple RTKs might result in
unexpected toxicities. Later, clinical experience with a second
indolinone (SU6668) with inhibitory activity against the family of RTKs
with split kinase domains We therefore undertook a series of experiments to determine the potency
of these compounds in the functional inhibition of a variety of cell
lines expressing mutant Kit using phosphorylation of the receptor, cell
viability, and apoptosis as read-outs for activity. Our studies
demonstrate that all 3 indolinones are capable of inhibiting
autophosphorylation of catalytic domain and JM domain mutant forms of
Kit. As such, these compounds may be useful in the treatment of
spontaneous cancers expressing Kit mutations.
Cell culture
Indolinone reagents The RTK inhibitors SU11652 (5-[(Z)-(5-chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-N-[2-(diethylamino)ethyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide), SU11654 (5-[(Z)-(5-fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-2,4-dimethyl-N-(2-pyrrolidin-1-ylethyl)-1H-pyrrole-3-carboxamide), and SU11655 (5-[(Z)-(5-chloro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-2,4-dimethyl-N-(2-pyrrolidin-1-ylethyl)-1H-pyrrole-3-carboxamide) were provided by Sugen. Fresh stock solutions of inhibitor (10 mM) were prepared before each experiment by dissolving approximately 4 mg in 1 mL dimethyl sulfoxide (DMSO). This stock solution was then used for further dilution in DMSO.Antibodies For flow cytometry, the Ack45 anti-Kit monoclonal antibody conjugated to phycoerythrin was used at a dilution of 1:200 (BD PharMingen, San Diego, CA). For immunoprecipitation and Western blotting of Kit, the Ab-1 rabbit polyclonal anti-Kit antibody was used (Oncogene Research Products, Boston, MA). A peroxidase-conjugated antiphosphotyrosine antibody (RC20; BD Transduction Laboratories, San Diego, CA) was used to detect phosphorylation of Kit at a concentration of 1:2500. An anti-PARP monoclonal antibody (BD Transduction Laboratories) was used to identify PARP cleavage after indolinone treatment at a concentration of 1:2000. Anti-propidium iodide (PI) 3-kinase and anti-SHP-1 monoclonal antibodies (BD Transduction Laboratories) were used to identify coprecipitation with Kit. Peroxidase-conjugated mouse antirabbit and rat antimouse antibodies (Pierce, Rockford, IL) were used at dilutions of 1:40 000.Cell viability and proliferation The BR, C2, and P815 cells were counted and resuspended in 24-well plates at a concentration of 0.5 × 106 cells/well (BR and C2) or 0.25 × 106 cells/well (P815) in complete medium. Cells were left untreated, or SU11652, SU11654, or SU11655 was added to a final concentration of 0.01 to 1 µM. Cells were collected after 24, 48, and 72 hours of culture and were counted to determine the number of cells surviving.Propidium iodide staining and flow cytometry The BR, C2, and P815 cells were counted and resuspended in 24-well plates at a concentration of 0.5 × 106 cells/well (BR and C2) or 0.25 × 106 cells/well (P815) in complete medium. Cells were left untreated, or SU11652, SU11654, or SU11655 was added to a final concentration of 0.01 to 1 µM. Cells were collected after 24, 48, and 72 hours of culture, washed twice with 0.1% glucose-phosphate-buffered saline (PBS), fixed in 1 mL cold 70% ethanol, and stored at 4°C. Before flow cytometric analysis, the cells were centrifuged, and 0.5 mL PI staining solution was added (50 µg/mL PI and 10 µg/mL RNAse in 0.1% glucose-PBS).Immunoprecipitation and Western blotting For analysis of PARP cleavage, cell lines were either left untreated or were incubated with SU11652, SU11654, or SU1165 at concentrations of 0.01 to 1 µM. After 6, 24, 48, or 72 hours, the cells were collected, washed once in PBS, and resuspended in lysis buffer consisting of 20 mM Tris-HCl, pH 8.0, 137 mM NaCl, 10% glycerol, 1% IGEPAL CA-630, 10 mM EDTA, 1 µg/mL aprotinin, 1 µg/mL leupeptin, 1 µg/mL pepstatin A, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na orthovanadate, and 10 mM Na fluoride for 1 hour at 4°C. Lysates were then spun, and protein was quantitated. Approximately 40 µg protein per sample was loaded onto a 12% polyacrylamide gel and transferred to polyvinylidene difluoride (PVDF) membrane after electrophoresis, then Western blotting was performed for PARP.For analysis of Kit autophosphorylation, cell lines were serum starved
for 2 hours, then left untreated or incubated with SU11652, SU11654, or
SU11655 at concentrations of 0.01 to 1 µM for 2 hours. Cells were
collected, washed once in PBS, and resuspended in lysis buffer for 1 hour at 4°C. The cell line expressing wild-type (WT) Kit (C57) was
incubated with recombinant mouse SCF (rmSCF) (Sigma, St Louis, MO) at
100 µg/mL for 15 minutes before lysis. The supernatant was precleared
with Protein A Sepharose 4 Fast Flow beads (Pierce) for 1 hour at
4°C, then incubated with 2 µg anti-Kit overnight at 4°C. The
Protein A beads were added to the supernatant and incubated for 1 hour
at 4°C, and the beads were collected and washed before the addition
of the loading buffer. Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) was performed using 7.5% or 10% gels,
samples were transferred to PVDF membrane, and the membranes were
incubated with either anti-PI 3-kinase, anti-SHP-1, or
antiphosphotyrosine antibodies. Membranes were developed using
SuperSignal ECL (Pierce), then stripped using a buffer consisting of
2% wt/vol SDS, 62.5 mM Tris HCl, pH 6.8, and 100 mM
Mutation in the juxtamembrane domain or catalytic domain of Kit leads to constitutive phosphorylation and association of PI 3-kinase Mutations in the kinase domain of Kit arise spontaneously in dogs and humans with mast cell cancer.17-23,26,62 In humans, these mutations occur primarily in the catalytic domain of Kit and have been associated with aggressive mastocytosis.19-23 In dogs, Kit mutations occur almost exclusively in exons 11 and 12 and consist of internal tandem duplications, similar to those reported in the Flt3 gene of human patients with acute myeloid leukemia (AML).26,63-65 Interestingly, gastrointestinal stromal tumors in humans frequently have mutations in Kit, although these consist of deletions in exon 11 rather than duplications.44,45,48 It has previously been demonstrated that mutations in the catalytic domain or JM domain lead to autophosphorylation of Kit in the absence of SCF stimulation.13-15,26,44,62 We were interested to know whether such mutations resulted in the constitutive association of PI 3-kinase because this downstream signaling molecule has been implicated in malignant transformation. In addition, previous studies have indicated that constitutively active forms of Kit lead to an increased rate of SHP-1 degradation and, thus, a markedly decreased level of protein associated with Kit.66 We initially believed that tandem duplications in the JM domain of Kit would lead to conformational changes and prevent SHP-1 binding, thereby resulting in the observed constitutive phosphorylation.We used 4 malignant mast cell lines expressing various forms of Kit to
begin to investigate the effects of catalytic domain and JM domain
mutations on cell surface expression of Kit and the association of
mutant Kit with PI 3-kinase and SHP-1. The C57 mouse mast cell line
expresses WT Kit, the P815 mouse mast cell line has a point mutation in
the catalytic domain (Asp816Tyr) and WT Kit, the C2 canine mast cell
line has a tandem duplication in exon 11 of the JM domain and does not
express WT Kit, and the BR canine mast cell line has a point mutation
in exon 11 (Leu575Pro) and WT Kit (Figure
1A). We first wanted to know whether the
presence of a mutation in either the catalytic domain or the JM domain led to alterations in cell surface expression of Kit. As can be seen in
Figure 1B, the expression of Kit was similar among all cell lines,
regardless of the presence or absence of a mutation or nature of the
mutation.
The effect of various Kit mutations on downstream signaling events that regulate cell growth and function is not completely characterized. However, it is now known that the activation of PI 3-kinase by Kit is important for malignant transformation.67,68 As previously reported, Kit autophosphorylation was present in the absence of SCF stimulation in the cell lines with mutations in the catalytic or JM domains. Additionally, we found that PI 3-kinase was constitutively associated with mutant Kit in the presence of mutation in either the JM or the catalytic domain but, as expected, not in the presence of WT Kit (Figure 1C). Interestingly, the point mutation in the JM domain led to slightly less autophosphorylation of Kit than the other 2 mutants and to a lower level of PI 3-kinase association, suggesting that point mutations in the JM domain may be less likely to result in transforming events. The tyrosine phosphatase SHP-1 is known to bind to Kit in the JM domain
and is believed to negatively regulate receptor function. We initially
thought that mutations in the JM domain, particularly tandem
duplications, might disrupt the conformation of this portion of the
receptor, thereby inhibiting SHP-1 association. Interestingly, we found
that SHP-1 remained associated with Kit in the presence or absence of
mutation in the JM or the catalytic domain. It is therefore likely that
the activation of Kit as a consequence of a tandem duplication in the
JM domain is secondary to the dimerization of receptor in the absence
of ligand binding rather than the loss of tyrosine phosphatase
function. Indeed, spontaneous receptor dimerization of Kit has been
demonstrated to occur in the presence of deletions and point mutations
in the JM domain.69,70 Moreover, a recent study identified
critical residues in the Kit intracellular JM domain that exist in a
putative Indolinone compounds inhibit the growth of mast cell lines expressing mutant Kit through cell cycle arrest and apoptosis To identify an RTK inhibitor capable of blocking the function of all forms of mutant Kit, we investigated the effects of 3 indolinones that act as competitive inhibitors with respect to ATP at the intracellular catalytic domain of the receptor.59,60,72-76 These indolinones, denoted SU11652, SU11654, and SU11655 (Sugen), inhibit several members of the split kinase family of RTKs including VEGFR, FGFR, PDGFR, and Kit (Figure 2, Table 1). The core structure of these indolinones is predicted to bind to Kit in a fashion similar to that observed in the SU5402/SU6668 cocrystal structure with FGFR1. The functional group off the pyrrole ring extends to the opening of the ATP binding site, which is hydrophilic and exposed to solvent.73 This is in contrast to the crystal structure of an STI571 analog in Abl, in which the inhibitor protrudes to the hydrophobic end of the ATP binding site by pushing up the flexible activation loop.77 To determine whether these compounds were capable of disrupting various forms of mutant Kit, we used the cell lines described above, each of which has a unique mutation in c-kit leading to constitutive phosphorylation in the absence of SCF stimulation.
To assess the effects of the indolinones on cell proliferation, BR, C2,
or P815 cells were cultured with SU11652, SU11654, or SU11655 at a
variety of concentrations, and the number of remaining cells was
determined at 24, 48, and 72 hours of culture. Interestingly, the 2 cell lines expressing Kit JM mutations (BR and C2) required lower
concentrations of indolinones for the inhibition of cell proliferation
than did the cell line expressing a catalytic domain mutation (P815)
(Figure 3). Both cell lines demonstrated
growth inhibition at concentrations as low as 0.01 µM. However, the
effect of indolinone treatment was more profound on the P815 line
because no live cells were noted after 24 hours of culture at 0.5 µM
(Figure 3). The cell line expressing WT Kit (C57) is a growth
factor-independent mast cell line and could not be assessed in this
model because additional genomic mutations render it relatively
resistant to the effects of these indolinones. As such, the inhibition
of cell proliferation was only noted at very high indolinone
concentrations (10 µM) (data not shown).
In the cell survival assays detailed above, we observed a decrease in
absolute cell numbers and morphologic features consistent with
apoptosis. To distinguish between cell cycle arrest and cell death, we
evaluated the cells by PI staining after treatment with SU11652,
SU11654, or SU11655. Cells were treated with various concentrations of
drug for 24, 48, or 72 hours, then collected for flow cytometric
analysis. The cell line expressing the catalytic domain mutation (P815)
exhibited rapid apoptosis within 24 hours at concentrations between
0.25 and 0.5 µM. Cell cycle arrest was not noted before apoptosis,
and apoptosis did not occur at concentrations lower than 0.25 µM (Figure 4). The cell line expressing
a JM tandem duplication (C2) exhibited initial cell cycle arrest
at concentrations between 0.01 and 0.1 µM after 24 hours of culture.
Apoptosis was then noted by 48 to 72 hours of drug treatment,
particularly at drug concentrations of 0.1 µM and above.
Interestingly, the cell line expressing the JM point mutation (BR)
exhibited far less apoptosis after indolinone treatment than the other
2 cell lines expressing Kit mutants, with cell death noted primarily at
concentrations of 1 µM. Instead, cell cycle arrest was the principle
effect of drug treatment on this cell line. As in the previous
experiment, the effects of the 3 indolinones on cell cycling and
apoptosis were not evaluated in C57 cells because this growth
factor-independent mast cell line expresses additional genomic
mutations that render it relatively resistant to the effects of these
compounds.
To more specifically evaluate the timing of apoptosis after
indolinone treatment, we used PARP cleavage as a sensitive indicator of
the apoptotic cascade induced through caspase 3 activation. In
agreement with the PI staining results, we found that the catalytic domain mutant underwent complete PARP cleavage within 24 hours of
indolinone exposure at 0.5 µM, with some activity observed at 0.25 µM (Figure 5). After 48 hours of
exposure to SU11652, SU11654, and SU11655, the catalytic domain mutant
exhibited complete PARP cleavage at concentrations of 0.25 µM. In
contrast, all 3 indolinones induced PARP cleavage in the JM tandem
duplication mutant by 24 hours of culture with 1 µM drug and by 48 hours of culture with 0.01 µM drug. Interestingly, the JM point
mutant required at least 72 hours of culture to undergo a similar
degree of PARP cleavage. Therefore, although SU11652, SU11654, and
SU11655 were capable of inducing cell cycle arrest and apoptosis in
cell lines expressing various mutations in Kit, these effects required different concentrations of compound and occurred at different time
points after drug exposure. These results support the notion that the 3 Kit mutants result in unique patterns of downstream signaling events
and may contribute to malignant transformation through different
effects on normal mast cells.
Indolinone compounds inhibit autophosphorylation of mutant Kit in a dose-dependent manner To determine whether the observed cell cycle arrest correlated with the disruption of Kit phosphorylation, we evaluated the effects of indolinone treatment on the phosphorylation status of Kit derived from the previously described cell lines. Inhibition of WT Kit phosphorylation was dose dependent, occurring at concentrations as low as 0.01 µM in the presence of SCF stimulation (Figure 6). In agreement with the previous results regarding cell cycle arrest and apoptosis, the catalytic domain mutant required higher concentrations of all 3 indolinones for inhibition, with an IC50 of approximately 0.25 to 0.5 µM. Finally, SU11652, SU11654, and SU11655 inhibited autophosphorylation of both JM mutants with an IC50 of 0.01 to 0.1 µM. Although autophosphorylation of the Kit with the JM point mutation was nearly completely inhibited at 0.01 µM of all 3 indolinones, the cell line expressing this mutant (BR) did not undergo apoptosis at this concentration of drug. This suggests that other mutations are present in the BR line that contribute to growth factor independence and cell survival. Reprobing of all blots for Kit revealed no changes in the expression of Kit protein in cells treated with the indolinones. As such, the dose-dependent decrease in the autophosphorylation of Kit was secondary to the inhibition of kinase activity rather than the down-regulation of protein expression. It is therefore probable that the observed biological effects of SU11652, SU11654, and SU11655 on the malignant mast cell lines are the direct result of the inhibition of Kit signaling.
Mutations in the proto-oncogene c-kit occur in a variety of neoplastic diseases, particularly those associated with mast cells in dogs and humans. These mutations consist of point mutations in the catalytic domain, deletions, point mutations, and tandem duplications in the JM domain, and less commonly, point mutations in the extracellular domain. Most of these mutations lead to constitutive phosphorylation of Kit in the absence of ligand binding. Interestingly, neoplastic diseases expressing activating mutations in Kit (mast cell tumors and GISTs) are relatively resistant to standard chemotherapeutic regimens. As such, novel approaches to the treatment of these diseases are critical for improved therapeutic outcome. The recent success of the RTK inhibitor, STI571, in the treatment of chronic myelogenous leukemia has demonstrated that identification of particular aberrant signaling pathways in neoplastic cells and subsequent targeting of these pathways with specific inhibitors (so-called targeted therapy) may yield substantial anticancer benefits. Ideally, an effective RTK inhibitor would exhibit good oral bioavailability, a large volume of distribution, an elimination half-life that permits daily dosing, and the ability to block the function of a restricted set of proteins. We chose to investigate the potential usefulness of 3 multitargeted indolinones in the inhibition of dysfunctional Kit signaling because these compounds have a number of such desirable features. We used 4 mast cell lines expressing either WT Kit or a variety of Kit mutants to investigate the ability of 3 indolinones (denoted SU11652, SU11654, and SU11655) to disrupt Kit function. These compounds were effective in blocking cell proliferation in all cell lines expressing mutant Kit (Figure 3). For lines expressing a JM Kit mutation, treatment with all 3 indolinones initially led to cell cycle arrest, followed by apoptosis after 24 to 72 hours of drug exposure (Figures 4, 5). The cell line with the catalytic domain mutant exhibited significant apoptosis after 24 hours of treatment, with no cells surviving at higher concentrations of each drug. The concentrations of SU11652, SU11654, and SU11655 necessary for inducing cell cycle arrest and apoptosis correlated directly with those required for the inhibition of Kit phosphorylation. Our results demonstrate that these indolinones are effective at inhibiting the function of WT Kit; the IC50 was 0.01 to 0.1 µM, translating to a target plasma concentration of 4 to 40 ng/mL (Figure 6). Both JM mutants were also inhibited by SU11652, SU11654, and SU11655 with a similar IC50 (Figure 6). Interestingly, the catalytic domain mutant required a higher concentration of drug for maximal inhibition, with an IC50 of 0.25 to 0.5 µM (Figure 6). This is in agreement with a prior study examining an earlier indolinone (SU6577), in which higher concentrations of drug were necessary to block the catalytic domain mutant when compared with a JM domain mutant.78 Differences in levels of protein are unlikely to be responsible for this discrepancy because cell surface expression of Kit is comparable among the various mutants (Figure 1). Given that activating mutations in the catalytic domain of Kit induce significant PI 3-kinase activity and are potent inducers of malignant transformation, it is possible that this form of mutation may result in greater constitutive activation of Kit when compared with the JM mutations. In conclusion, our data demonstrate that, in contrast to other tyrosine
kinase inhibitors, the multitargeted indolinones SU11652, SU11654, and
SU11655 are potent inhibitors of WT and mutant forms of Kit at
concentrations of drug that are readily achievable in vivo (Table
2).52,53,79,80 Although we
have focused our investigations on the application of these compounds
to malignant mast cell disease, aberrant Kit function (through
mutation, inappropriate expression, and generation of autocrine loops
of growth factor stimulation) has been documented in several other
neoplastic disorders including GISTs, leukemias, small cell lung
carcinoma, genitourinary tumors, and glioblastoma.81-85 It
is possible that these indolinones will have broad applicability in the
treatment of a wide variety of cancers.
We thank Dr Alison L. Hannah for helpful discussions and critical review of the manuscript and Dr Chris Liang for structural information relevant to the indolinones.
Submitted January 2, 2002; accepted March 4, 2002.
Prepublished online as Blood First Edition Paper, April 17, 2002; DOI 10.1182/blood-2001-12-0350.
Supported by grant 0038F from the Center for Companion Animal Health at the School of Veterinary Medicine, University of California at Davis.
Several of the authors (N.S., D.B.M., G.M., J.M.C.) are employed by a company (Sugen, Inc) whose potential product was studied in the present work.
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: Cheryl A. London, Department of Surgical and Radiological Sciences, School of Veterinary Medicine, 2112 Tupper Hall, One Shields Ave, University of California at Davis, Davis, CA 95616; e-mail: calondon{at}ucdavis.edu.
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  S. Letard, Y. Yang, K. Hanssens, F. Palmerini, P. S. Leventhal, S. Guery, A. Moussy, J.-P. Kinet, O. Hermine, and P. Dubreuil Gain-of-Function Mutations in the Extracellular Domain of KIT Are Common in Canine Mast Cell Tumors Mol. Cancer Res., July 1, 2008; 6(7): 1137 - 1145. [Abstract] [Full Text] [PDF]
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M. K. Callahan, M. Garg, and P. K. Srivastava Heat-shock protein 90 associates with N-terminal extended peptides and is required for direct and indirect antigen presentation PNAS, February 5, 2008; 105(5): 1662 - 1667. [Abstract] [Full Text] [PDF]
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N. P. Shah, F. Y. Lee, R. Luo, Y. Jiang, M. Donker, and C. Akin Dasatinib (BMS-354825) inhibits KITD816V, an imatinib-resistant activating mutation that triggers neoplastic growth in most patients with systemic mastocytosis Blood, July 1, 2006; 108(1): 286 - 291. [Abstract] [Full Text] [PDF]
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A. S. Corbin, S. Demehri, I. J. Griswold, Y. Wang, C. A. Metcalf III, R. Sundaramoorthi, W. C. Shakespeare, J. Snodgrass, S. Wardwell, D. Dalgarno, et al. In vitro and in vivo activity of ATP-based kinase inhibitors AP23464 and AP23848 against activation-loop mutants of Kit Blood, July 1, 2005; 106(1): 227 - 234. [Abstract] [Full Text] [PDF]
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A. S. Corbin, I. J. Griswold, P. La Rosee, K. W. H. Yee, M. C. Heinrich, C. L. Reimer, B. J. Druker, and M. W. N. Deininger Sensitivity of oncogenic KIT mutants to the kinase inhibitors MLN518 and PD180970 Blood, December 1, 2004; 104(12): 3754 - 3757. [Abstract] [Full Text] [PDF]
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R. Grundler, C. Thiede, C. Miething, C. Steudel, C. Peschel, and J. Duyster Sensitivity toward tyrosine kinase inhibitors varies between different activating mutations of the FLT3 receptor Blood, July 15, 2003; 102(2): 646 - 651. [Abstract] [Full Text] [PDF]
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C. A. London, A. L. Hannah, R. Zadovoskaya, M. B. Chien, C. Kollias-Baker, M. Rosenberg, S. Downing, G. Post, J. Boucher, N. Shenoy, et al. Phase I Dose-Escalating Study of SU11654, a Small Molecule Receptor Tyrosine Kinase Inhibitor, in Dogs with Spontaneous Malignancies Clin. Cancer Res., July 1, 2003; 9(7): 2755 - 2768. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
, although the effectiveness of this therapy has been
variable.
vascular endothelial growth factor
receptor-2 (VEGFR2), fibroblast growth factor-receptor (FGFR), and
platelet-derived growth factor-receptor (PDGFR), all thought to
materially contribute to the process of neovascularization
-mercaptoethanol for 1 minute at 50°C. Membranes were then
reprobed for Kit using the Ab-1 anti-Kit antibody.
