EXEL-0862, a novel tyrosine kinase inhibitor, induces apoptosis in

advertisement
NEOPLASIA
EXEL-0862, a novel tyrosine kinase inhibitor, induces apoptosis in vitro
and ex vivo in human mast cells expressing the KIT D816V mutation
Jingxuan Pan,1 Alfonso Quintás-Cardama,1 Hagop M. Kantarjian,1 Cem Akin,2 Taghi Manshouri,1
Peter Lamb,3 Jorge E. Cortes,1 Ayalew Tefferi,4 Francis J. Giles,1 and Srdan Verstovsek1
1The University of Texas M. D. Anderson Cancer Center, Houston, TX; 2University of Michigan, Ann Arbor, MI; 3Exelixis,
San Francisco, CA; 4Division of Hematology, Mayo Clinic College of Medicine, Rochester, MN.
Gain-of-function mutations of the receptor tyrosine kinase KIT play a key role in
the pathogenesis of systemic mastocytosis (SM), gastrointestinal stromal tumors
(GISTs), and some cases of acute myeloid
leukemia (AML). Whereas KIT juxtamembrane domain mutations seen in most
patients with GIST are highly sensitive to
imatinib, the kinase activation loop mutant D816V, frequently encountered in SM,
hampers the binding ability of imatinib.
We investigated the inhibitory activity of
the novel tyrosine kinase inhibitor EXEL0862 against 2 subclones of human mast
cell line-1 (HMC-1)—HMC-1.1, harboring
the juxtamembrane domain mutation
V560G, and HMC-1.2, carrying V560G and
the activation loop mutation D816V, found
in more than 80% of patients with SM.
EXEL-0862 inhibited the phosphorylation
of KIT in a dose-dependent manner and
decreased cell proliferation in both mast
cell lines with higher activity against HMC1.2 cells. The phosphorylation of KITdependent signal transducer and activator of transcription-3 (STAT3) and STAT5
was abrogated upon exposure to nanomolar concentrations of EXEL-0862. In addition, EXEL-0862 induced a time- and dosedependent proapoptotic effect in both
mast cell lines and caused a significant
reduction in mast-cell content in bone
marrow samples from patients with SM
harboring D816V and from those without
the D816V mutation. We conclude that
EXEL-0862 is active against KIT activation loop mutants and is a promising
candidate for the treatment of patients
with SM and other KIT-driven malignancies harboring active site mutations.
(Blood. 2007;109:315-322)
© 2007 by The American Society of Hematology
Introduction
Systemic mastocytosis (SM) is characterized by clonal proliferation of mast cells in the bone marrow, spleen, and other extracutaneous organs.1 Indolent and aggressive disease variants have been
described.2 Clinically, SM can manifest with mediator-related
symptoms or organomegaly. In patients with aggressive disease
variants, signs of organ dysfunction caused by mast-cell infiltration
are present. Patients with indolent SM (ISM) can be treated
successfully with antimediator drugs. By contrast, patients with
aggressive SM (ASM) or mast-cell leukemia (MCL) are candidates
for cytoreductive or targeted drugs. Current therapy for ASM and
MCL includes interferon-␣ and cladribine, but their efficacy is
limited and the prognosis for patients remains poor.3 Nearly all
patients with SM harbor the activating oncogenic mutation KIT
D816V, which involves the substitution of an aspartic residue at
codon 816 of the activation loop with a valine residue. This
mutation promotes receptor autophosphorylation without the requirement of stem-cell factor (SCF) stimulation.4-7
KIT is a 145-kDa transmembrane receptor tyrosine kinase of the
type III subgroup characterized by 5 extracellular immunoglobulinlike domains and a split tyrosine kinase domain.8 KIT-dependent
cell types include mast cells, hematopoietic stem cells, germ cells,
melanocytes, and interstitial cells of Cajal, among others.8-10 Upon
binding of SCF to the extracellular immunoglobulinlike domains,
KIT undergoes homodimerization and autophosphorylation at the
Y568 and Y570 tyrosine residues of the juxtamembrane domain.11
This leads to the phosphorylation and activation of multiple
signaling pathways such as Janus kinase/signal transducer and
activator of transcription (Jak-STAT), Src kinases, mitogenactivated protein (MAP) kinases, and phosphatidylinositol-3 (PI3)
kinase.11 Gain-of-function point mutations in the KIT kinase
domain result in ligand-independent constitutive activation of KIT
signaling, which leads to uncontrolled cell proliferation and
resistance to apoptosis.12 Activation of the KIT tyrosine kinase by
somatic mutation has been documented in a variety of human
malignancies, including SM, acute myeloid leukemia (AML), and
gastrointestinal stromal tumors (GISTs).9
Several small molecule tyrosine kinase inhibitors such as
imatinib and SU5614 have shown activity against the tyrosine
kinase activity of wild-type and some KIT mutants.13-15 However,
the inhibitory effect of these agents depends greatly upon the nature
of the KIT mutant isoform. For instance, imatinib has been
associated with sustained objective responses in more than 50% of
patients with metastatic GIST bearing the juxtamembrane KIT
mutation V560G.15,16 However, mutations mapping to the KIT
kinase domain17 render imatinib completely ineffective.18 The
latter are best exemplified by the D816V mutation, which involves
the substitution of aspartate to valine in codon 816 in the activation
loop17 lying at the entrance to the KIT enzymatic pocket, thus
interfering with imatinib binding.18 Neither imatinib18 nor the
chemically related tyrosine kinase inhibitor AMN10713 has significant in vitro activity against the D816V KIT mutants. Thus, drugs
Submitted April 3, 2006; accepted July 17, 2006. Prepublished online as Blood
First Edition Paper, August 15, 2006; DOI 10.1182/blood-2006-04-013805.
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The publication costs of this article were defrayed in part by page charge
© 2007 by The American Society of Hematology
BLOOD, 1 JANUARY 2007 䡠 VOLUME 109, NUMBER 1
315
316
BLOOD, 1 JANUARY 2007 䡠 VOLUME 109, NUMBER 1
PAN et al
specifically targeting the D816V KIT mutant hold great promise for
the treatment of neoplastic disorders harboring this mutation.
We report herein that the novel tyrosine kinase inhibitor
EXEL-0862 kills mast cell lines harboring the juxtamembrane
V560G KIT mutant and, significantly more, those bearing the
imatinib-resistant kinase domain D816V KIT mutation. It also
kills bone marrow mast cells in patients with SM. Furthermore,
EXEL-0862 exerts a strong inhibitory effect on the KITdependent downstream signaling pathways STAT3 and STAT5
and induces apoptosis in human mast cells harboring the D816V
KIT mutant isoform.
viability. The assay was performed according to the manufacturer’s
recommendations. Briefly, cells were seeded in triplicate in 96-well
microtiter plates (Falcon, Franklin Lakes, NJ), incubated in the presence of
different EXEL-0862 concentrations for 72 hours, and proliferation was
measured as a percentage of the proliferation of untreated cells. Four hours
before culture termination, 20 ␮L MTS solution was added to the culture.
During the incubation period, the MTS solution was reduced only by viable
cells into an insoluble colored formazan. Absorbance or optical density was
read on a 96-well plate reader at a single wavelength of 595 nm. Drug
concentrations resulting in 20%, 50%, and 80% inhibition of cell proliferation (IC) were determined.
Cell-cycle analysis by flow cytometry
Materials and methods
Reagents and antibodies
EXEL-0862 (WO2004050681 A2) was obtained from Exelixis (South San
Francisco, CA) and was stored as a 10-mM stock solution in dimethyl
sulfoxide (DMSO). Drug stock dilutions were stored at ⫺20°C. Working
dilutions were prepared in 10% media with freshly thawed aliquots for
immediate use in experiments. Recombinant human SCF was purchased
from Peprotech (Rocky Hill, NJ). Antibodies and their sources were as
follows: antibodies against Mcl-1 (S-19), antiphosphotyrosine (py99), and
protein-A/G agarose, were from Santa Cruz Biotechnology (Santa Cruz,
CA); antibodies against poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) and anti-CD117 conjugated with phycoerythrin were from
Becton-Dickson Biosciences PharMingen (San Diego, CA); rabbit polyclonal antibodies against caspase-3, caspase-9, XIAP, and Bax were from
Cell Signaling Technology (Beverly, MA); mouse monoclonal antibody
specific against phosphotyrosine 705 of Stat3 (clone 9E12), rabbit polyclonal anti-Stat3, mouse monoclonal anti–phospho-Stat5A/B (Y694/Y699;
clone 8-5-2), and rabbit polyclonal anti-Stat5A were from Upstate Technology (Lake Placid, NY); mouse anti–human-KIT (CD117) monoclonal
antibody was from R&D Systems (Minneapolis, MN); mouse monoclonal
antibody against actin was from Sigma (St Louis, MO); and anti–mouse
immunoglobulin G and anti–rabbit immunoglobulin G horseradish peroxidase–conjugated antibodies were from Amersham Biosciences (Arlington
Heights, IL).
After drug treatment, cells were collected, washed in Ca2⫹-free phosphatebuffered saline (PBS), and fixed overnight in 70% cold ethanol at ⫺20°C.
The cells were then washed twice in cold PBS and labeled with propidium
iodide (PI) for 1 hour in the dark. Cell-cycle distribution, including the
percentage of cells in sub-G1 phase, was determined using a FACSCalibur
(BD Biosciences, San Jose, CA) flow cytometer equipped with CellQuest
(BD Biosciences) software.
Measurement of mitochondrial transmembrane potential (⌬⌿m)
After treatment with EXEL-0862, changes in inner mitochondrial transmembrane potential in HMC-1.1 and HMC-1.2 cells were examined by flow
cytometry following incubation with submicromolar concentrations of
MitoTracker (Molecular Probes, Eugene, OR) probes. Briefly, cells were
stained with 2 probes: MitoTracker Red (chloromethyl-X-rosamine
[CMXRos]; Molecular Probes, Eugene, OR) and MitoTracker Green FM
(MTGreen; Molecular Probes). Cells were washed in Ca2⫹-free PBS,
stained with MitoTracker dyes, and incubated at 37°C for 1 hour in the dark.
MitoTracker probes passively diffuse across the plasma membrane and
accumulate in mitochondria. CMXRos is taken up by mitochondria as a
result of the ⌬⌿m and reacts with thiol residues to form covalent thiol ester
bonds. MTGreen FM preferentially accumulates in mitochondria regardless
of the mitochondrial membrane potential, which makes it a useful tool for
determining mitochondrial mass. MTGreen FM is a mitochondrionselective probe that contains a thiol-reactive chloromethyl moiety and
becomes fluorescent in the lipid environment of mitochondria. Samples
were analyzed in a flow cytometer and analyzed using CellQuest (BD
Biosciences) software.
Kinase IC50 determinations
Recombinant human kinase domains from KIT, VEGFR2, FGFR1, FLT3,
PDGFR␤, and IRK were obtained from ProQuinase (Freiberg, Germany).
Recombinant human KIT kinase domain containing the D816V mutation
was purified from baculovirus-infected Sf9 cells. Kinase activity was
monitored by measuring ATP consumption using luciferase as a readout
(VEGFR2, FLT3, IRK, PDGFR␤) or by detecting peptide phosphorylation
using Alphascreen (Perkin Elmer, Wellesley, MA) (KIT, KIT D816V,
FGFR1). All IC50 determinations were performed at an ATP concentration
equal to the Michaelis-Menten dissociation constant (Km) for each kinase.
Cell culture
Two subclones of the human mast cell line-1 (HMC-1) were used:
HMC-1.1, which harbors the mutation V560G in the juxtamembrane
domain of KIT, and HMC-1.2, which is a subclone of the HMC-1.1 cell line
with the SM-associated D816V kinase domain mutation on the same allele
as the original V560G mutation.19 Both HMC-1 subclones were kindly
provided by Dr Joseph Butterfield (Mayo Clinic, Rochester, MN) and were
maintained in Iscove modified Dulbecco medium (IMDM; Invitrogen,
Carlsbad, CA) supplemented with 10% fetal calf serum (FCS; Hyclone,
Logan, UT) and 1.2 mM ␣-thioglycerol (Sigma).
Cell proliferation inhibition assay
The MTS assay (CellTiter 96Aqueous One Solution reagent; Promega,
Madison, WI) was used to evaluate the effect of EXEL-0862 on mast-cell
Apoptosis assay
To determine the proportion of apoptotic HMC-1.1 and HMC-1.2 cells after
incubation in the presence of EXEL-0862, cells were pelleted, washed in
Ca2⫹-free PBS, and resuspended in 100 ␮L annexin V binding buffer
(10 mM 4-[2-hydroxyethyl]-1-piperazineethane-sulfonic acid, [pH 7.4];
0.15 M NaCl; 5 mM KC1; 1 mM MgCl2; 1.8 mM CaCl2) before the
fluorogenic substrate annexin V–fluorescein isothiocyanate (Sigma) was
added. Next, cells were incubated for 15 minutes at room temperature in the
dark. After incubation, cells were washed in 2 mL Ca2⫹-free PBS and
resuspended in 0.5 mL binding buffer. PI was added to permit identification
and exclusion of cells that had lost membrane integrity during analysis.
Binding of annexin V to apoptotic cells was determined with a flow
cytometer, and the resultant data were analyzed with CellQuest (BD
Biosciences) software.
Preparation of cell lysates, SDS-PAGE, and immunoblotting
Preparation of total cell lysates. Control cells and cells treated with
EXEL-0862 were rinsed with PBS and then lysed with RIPA buffer (1 ␣
PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented
with freshly added 10 mM ␤-glycerophosphate, 1 mM sodium orthovanadate, 10 mM NaF, 1 mM phenylmethylsulfonyl fluoride, and 1 ⫻ Roche
complete Mini protease inhibitor cocktail (Roche, Indianapolis, IN). The
DNA in the lysates was sheared by rapidly passing the lysate 10 times
through a 23-gauge needle or by sonication with eight 1-second bursts at
medium power.
BLOOD, 1 JANUARY 2007 䡠 VOLUME 109, NUMBER 1
Cytosolic fraction extraction. Control cells and cells treated with
EXEL-0862 were washed twice with ice-cold PBS. Cell pellets were mildly
resuspended with digitonin extraction buffer (10 mM PIPES [pH 6.8],
0.015% [wt/vol] digitonin, 300 mM sucrose, 100 mM NaCl, 3 mM MgCl2,
5 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride supplemented with
freshly added phosphatase inhibitors and protease inhibitors, as described in
the preceding paragraph. After incubation on ice for 10 minutes, samples
were centrifuged at 20 000g (14 000 rpm) for 10 minutes. Supernatants
containing cytosolic protein were transferred to a clean tube. Protein
concentration was determined in the final supernatant using the Bio-Rad
protein assay dye reagent, following the manufacturer’s instructions
(Bio-Rad, Hercules, CA). Samples were then stored in aliquots at ⫺80°C.
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDSPAGE) and immunoblotting were described previously.20
Immunoprecipitation
Immunoprecipitation was carried out according to the methods described previously.21 In brief, total cell lysates obtained from control or
treated cells were mixed with 3 ␮g mouse anti–human KIT (CD117)
monoclonal antibody overnight at 4°C, followed by the addition of
50 ␮L protein-A/G agarose slurry, and incubated for 3 hours. The
immunoprecipitated complex was washed 3 times and then subjected to
Western blotting.
Immunofluorescence staining
HMC-1.2 cells were first starved in serum-free medium for 24 hours and
then stimulated with SCF (100 ng/mL, final concentration) in 20% serum
for 1 hour. EXEL-0862 (0.1 or 1.0 ␮M) was added to the culture 20 minutes
before the addition of SCF. Next, cells were harvested, washed twice with
PBS, and cytospun onto glass slides. After fixation with 3% paraformaldehyde for 20 minutes, cells were permeabilized with 1% Triton X-100 and
0.5% NP-40 for 10 minutes. Immunofluorescence staining was performed
as described previously.20 Cells were mounted onto slides with Pro-Long
Gold Antifade reagent with DAPI (P36935; Molecular Probes).
Patient samples and evaluation of mast cells
Bone marrow cells were obtained by aspiration from 6 patients undergoing
evaluation for SM. Four patients (3 men, 1 woman; age range, 50-70 years)
were diagnosed with ISM according to the WHO classification, and 2 others
had no diagnostic evidence of SM. Three patients with SM carried the
D816V KIT mutation, and 1 carried wild-type KIT. No end-organ involvement was apparent in any of these patients. All had 10% or less bone
marrow cellularity involvement by neoplastic mast cells, and mast cells
coexpressed CD117, CD2, and CD25 in all. The research protocol was
approved by the M. D. Anderson Cancer Center institutional review board
(Houston, TX). Patients provided written informed consent in accordance
with the Declaration of Helsinki.
Bone marrow mononuclear cells were separated by Histopaque (density
1.077) gradient centrifugation. Contaminating red cells were lysed in 0.8%
ammonium chloride solution (StemCell Technologies, Vancouver, BC,
Canada) for 10 minutes. The presence of the D816V KIT point mutation in
bone marrow samples was detected by reverse transcriptase–polymerase
chain reaction and restriction fragment length polymorphism analysis, as
described previously,21,22 and was confirmed by direct sequencing in all
patients. Mononuclear cells were cultured in Stem-Pro serum-free medium
(Invitrogen) supplemented with recombinant human SCF (100 ng/mL) for
7 days in the presence or absence of EXEL-0862 and then stained with
anti–CD117-phycoerythrin (PE) for 30 minutes and washed. Detection of
mast cells was performed by detection of surface CD117 expression by flow
cytometry, as previously described.21
Statistical analysis
GraphPad Prism software (GraphPad Software, San Diego, CA) was used to
conduct the statistical analysis. P values of less than .05 were considered
statistically significant.
HMCs WITH D816V MUTATION SENSITIVE TO EXEL-0862
317
Results
EXEL-0862 inhibits cell proliferation and KIT phosphorylation
at nanomolar concentrations
Mutations within the activation loop of the KIT kinase lead to
ligand-independent activation and increased catalytic activity of
KIT.8-10 EXEL-0862 is a novel kinase inhibitor optimized for
activity against fibroblast growth factor receptors (FGFRs), vascular endothelial growth factor receptors (VEGFRs), platelet-derived
growth factor receptors (PDGFRs), and FLT3 (Table 1). EXEL0862 is also a potent inhibitor of wild-type KIT (IC50, 8.5 nM) and
retains significant activity against KIT bearing the D816V mutation
(IC50, 42 nM). We examined the effect of EXEL-0862 on KIT
phosphorylation in HMC-1.2 cells harboring the KIT loop activation mutant D816V, which confers resistance to imatinib. The
inability of imatinib to bind this mutant isoform was attributed to
allosteric conflict between the imatinib structure and the open
conformation of the KIT activation loop. HMC-1.1 cells harboring
the juxtamembrane KIT V560G mutant are sensitive to imatinib
and were used as a control.
We first investigated HMC-1.1 and HMC-1.2 cell viability on
exposure to EXEL-0862 by the MTS assay. Three independent
samples were measured for each data point. The viability of
HMC-1.1 and HMC-1.2 cells was markedly reduced after 72 hours
of exposure to increasing concentrations of EXEL-0862 up to
1 ␮M (Figure 1A), indicating that EXEL-0862 inhibited cell
viability at nanomolar concentrations. The IC50 for EXEL-0862
was significantly higher in HMC-1.1 cells (approximately 510 nM)
than in HMC-1.2 cells (approximately 350 nM) (F test; P ⬍ .001;
Table 2). Interestingly, the IC50 of imatinib for cellular proliferation
of HMC-1.2 cells (greater than 10 000 nM)18 was approximately
30-fold higher than that of EXEL-0862. Overall, our results
indicated that EXEL-0862 might be more active against D816V
HMC-1.2 cells than against HMC-1.1 cells.
After treatment of HMC-1.2 cells with escalating concentrations of EXEL-0862, cell lysates were immunoprecipitated with
mouse anti–human KIT monoclonal antibody, and KIT phosphorylation was detected with specific antibodies against phosphotyrosine. The level of phosphorylated KIT was significantly higher in
untreated HMC-1.2 cells than in untreated HMC-1.1 cells (Figure
1C), which is consistent with previous reports demonstrating that
the mutation D816V elicits KIT activation.12 Treatment with
EXEL-0862 decreased the phosphorylation of KIT in a concentration-dependent manner, indicating that EXEL-0862 substantially
inhibited the activation of KIT in D816V-expressing cells. When
the membrane was stripped and reprobed with anti-KIT antibody,
we observed that the amount of total KIT was unchanged,
suggesting that EXEL-0862 abolished KIT phosphorylation without altering KIT expression.
Table 1. Inhibitory concentrations of EXEL-0862 against several
protein tyrosine kinases
Kinase
KIT
KIT D816V
IC50, nM
8.5
42
PDGFR␤
1.1
VEGFR2
2.0
FGFR1
4.0
FLT3
InsulinR
1.5
1603
318
BLOOD, 1 JANUARY 2007 䡠 VOLUME 109, NUMBER 1
PAN et al
nucleus in control (starved) HMC-1.2 and HMC-1.1 cells, albeit
dominantly in the cytoplasm. After stimulation with SCF and
serum, STAT3 was localized mainly within the cell nuclei, as
shown by its colocalization with the nuclear DAPI (4⬘,6diamidino-2-phenylindole) stain (Figure 2D). In contrast, after
treatment with EXEL-0862, most STAT3 remained localized to
the cytoplasm, even after stimulation with SCF, suggesting that
EXEL-0862 abrogated STAT3 nuclear translocation. In aggregate, these data suggest that EXEL-0862 exerts a potent
inhibitory effect on STAT3 and STAT5 phosphorylation that
results in the inhibition of nuclear translocation.
EXEL-0862 induces apoptosis without significantly affecting
cell-cycle distribution
Figure 1. EXEL-0862 inhibits proliferation of human mast cells bearing D816V
mutation and KIT phosphorylation. (A) Dose-response curves of HMC-1.1 and
HMC-1.2 cells treated with EXEL-0862. Cells were exposed in vitro for 72 hours to
increasing concentrations of EXEL-0862, and the MTS assay was used to evaluate
growth inhibition. Graphs show data from a representative experiment performed in
triplicate; error bars represent standard deviation. (B) KIT phosphorylation was
inhibited by EXEL-0862 in a dose-dependent manner in HMC-1.1 and HMC-1.2 cells
when exposed at the indicated concentrations for 3 hours. EXEL-0862 ICs were
based on the MTS assay, as shown in panel A. Cell lysates were immunoprecipitated
with antibody against KIT. Western blot analysis was performed with antiphosphotyrosine antibody.
EXEL-0862 inhibits the activation of STAT3 and STAT5
Because EXEL-0862 inhibited KIT phosphorylation, we reasoned
that downstream signal transduction events critical in promoting
KIT-mediated cell survival, such as STAT3 and STAT5 activation,23,24 might also be inhibited by EXEL-0862. HMC-1.2 cells
were exposed to various concentrations of EXEL-0862 for 24 hours.
Phosphorylation of STAT3 and STAT5 was evaluated with their
respective phosphospecific antibodies by Western blot. Phosphorylated STAT3 and STAT5 were detectable in untreated HMC-1.1 and
HMC-1.2 cells (Figure 2A). With the addition of EXEL-0862 at
concentrations ranging between 0.35 ␮M and 1.0 ␮M, STAT3 and
STAT5 were completely dephosphorylated in HMC-1.2 cells. In
HMC-1.1 cells, treatment with EXEL-0862 at the same dose range
rendered complete inhibition of phosphorylation of STAT5. However, complete dephosphorylation of STAT3 was observed only on
treatment with EXEL-0862 at 1.0 ␮M (Figure 2A). Time-course
studies revealed that the inhibition of STAT5 phosphorylation
occurred rapidly and was detectable as early as 4 hours after
treatment with EXEL-0862 at 0.35 ␮M (Figure 2B).
Given that phosphorylation is critical for STAT3 and STAT5
dimerization, nuclear translocation, and DNA binding, we investigated whether EXEL-0862 affected the phosphorylation and nuclear
translocation of STAT3 and STAT5 on cytokine stimulation.
Western blot analysis (Figure 2C) revealed the presence of
phosphorylated STAT3 and STAT5 in starved HMC-1.2 and
HMC-1.1 cells, suggesting that the phosphorylation of STATs was
independent of exogenous cytokines and was likely mediated by
the gain-of-function D816V KIT and V560G mutants, resulting in
constitutively activated STAT3 and STAT5 in a ligand-independent
manner. Of note, STAT3 and STAT5 phosphorylation was further
enhanced on SCF stimulation (Figure 2C). The level of phosphorylated STAT3 and STAT5 in the presence of EXEL-0862 was lower
than at baseline (lanes 3-4 vs lane 1; Figure 2C). Indirect
immunofluorescence staining with anti–total STAT3 antibody
(Figure 2D) revealed that STAT3 was localized in cytoplasm and
We next analyzed the capacity of EXEL-0862 to induce apoptosis
in KIT mutant cell lines. When HMC-1.1 and HMC-1.2 cells were
exposed to escalating doses of EXEL-0862, a dose-dependent
specific cleavage of PARP, which is widely accepted as a specific
marker of apoptosis, was observed (Figure 3A). Further, a timedependent cleavage of PARP was demonstrated in both human
mast cell lines treated at a fixed EXEL-0862 concentration
(0.35 ␮M) and harvested at various time points (Figure 3B). Flow
cytometry analysis of cells subjected to annexin V/PI double
staining revealed that the percentage of annexin V–positive cells
increased after treatment with EXEL-0862, further supporting that
EXEL-0862 induced cell death by apoptosis (Figures 3C-D).
Exposure of HMC-1.1 and HMC-1.2 cells to increasing concentrations of EXEL-0862 for 24 hours did not result in significant
cell-cycle disturbance, as evidenced by flow cytometry analysis
(Figure 4). More important, increasing doses of EXEL-0862
enhanced the accumulation of HMC-1.1 and HMC-1.2 cells in the
sub-G1 cell-cycle phase, indicating apoptotic cells.
EXEL-0862 induces mitochondrial damage, cytochrome c
release, and activation of caspase-3 and caspase-9
Modifications in mitochondrial transmembrane potential (⌬⌿m)
and cytochrome c translocation from mitochondria to the cytoplasm have been correlated with the induction of cell death by
apoptosis. Thus, to gain further insight into the mechanism of
apoptosis induced by EXEL-0862, we first analyzed whether
EXEL-0862 induces mitochondrial changes in HMC-1.1 and
HMC-1.2 cells. We measured by flow cytometry the mitochondrial
uptake of CMXRos and MTGreen double staining. Figure 5A
illustrates the graphs in which fluorescence of CMXRos and
MTGreen is plotted against the percentage of positive cells over
time. Treatment of HMC-1.1 and HMC-1.2 with EXEL-0862 at
IC80 concentrations produced a time-dependent increase in the
proportion of cells with altered ⌬⌿m in HMC-1.1 and HMC-1.2
cells. Paradoxically, when IC50 concentrations were used, a marked
alteration in ⌬⌿m over time was seen in HMC-1.2, whereas the
opposite effect occurred in HMC-1.1 cells. Next, we compared the
Table 2. Inhibitory concentration (IC) values for EXEL-0862
Cells
HMC-1.1
HMC-1.2
IC20, nM
309
142
IC50, nM
514
353
IC80, nM
717
564
Shown are the mean values of 3 independent experiments, each performed in
triplicate.
BLOOD, 1 JANUARY 2007 䡠 VOLUME 109, NUMBER 1
HMCs WITH D816V MUTATION SENSITIVE TO EXEL-0862
319
Figure 2. Activation of STAT3 and STAT5 is blocked by
EXEL-0862. (A) HMC-1.1 and HMC-1.2 cells were treated with
EXEL-0862 at concentrations ranging from 0.35 ␮M to 1.0 ␮M for
24 hours. Cell lysates were then analyzed by Western blot with
phosphospecific antibodies, as indicated. (B) Time-course study
of STAT3 and STAT5 phosphorylation inhibition. Cells were
exposed to EXEL-0862 at 0.35 ␮M for 24 hours. Phosphorylation
of STAT3 and STAT5 was analyzed by Western blot. (C-D) EXEL
inhibited the phosphorylation of STAT3 and STAT5 and their
nuclear translocation on SCF stimulation. HMC-1.2 and HMC-1.1
cells were starved in serum-free medium for 24 hours. SCF (100
ng/mL, final concentration) in 20% serum was added for 1 hour.
Cells were harvested to prepare cell lysates for Western blot (C)
or to be fixed in 3% paraformaldehyde for immunofluorescence
staining with FITC and/or DAPI (D; arrows indicate cytoplasmic
[CONTL and EXEL⫹SCF] or nuclear [SCF] STAT3 localization).
EXEL-0862 (1.0 ␮M) was added to the culture 20 minutes before
the addition of SCF. Cells were visualized with a 40 ⫻/0.9
objective lens mounted on an Olympus BX60 epifluorescence
microscope (Olympus, Melville, NY), and the images were recorded with an Optronics CCD camera and Fluoview software
version 4.3 (Olympus, Center Valley, PA).
release of cytochrome c in untreated cells and cells treated with
EXEL-0862 at 0.35 ␮M for 24 hours. To this end, cells were
harvested, washed with PBS, and resuspended with digitonin
extraction buffer to obtain the cytosolic fraction. Cytochrome c
could not be detected in untreated cells, as measured by immunoblotting with anti–cytochrome c antibody (Figure 5B). Treatment
with EXEL-0862 significantly enhanced cytochrome c release. Of
note, this effect was markedly higher in HMC-1.2 cells than in
HMC-1.1 cells.
Western blot analysis was performed on HMC-1.1 and HMC1.2 cell lysates obtained after treatment with EXEL-0862 at 0.35,
0.7, and 1.0 ␮M. Decreasing levels of caspase-3 and caspase-9
were observed with higher concentrations of EXEL-0862, reflecting increased cleavage of caspase-3 and caspase-9 (Figure 5C),
which occurred in parallel with increased cytochrome c release.
Additionally, we explored the effect of EXEL-0862 at 0.35 ␮M for
4 hours, 24 hours, and 48 hours on the expression of other
apoptosis-related proteins. Cell lysates were subjected to SDS-
Figure 3. EXEL-0862 induces apoptosis in human mast cells expressing D816V
KIT. (A) EXEL-0862 induced PARP cleavage in a dose-dependent manner. HMC-1.1
and HMC-1.2 cells were exposed to EXEL-0862 at the indicated concentrations for
24 hours. Cell lysates were analyzed by Western blot with antibody against PARP. (B)
EXEL-0862 induced PARP cleavage in a time-dependent manner. Cells were treated
with 0.35 ␮M for 4, 24, and 48 hours, respectively. PARP was analyzed by Western
blot. (C-D) Apoptosis analysis by flow cytometry of HMC-1.1 and HMC-1.2 cells
exposed to escalating concentrations of EXEL-0862 for 24 hours and subjected to
annexin V-PI double staining. Cells stained with annexin V were defined as apoptotic.
Ratios between apoptotic cells and total cells are plotted and represent the
mean ⫾ SEM of experiments performed in duplicate.
PAGE, and immunoblots revealed no significant changes in the
expression of Bcl-2, Bax, Mcl-1, and X-linked inhibitor of
apoptosis protein (XIAP) (Figure 5D). Overall, these data suggest
that EXEL-0862 may trigger mast-cell apoptosis through direct
mitochondrial damage with the release of cytochrome c and
caspase activation.
Ex vivo effect of EXEL-0862 on primary mast cells from patients
with SM
Our in vitro results prompted us to assess the efficacy of EXEL0862 on neoplastic mast cells of patients with SM. We evaluated
the ex vivo antineoplastic effect of EXEL-0862 in bone marrow
mast cells obtained from patients with SM bearing the D816V KIT
mutant in short-term cultures. Human mast cells express a high
level of surface CD117 (KIT) and exhibit marked granularity,25
which make this cell population readily identifiable by flow
cytometry. Treatment with EXEL-0862 at doses ranging from 0.1
to 1.0 ␮M for 7 days in the presence of SCF resulted in a significant
reduction in the mast-cell percentage in bone marrow from patients
with SM. Figure 6A illustrates the percentage of mast-cell viability
Figure 4. Cell-cycle distribution in human mast cells after treatment with
EXEL-0862. HMC-1.1 and HMC-1.2 cells were exposed to increasing concentrations
of EXEL-0862 for 24 hours. Then cells were fixed in ethanol and analyzed by flow
cytometry. Accumulation of HMC-1.1 and HMC-1.2 cells in sub-G1 phase was
observed with increasing doses of EXEL-0862.
320
PAN et al
Figure 5. EXEL-0862 leads to mitochondrial damage, cytochrome c release,
and activation of caspase-3 and -9. (A) Mitochondrial potential damage was elicited
by EXEL-0862. Cells were exposed to EXEL-0862, stained with CMXRos and
MTGreen, and immediately analyzed by flow cytometry. Histograms represent the
population of cells with damaged mitochondrial potential. Values represent the
mean ⫾ SEM values from duplicate experiments. (B) EXEL-0862 treatment induced
cytochrome c release into the cytosol. Cells were treated with 0.35 ␮M EXEL for
24 hours, and the cytosolic fraction was extracted with digitonin buffer. Cytochrome c
was detected with immunoblots. (C) EXEL-0862 activated caspase-3 and -9. Cells
were exposed to EXEL-0862 at the indicated concentrations for 24 hours. Cell lysates
were analyzed by Western blot with antibodies against caspase-9 and caspase-3. (D)
Cells were treated with 0.35 ␮M EXEL-0862 for 24 hours. Expression of apoptosisrelated proteins was analyzed by Western blot.
normalized to the results obtained in cells incubated in the absence
of EXEL-0862. Treatment with EXEL-0862 at 1.0 ␮M for 7 days
led to almost 90% reduction in bone marrow mast cells compared
with bone marrow incubated without EXEL-0862 (P ⬍ .005). As a
control and comparison for the ex vivo studies, we tested the effect
of the drug on CD34⫹-derived mast cells without D816V KIT
mutation in samples from 3 donors (2 healthy adults and 1 patient
with ISM who carried wild-type KIT). EXEL-0862 significantly
reduced the viability of mast cells at 1 ␮M (P ⬍ .05), as evidenced
by annexin V binding (Figure 6B), suggesting that EXEL-0862 also
kills mast cells that depend on wild-type KIT for survival. This
result is consistent with the in vitro inhibitory effect of EXEL-0862
on wild-type KIT (Table 1).
BLOOD, 1 JANUARY 2007 䡠 VOLUME 109, NUMBER 1
of HMC-1.1 and HMC-1.2 with IC50 values of 514 and 353 nM,
respectively. In addition, EXEL-0862 inhibited phosphorylation of
the KIT-dependent downstream signaling molecules STAT3 and
STAT5, and, consistent with findings on proliferation assays,
EXEL-0862 promoted apoptosis in vitro and ex vivo in cells
carrying the D816V KIT mutant isoform.
Imatinib, which is approved for the treatment of patients with
chronic myelogenous leukemia (CML) and metastatic GIST,
exhibits a significant inhibitory effect on HMC-1.1 cells harboring
KIT V560G but fails to inhibit the growth of HMC-1.2 cells
carrying KIT D816V, translating into a lack of significant clinical
response in patients with SM harboring KIT loop activation
mutations.1,4,13,14 Like imatinib, AMN107 exhibited a weak inhibitory effect on HMC-1.2 cell growth and phosphorylation (IC50,
approximately 1-5 ␮M) after exposure to this agent for 48 hours in
the MTS assay. To overcome this problem, several small molecule
tyrosine kinase inhibitors have been tested against D816V KIT,
including MLN518,26 PD-180970,26 PKC412,27 BMS-354825,28
AP-23464,29 and AP-23848.29 MLN518 and PD180970 demonstrated activity against cell lines expressing juxtamembrane mutant
KIT, but only MLN518, at nanomolar concentrations, targeted
active-site mutant cell lines. PKC412, AP-23464, and AP-23848
have in vitro inhibition effects on activation-loop mutations of KIT
in nanomolar ranges. Recently, it was reported that the dual Abl/Src
kinase inhibitor dasatinib (formerly BMS-354825) induces apoptosis in mast cells expressing KIT activation loop mutations,
including D816V. PKC412 has produced a transient clinical
response in a patient with MCL harboring this mutation. The
potential clinical benefit and the toxicity profile of these compounds remain to be determined in clinical trials.
In the study reported here, exposure of HMC-1.2 cells harboring KIT D816V to nanomolar concentrations of EXEL-0862
caused a significant inhibition of KIT phosphorylation. In fact, the
IC50 values in a 72-hour MTS assay were significantly lower in
HMC-1.2 (353 nM) than in HMC-1.1 (514 nM) cells, indicating
that EXEL-0862 might be more potent against imatinib-resistant
cells bearing KIT activation loop mutations involving codon
D816.18 Furthermore, ex vivo exposure of bone marrow mast cells
from patients with SM bearing KIT D816V to EXEL-0862 resulted
in a dramatic reduction of bone marrow mast-cell content, suggesting that therapeutic inhibition of KIT kinase by compounds such as
EXEL-0862 could be effective for patients with SM associated
with the gain-of-function KIT D816V mutation.
Discussion
KIT mutations at codon 816 have been reported in a variety of
malignancies, including SM. More than 80% of patients with SM
harbor the KIT D816V mutant isoform.4,5,18 This mutation constitutively activates KIT tyrosine kinase and stabilizes the activation
loop in the active conformation, thus precluding the binding of
kinase inhibitors such as imatinib and AMN107, with selective
affinity for the open configuration of the kinase domain. Hence,
KIT D816V has been proposed as a key therapeutic target for the
treatment of SM. We investigated the activity of the novel tyrosine
kinase inhibitor EXEL-0862 against the mast cell lines HMC-1.1
and HMC-1.219 and against bone marrow cells from patients with
SM harboring KIT D816V to predict whether this compound might
have clinical activity in patients with SM. That HMC-1.1 and
HMC-1.2 differ only by the presence of the D816V mutation makes
them ideal models for the study of the activity of novel agents
against this kinase domain mutation. We documented that EXEL0862 potently inhibited cell proliferation and KIT phosphorylation
Figure 6. Ex vivo EXEL-0862 treatment inhibits the viability of primary bone
marrow mast cells. (A) Bone marrow mononuclear cells from 3 patients with ISM
who carried D816V KIT were incubated for 7 days with EXEL-0862 at 0.1 ␮M and
1 ␮M. Then, the mast-cell content in cultures was determined by flow cytometry. (B)
Mast cells without D816V KIT mutation in samples from 3 donors were derived from
bone marrow CD34⫹ cells by culture in Stem-Pro and SCF (100 ng/mL) and IL-3
(30 ng/mL, only for the first week) and weekly hemirepletion of the culture medium for
approximately 9 to 14 weeks. Mast cells were then incubated with or without
EXEL-0862 for 48 hours at the indicated concentrations. Viable cells, defined as
Annexin-V-FITC–negative cells, were measured by flow cytometry. Data are expressed as percentage of mast-cell survival normalized to results obtained in cells
incubated in the absence of drug (control). Each point represents the mean ⫾ SEM
values from 3 patients with ISM. *P ⬍ .05; **P ⬍ .005; Student t test.
BLOOD, 1 JANUARY 2007 䡠 VOLUME 109, NUMBER 1
HMCs WITH D816V MUTATION SENSITIVE TO EXEL-0862
Both the juxtamembrane V560G and the kinase domain D816V
mutations give rise to constitutively activated KIT isoforms that
preferentially phosphorylate STAT3 and STAT5, thus leading to
cell proliferation. STAT proteins act as transcription factors capable
of delivering a rapid and direct signal from extracellular stimuli to
the nucleus in normal and malignant cells.30-32 STAT5 knockout
mice exhibit mast-cell deficiency,33 indicating that STAT5 might be
a key regulator of mast-cell development, proliferation, and
survival. Activation of STAT3/STAT5 is a key event in KITmediated cell proliferation and inhibition of apoptosis. Our data
demonstrate the presence of constitutive STAT3 and STAT5
phosphorylation in untreated starved HMC-1.2 and HMC-1.1 mast
cells (Figure 2A), corroborating previous reports suggesting that
STAT3 and STAT5 are aberrantly phosphorylated by KIT D816V,
V560G, and other KIT-activating mutations.34-37 This has been further
supported by the observation that 10 of 14 KIT mutations (including
D816V and V560G) conferred interleukin 3–independent growth and
were associated with constitutive phosphorylation of tyrosine residues in
STAT3 and STAT5.36 This might explain our observation regarding the
phosphorylation of STAT3 and STAT5 (Figure 2C), which is consistent
with the existence of a limited amount of STAT3 in the nucleus of
starved HMC-1.1 and HMC-1.2 cells (Figure 2D). The cytoplasmic
STAT3 in starved cells might reflect incomplete phosphorylation in the
absence of growth factor (SCF), even though there is constitutive
activation by the mutant KIT. Upon SCF stimulation, STAT3 is fully
phosphorylated, and more STAT3 molecules are translocated into the
nucleus, a process blocked by EXEL-0862. Because EXEL-0862
inhibits STAT3/STAT5 phosphorylation before the occurrence of apoptosis, the former process may contribute to the proapoptotic effect of
EXEL-0862 on HMC-1.2 and HMC-1.1 cells.
EXEL-0862–induced apoptosis was more pronounced in HMC1.2 D816V-bearing cells than in HMC-1.1 cells expressing wildtype KIT and appeared to be mediated by activation of downstream
321
elements of the intrinsic apoptotic pathway (eg, cytochrome
c/caspase-9/caspase-3). In addition, the alteration in ⌬⌿m and the
release of cytochrome c into the cytoplasm appear to indicate that
EXEL-0862 may promote mitochondrial damage through an as yet
unknown mechanism. Notably, EXEL-0862–induced apoptosis
was not associated with a change in the expression of other
apoptosis-related proteins that have been associated with resistance
to chemotherapy, such as Bcl-2, Mcl-1, XIAP, and Bax.
In summary, our in vitro and ex vivo data demonstrate that
EXEL-0862 can effectively target juxtamembrane and, more
remarkably, activation loop mutants of KIT, including the imatinibresistant KIT D816V mutation. This suggests that EXEL-0862 may
have clinical efficacy against human neoplasms driven by gain-offunction KIT mutations.
Acknowledgments
This work was supported in part by the Joe W. and Dorothy Dorsett
Brown Foundation (Metairie, LA). The flow cytometry core
facility at M. D. Anderson Cancer Center is supported by National
Cancer Institute Cancer Center Core grant CA16672.
Authorship
Conflict-of-interest disclosure: P.L. is an employee of Exelixis,
which is the owner of EXEL-0862, the compound studied and
reported in this work.
Correspondence: Srdan Verstovsek, Department of Leukemia, University of Texas M. D. Anderson Cancer Center, Unit
428, 1515 Holcombe Blvd, Houston, TX 77030; e-mail:
sverstov@mdanderson.org.
References
1. Valent P, Akin C, Sperr WR, et al. Mastocytosis:
pathology, genetics, and current options for
therapy. Leuk Lymphoma. 2005;46:35-48.
2. Valent P, Horny HP, Li CY, et al. Mastocytosis
(mast cell disease). In: Jaffe ES, et al, eds. World
Health Organization (WHO) Classification of Tumors: Pathology and Genetics. Tumors of Hematopoietic and Lymphoid Tissues. Lyon, France:
IARC Press; 2001:291-302.
3. Hennessy B, Giles F, Cortes J, et al. Management of patients with systemic mastocytosis: review of M. D. Anderson Cancer Center experience. Am J Hematol. 2004;77:209-214.
4. Pardanani A. Systemic mastocytosis: bone marrow pathology, classification, and current therapies. Acta Haematol. 2005;114:41-51.
5. Akin C. Clonality and molecular pathogenesis of
mastocytosis. Acta Haematol. 2005;114:61-69.
6. Pan J, Xu G, Yeung SC. Cytochrome c release is
upstream to activation of caspase-9, caspase-8,
and caspase-3 in the enhanced apoptosis of anaplastic thyroid cancer cells induced by manumycin and paclitaxel. J Clin Endocrinol Metab. 2001;
86:4731-4740.
7. Hirota S, Isozaki K, Moriyama Y, et al. Gain-offunction mutations of c-kit in human gastrointestinal stromal tumors. Science. 1998;279:577-580.
8. Miettinen M, Lasota J. KIT (CD117): a review on
expression in normal and neoplastic tissues, and
mutations and their clinicopathologic correlation.
Appl Immunohistochem Mol Morphol. 2005;13:
205-220.
9. Heinrich MC, Blanke CD, Druker BJ, Corless CL.
Inhibition of KIT tyrosine kinase activity: a novel
molecular approach to the treatment of KITpositive malignancies. J Clin Oncol. 2002;20:
1692-1703.
10. Lennartsson J, Jelacic T, Linnekin D, Shivakrupa
R. Normal and oncogenic forms of the receptor
tyrosine kinase kit. Stem Cells. 2005;23:16-43.
11. Mol CD, Lim KB, Sridhar V, et al. Structure of a
c-kit product complex reveals the basis for kinase
transactivation. J Biol Chem. 2003;278:3146131464.
12. Furitsu T, Tsujimura T, Tono T, et al. Identification
of mutations in the coding sequence of the protooncogene c-kit in a human mast cell leukemia cell
line causing ligand-independent activation of c-kit
product. J Clin Invest. 1993;92:1736-1744.
13. Gleixner KV, Mayerhofer M, Aichberger KJ, et al.
PKC412 inhibits in vitro growth of neoplastic human mast cells expressing the D816V-mutated
variant of KIT: comparison with AMN107, imatinib,
and cladribine (2CdA), and evaluation of cooperative drug effects. Blood. 2006;107:752-759.
14. Valent P, Ghannadan M, Akin C, et al. On the way
to targeted therapy of mast cell neoplasms: identification of molecular targets in neoplastic mast
cells and evaluation of arising treatment concepts. Eur J Clin Invest. 2004;34(suppl 2):41-52.
15. Savage DG, Antman KH. Imatinib mesylate—a
new oral targeted therapy. N Engl J Med. 2002;
346:683-693.
16. Frost MJ, Ferrao PT, Hughes TP, et al. Juxtamembrane mutant V560GKit is more sensitive
to imatinib (STI571) compared with wild-type c-kit
whereas the kinase domain mutant D816VKit is
resistant. Mol Cancer Ther. 2002;1:1115-1124.
17. Longley BJ Jr, Metcalfe DD, Tharp M, et al. Acti-
vating and dominant inactivating c-KIT catalytic
domain mutations in distinct clinical forms of human mastocytosis. Proc Natl Acad Sci U S A.
1999;96:1609-1614.
18. Ma Y, Zeng S, Metcalfe DD, et al. The c-KIT mutation causing human mastocytosis is resistant to
STI571 and other KIT kinase inhibitors; kinases
with enzymatic site mutations show different inhibitor sensitivity profiles than wild-type kinases
and those with regulatory-type mutations. Blood.
2002;99:1741-1744.
19. Butterfield JH, Weiler D, Dewald G, Gleich GJ.
Establishment of an immature mast cell line from
a patient with mast cell leukemia. Leuk Res.
1988;12:345-355.
20. Pan J, She M, Xu ZX, Sun L, Yeung SC. Farnesyltransferase inhibitors induce DNA damage via
reactive oxygen species in human cancer cells.
Cancer Res. 2005;65:3671-3681.
21. Akin C, Brockow K, D’Ambrosio C, et al. Effects
of tyrosine kinase inhibitor STI571 on human
mast cells bearing wild-type or mutated c-kit. Exp
Hematol. 2003;31:686-692.
22. Akin C, Fumo G, Yavuz AS, et al. A novel form of
mastocytosis associated with a transmembrane
c-Kit mutation and response to imatinib. Blood.
2004;103:3222-3225.
23. Bromberg JF, Wrzeszczynska MH, Devgan G, et
al. Stat3 as an oncogene. Cell. 1999;98:295-303.
24. Debierre-Grockiego F. Anti-apoptotic role of
STAT5 in haematopoietic cells and in the pathogenesis of malignancies. Apoptosis. 2004;9:717728.
25. Fumo G, Akin C, Metcalfe DD, Neckers L. 17allylamino-17-demethoxygeldanamycin (17-AAG)
322
BLOOD, 1 JANUARY 2007 䡠 VOLUME 109, NUMBER 1
PAN et al
is effective in down-regulating mutated, constitutively activated KIT protein in human mast cells.
Blood. 2004;103:1078-1084.
and in vivo activity of ATP-based kinase inhibitors
AP23464 and AP23848 against activation-loop
mutants of Kit. Blood. 2005;106:227-234.
nal transduction by several KIT juxtamembrane
domain mutations. Oncogene. 2003;22:47104722.
26. Corbin A, Griswold IJ, La Rosee P, et al. Sensitivity of oncogenic KIT mutants to the kinase inhibitors MLN518 and PD180970. Blood. 2004;104:
3754-3757.
30. Inghirami G, Chiarle R, Simmons WJ, Piva R,
Schlessinger K, Levy DE. New and old functions
of STAT3: a pivotal target for individualized treatment of cancer. Cell Cycle. 2005;4:1131-1133.
27. Gotlib J, Berube, C, Growney JD, et al. Activity of
the tyrosine kinase inhibitor PKC412 in a patient
with mast cell leukemia with the D816V KIT mutation. Blood. 2005;106:2865-2870.
31. Battle TE, Frank DA. The role of STATs in apoptosis. Curr Mol Med. 2002;2:381-392.
35. Ning ZQ, Li J, Arceci RJ. Signal transducer and
activator of transcription 3 activation is required
for Asp(816) mutant c-Kit-mediated cytokineindependent survival and proliferation in human
leukemia cells. Blood. 2001;97:3559-3567.
28. Shah N, Lee FY, Luo R, et al. Dasatinib (BMS354825) inhibits KITD816V, an imatinib-resistant
activating mutation that triggers neoplastic growth
in the majority of patients with systemic mastocytosis. Blood. 2006;108:286-291.
29. Corbin A, Demehri S, Griswold IJ, et al. In vitro
32. Moriggl R, Sexl V, Kenner L, et al. Stat5 tetramer
formation is associated with leukemogenesis.
Cancer Cell. 2005;7:87-99.
33. Ikeda K, Nakajima H, Suzuki K, Watanabe N,
Kagami S, Iwamoto I. Stat5a is essential for the
proliferation and survival of murine mast cells. Int
Arch Allergy Immunol. 2005;137(suppl 1):45-50.
34. Casteran N, De Sepulveda P, Beslu N, et al. Sig-
36. Growney JD, Clark JJ, Adelsperger J, et al. Activation mutations of human c-KIT resistant to imatinib mesylate are sensitive to the tyrosine kinase
inhibitor PKC412. Blood. 2005;106:721-724.
37. Cammenga J, Horn S, Bergholz U, et al. Extracellular KIT receptor mutants, commonly found in
core binding factor AML, are constitutively active
and respond to imatinib mesylate. Blood. 2005;
106:3958-3961.
Download