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PLENARY PAPER
From Johns Hopkins University School of Medicine,
Departments of Oncology and Pediatrics, Baltimore, MD; and Cephalon,
West Chester, PA.
Constitutively activating internal tandem duplication (ITD) and
point mutations of the receptor tyrosine kinase FLT3 are present in up
to 41% of patients with acute myeloid leukemia (AML). These FLT3/ITD
mutations are likely to be important because their presence is
associated with a poor prognosis. Both types of mutations appear to
activate the tyrosine kinase activity of FLT3. We describe here the
identification and characterization of the indolocarbazole derivative
CEP-701 as a FLT3 inhibitor. This drug potently and selectively
inhibits autophosphorylation of wild-type and constitutively activated
mutant FLT3 in vitro in FLT3/ITD-transfected cells and in human
FLT3-expressing myeloid leukemia-derived cell lines. We demonstrate
that CEP-701 induces a cytotoxic effect on cells in a dose-responsive
fashion that parallels the inhibition of FLT3. STAT5 and ERK1/2,
downstream targets of FLT3 in the signaling pathway, are inhibited in
response to FLT3 inhibition. In primary leukemia blasts from AML
patients harboring FLT3/ITD mutations, FLT3 is also inhibited, with an
associated cytotoxic response. Finally, using a mouse model of FLT3/ITD
leukemia, we demonstrate that the drug inhibits FLT3 phosphorylation in
vivo and prolongs survival. These findings form the basis for a planned
clinical trial of CEP-701 in patients with AML harboring FLT3-
activating mutations.
(Blood. 2002;99:3885-3891) The receptor tyrosine kinase FLT3 (FMS-like
tyrosine kinase 3) appears to play an important role in the development
of hematopoietic stem cells, dendritic cells, B progenitor cells, and
natural killer (NK) cells, as demonstrated by targeted disruption
studies.1,2 In hematopoietic cells, FLT3 expression is
normally restricted to the CD34+ fraction of human bone
marrow and a smaller fraction of CD34 FLT3 is aberrantly expressed on the malignant cells in the majority of
patients with of acute myeloid leukemia (AML).12,13 Two
distinct types of FLT3 mutations have now been identified in the subset
of AML cases that expresses FLT3: internal tandem duplication (ITD)
mutations within the juxtamembrane region and point mutations at
aspartate 835 (Asp835) within the kinase
domain.14,15 Both types of mutations constitutively
activate the tyrosine kinase activity of FLT3 in experimental
systems.15,16 The incidence of FLT3-activating mutations
in AML appears to be up to 41% (17%-34% FLT3/ITD mutations, 7%
Asp835 mutations).17 Current therapy using cytotoxic
agents such as cytosine arabinoside (Ara C) and daunorubicin cures
approximately 30% of patients with AML, but most published studies
demonstrate that the FLT3-activating mutations are associated with a
distinctly worse prognosis.15,16,18,19 In one study, the
cure rate with a FLT3 mutation was reported to be as low as 7%
compared to a cure rate of 44% without a mutation.20 New
therapies are clearly needed for the estimated 200 000 cases of AML
that occur each year worldwide.21 Recent clinical
successes with the BCR-ABL inhibitor STI-571 (imatinib mesylate
[Gleevec]; Novartis, Basel, Switzerland) in chronic
myelogenous leukemia (CML) suggest that therapies targeted to specific
activated kinases may be an effective approach in certain
malignancies.22 FLT3 represents a rational potential
target in AML.
Reagents
Cell culture
Patient samples Bone marrow samples were collected with informed consent from patients with de novo AML as part of a Johns Hopkins institutional review board-approved protocol. Mononuclear cells were separated and stored frozen as described previously.23Mice Ten-week-old male Balb/c mice (Harlan, Indianapolis, IN), were maintained 5 per cage under temperature- and humidity-controlled conditions with food and water ad libitum. Animals were quarantined for 1 week prior to experimental manipulation. For harvesting splenic leukocytes, mice were killed by cervical dislocation. Immediately after death, spleens were removed, transferred to ice-cold phosphate-buffered saline (PBS), and forced through a 40-µM nylon cell strainer (Becton Dickinson, Franklin Lakes, NJ). Erythrocytes were lysed in erythrocyte lysis buffer (0.155 M NH4Cl, 0.01 M KHCO3, 0.1 M EDTA), then the remaining leukocytes were washed in PBS and lysed in detergent buffer as previously described.23Reverse transcription-polymerase chain reaction Total RNA was isolated using RNeasy columns (Qiagen, Valencia, CA) and reverse transcribed. The resulting complimentary DNA (cDNA) (or control lacking reverse transcriptase) was used as a template for polymerase chain reaction (PCR) with primers for the FLT3/ITD assay or Asp835 assay.14,15 PCRs for the Asp835 mutation were digested with EcoRV. Products were resolved on 2.5% agarose gels. FLT3/ITD mutations were identified as bands migrating above the expected 365-bp size of the wild-type FLT3 fragment, whereas Asp835 mutations were identified as PCR products lacking digestion at the expected EcoRV site.15BaF3 cell constructs Total RNA was extracted from AML patient samples and reverse transcription-PCR (RT-PCR) was performed with the one-step RT-PCR kit (Invitrogen) using the forward primer 5'-CAATTG-(MunI)-TGTCGAGCAGTACTCTAAACA-3' and the reverse primer 5'-GCATGC-(SphI)-ATCCTAGTACCTTCCCAAACTC-3' at 94°C, 55°C, and 72°C (1 minute each) for 40 cycles with a final extension of 10 minutes at 72°C.3,14 The PCR products were resolved on 2.5% agarose gels with ethidium bromide staining and the FLT3/ITD DNA fragments were isolated and sequenced by the dideoxy method. FLT3/ITD fragments were digested with MunI and SphI and ligated to the MunI- and SphI-digested full-length FLT3 cDNA to replace the same region in the wild-type FLT3 cDNA. These cDNAs were subcloned into the pCIneo expression vector (Promega, Madison, WI) and used for transfection. BaF3 cells were transfected by electroporation at 300 mV/960 µF (Bio-Rad, Richmond, CA). Transfected cells were cultured in IL-3-containing medium for 48 hours and then selected in 1 mg/mL Gly418 (Invitrogen) for 2 weeks. Transfected cell lines were then subcloned by limiting dilution. CMV.fms, containing the cDNA for human FMS, was a generous gift from Linzhao Cheng (Johns Hopkins University, Baltimore, MD). The cDNA of human KIT, in the vector pcDNA3.1(+)c-kit, was a gift from Robert Arceci (Johns Hopkins University). The cDNA of human PDGFR- , in the vector pcDNA3-PDGFR-, was a gift from Frank
Bohmer (Institute for Molecular Biotechnology, Jena, Germany).
Proliferation and apoptosis assays Cell proliferation assays using 3-4,5-dimethylthiazol-2,5-diphenyltetrazolium (MTT) and apoptosis assays with annexin V were performed as described previously.23 Briefly, for the MTT assay, 50 µL of 2 × medium was aliquoted into triplicate wells of 96-well plates for each CEP-701 concentration point. All wells contained the same concentration of DMSO. Fifty µL cell suspension in medium was added to each well (50 000 cells/well for cell lines, 200 000 cells/well for AML blasts). Plates were incubated for 48 hours (cell lines) or 72 hours (AML blasts) at 37°C, 5% CO2, then the MTT assay was performed according to the manufacturer's instructions (Roche, Indianapolis, IN). For the annexin V apoptosis assay, cells were incubated for 48 hours in medium with increasing CEP-701 concentration, then annexin V binding was performed and assessed by flow cytometry according to the manufacturer's instructions (Clontech, Palo Alto, CA).Immunoprecipitation and immunoblotting Immunoprecipitation and immunoblotting were performed as described previously.11 Briefly, cells were incubated in culture medium with CEP-701 for 1 hour at 37°C, then washed in ice-cold PBS, and lysed in detergent buffer. Cytokines, if used, were added for the last 15 minutes of incubation. 500 µg clarified lysate per sample was incubated at 4°C overnight with anti-FLT3 antibody (S18; Santa Cruz Biotechnology), then protein A agarose (Upstate Biotechnology) was added for an additional 2 hours. After electrophoresis and transfer to Immobilon membranes (Millipore, Bedford, MA), immunoblotting was performed using antiphosphotyrosine antibody 4G10 to assess phosphorylated FLT3, or with anti-FLT3 antibody to measure total FLT3. Protein bands were visualized using chemiluminescence (ECL; Amersham, Piscataway, NJ) and scanned with an AGFA Arcus 1200 laser scanner. Densitometric analysis was performed on a Macintosh G4 computer using the public domain NIH Image program (developed at the US National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/).
CEP-701 is a selective inhibitor of FLT3 To study the role of FLT3/ITD mutations in leukemia and to develop a screening assay for identifying FLT3 inhibitors, we generated FLT3/ITD model cell lines. Several FLT3/ITD mutations were cloned from RNA isolated from bone marrow samples from AML patients at our institution. These FLT3/ITD mutations were used to transfect BaF3 cells, a nonleukemic IL-3-dependent pro-B cell line derived from Balb/c mice, which lacks FLT3 expression.24 The resulting cloned cell lines (BaF3/ITD) proliferated in the absence of IL-3 and expressed constitutively phosphorylated FLT3. The BaF3/ITD cells were used to screen a library of small-molecule tyrosine kinase inhibitors to identify compounds with activity against FLT3. Cells were aliquoted into 96-well plates where each well contained a 100-nM concentration of different compounds. After 24 hours of incubation at 37°C, 5% CO2 in RPMI with 10% FBS, an MTT proliferation assay was performed to identify compounds that inhibited proliferation of the BaF3/ITD cells (data not shown).23 Selective inhibition of FLT3 in BaF3/ITD cells should revert the cells to IL-3-dependent growth. Therefore, to screen for compounds that were relatively selective for FLT3, the identical compounds were assayed on duplicate plates with the addition of 1 ng/mL IL-3. Rescue of growth by IL-3 demonstrates that normal proliferative pathways are still intact.One compound that demonstrated selectivity in this assay was the
indolocarbazole derivative, CEP-701. As an initial test of its potency
as a FLT3 inhibitor in cell cultures, we carried out dose-response
experiments in BaF3/ITD cells (Figure
1A). Autophosphorylation of FLT3 is
potently inhibited by CEP-701 with an inhibitory concentration of 50%
(IC50) of approximately 2 nM, a result that correlates closely with in vitro kinase assays (3 nM; data not shown). In addition, we transfected BaF3 cells with FLT3 constructs containing Asp835Tyr and Asp835His point mutations. These constitutively activated FLT3 receptors were likewise inhibited by CEP-701 with a
similar IC50 (data not shown). To confirm selectivity for
FLT3 in the cell-based assay, we tested CEP-701 against the activity of
the receptor tyrosine kinases most closely related to FLT3: PDGFR-
As another measure of selectivity for FLT3, the effect of CEP-701 on
parental BaF3 cells and BaF3/ITD cells was examined (Figure 2). CEP-701 inhibited the proliferation
of BaF3/ITD cells in a dose-dependent fashion with an IC50
of approximately 5 nM (Figure 2A). BaF3-derived cell lines expressing
Asp835 or FLT3/ITD mutants were likewise inhibited (data not shown). In
contrast to these results, parental BaF3 cells were not inhibited at
concentrations as high as 200 nM. Furthermore, the addition of IL-3
rescued the growth of BaF3/ITD cells in the presence of CEP-701. In
addition, the results of experiments measuring induction of apoptosis
by CEP-701 showed dose-response curves similar to the MTT assay results in Figure 2A (data not shown). The immunoblot in Figure 2B shows that
addition of IL-3 to the BaF3/ITD cells had no effect on FLT3 inhibition
by CEP-701. Thus, IL-3 "rescue" of BaF3/ITD cells from CEP-701-induced cytotoxicity is not mediated through any effect on
FLT3 phosphorylation. These results imply that CEP-701 does not
interfere with IL-3-mediated signal transduction leading to proliferation and survival in BaF3 cells, but only affects
FLT3-dependent signaling. Thus, CEP-701 is a relatively selective FLT3
inhibitor with cytotoxic effects on FLT3/ITD-transformed BaF3 cells.
CEP-701 is cytotoxic to FLT3-expressing leukemia cell lines CEP-701 was tested on several human myeloid leukemia-derived cell lines that naturally express FLT3 (Figure 3). RT-PCR was used as described to assay for the presence of FLT3 mutations in each cell line (data not shown).23 EOL-1 (eosinophilic leukemia)25 and BV173 (CML blast crisis)26 cells express wild-type FLT3 lacking any ITD or Asp835 mutation, and HEL cells (AML M6)27 completely lack FLT3 expression. MV4-11 cells (AML M5)28 express a typical FLT3/ITD mutation consisting of a 30-bp insertion at nucleotide 1857 (by RT-PCR and dideoxy sequencing; data not shown). Cell lines were assayed for the effect of CEP-701 on proliferation, apoptosis, and FLT3 phosphorylation. EOL-1 and MV4-11 cells displayed a cytotoxic dose-response to CEP-701 that closely paralleled the inhibition of FLT3 phosphorylation (Figure 3A,B). Cytotoxicity was observed once FLT3 phosphorylation was inhibited to a level less than roughly 30% of the untreated controls. BV173 cells demonstrated characteristic FLT3 inhibition in response to CEP-701, but showed no induction of apoptosis and only a modest inhibition of proliferation (Figure 3C). Thus, BV173 cells are not dependent on FLT3 signaling for survival. This may result from their expression of BCR/ABL,26 the phosphorylation of which is unaffected by 50 nM CEP-701 (data not shown). HEL cells, which lack FLT3 expression (Figure 3D), displayed no cytotoxicity to CEP-701 even at levels up to 500 nM (higher doses not shown). These data demonstrate that in some human leukemia cell lines that express FLT3, CEP-701 inhibits proliferation and induces apoptosis in a manner that parallels the inhibition of FLT3 phosphorylation.
It is still possible that the cytotoxicity observed in EOL-1 and MV4-11
cells was mediated through inhibition of other unknown protein kinases.
However, if these effects were due to FLT3 inhibition, then the
addition of exogenous FL to the EOL-1 cells (which express wild-type
FLT3 and are likely activated by endogenous expression of FL) should
shift the CEP-701 dose-response curves toward the right. As predicted
(Figure 4), both the cytotoxic effect and inhibition of FLT3 phosphorylation were shifted to a similar degree. This same shift was seen for apoptosis (data not shown). In the presence or absence of FL, cytotoxicity was observed in EOL-1 cells
once CEP-701 inhibited FLT3 phosphorylation to below 30% of baseline
(as measured by densitometry). Because FL will only stimulate FLT3,
this provides further support that the cytotoxic effect of CEP-701 is
mediated through direct inhibition of FLT3.
STAT5 and ERK1/2 are inhibited in response to FLT3 inhibition A number of proteins, including STAT5 and ERK1/2, have been identified as downstream components of the wild-type and mutationally activated FLT3 signaling pathway.9-11,29 These proteins are involved in the proliferative and antiapoptotic effects of FLT3 activation. To test whether or not activation of these effector proteins was affected by inhibiting FLT3 with CEP-701, we examined the phosphorylation status of STAT5 and ERK1/2 in each of the cell lines (Figure 5). The 3 CEP-701-responsive cell lines (BaF3/ITD, EOL-1, and MV4-11) all demonstrated a loss of ERK1/2 and STAT5 phosphorylation at increasing doses of CEP-701. In contrast, BV173 and HEL cells, which displayed minimal or no cytotoxic response to CEP-701, showed no change in STAT5 or ERK1/2 phosphorylation. This would be predicted for HEL cells, which lack FLT3 expression, and for BV173 cells, which express BCR/ABL (which results in STAT5 and ERK1/2 phosphorylation).30,31 These results further establish the relative selectivity of CEP-701 for FLT3 and illustrate a possible association between CEP-701-mediated cytotoxicity and inhibition of STAT5 and ERK1/2 activation.
Effects of CEP-701 on primary AML samples The ultimate goal of these studies is to assess the potential for use of CEP-701 in treating AML. Therefore, the next experiments were designed to study the effect of CEP-701 on primary human AML samples available from our institution's cell bank. RT-PCR analysis (data not shown; performed as described23) of the 8 samples chosen revealed that 4 of the samples expressed FLT3/ITD mutations and one contained a Asp835Tyr mutation. A CEP-701 dose-response MTT assay was performed in triplicate on all 8 samples (Figure 6). Three of 4 FLT3/ITD samples displayed a cytotoxic response to CEP-701, as did a single non-ITD sample. One of the FLT3/ITD samples, along with the Asp835 mutant sample and the other 2 wild-type samples, did not respond to CEP-701. We also tested these samples for response to CEP-701 in the presence of 100 ng/mL FL and the responding samples were identical (not shown).
FLT3 protein was present and phosphorylated in 6 of the 7 samples analyzed (Figure 6B). CEP-701 at 50 nM completely inhibited FLT3 phosphorylation in all of these samples, including the one that harbored a Asp835 mutation. An interesting result of this experiment was the observed lack of difference in FLT3 phosphorylation levels between leukemic cells expressing wild-type and mutant FLT3. Although we have not attempted to address the mechanism of FLT3 stimulation in the nonmutant primary samples, it is possible that the activity seen here was influenced by coexpression of FL by the blasts themselves.32 From these experiments on primary AML samples, we can draw some tentative conclusions: (1) CEP-701 potently inhibits FLT3 phosphorylation in vitro in the leukemic blasts from AML patients. This includes wild-type FLT3, as well as both ITD and Asp835 mutant forms. (2) CEP-701 is selectively cytotoxic to most samples of AML blasts harboring FLT3/ITD mutations, suggesting that patients with these mutations are good candidates for this therapy. This finding is consistent with the findings of our previous study using a less selective, less potent FLT3 inhibitor.23 STAT5 and ERK1/2 are involved in FLT3 signal transduction and are
constitutively activated in a significant fraction of AML samples,
particularly in cases with FLT3/ITD mutations.29,33,34 In
the leukemia cell lines we studied, a correlation was noted between
CEP-701-mediated cytotoxicity and inhibition of STAT5 and ERK1/2. For
5 of the 8 primary AML samples shown in Figure 6, we had sufficient
protein extract to analyze the phosphorylation status of STAT3, STAT5,
and ERK1/2 by immunoblotting (Figure 7). STAT5 phosphorylation (Figure 7A) was present and inhibited by 50 nM
CEP-701 in 4 of the 5 samples, including the responding samples. By
comparison, STAT3 (Figure 7B), which mediates signaling from
constitutively activated KIT in AML cells,35 was present in all 5 samples, but its phosphorylation was not affected. ERK1/2 phosphorylation (Figure 7C) was affected in some samples, but the
degree of inhibition was less dramatic in all but a single sample.
STAT5 may mediate survival signals in these blasts in a manner similar
to that observed in BCR-ABL+ cells and erythroid
precursors.36,37 Inhibition of STAT5 phosphorylation by
CEP-701-mediated inhibition of FLT3 activation, therefore, may lead to
a cytotoxic response in some blast samples.
CEP-701 inhibits FLT3 in vivo and prolongs survival in a mouse model of FLT3/ITD leukemia Having established CEP-701 as a potent and relatively selective FLT3 inhibitor with in vitro activity against leukemia cells, the next step was to test the compound for in vivo activity. Balb/c mice injected with cells expressing constitutively activated FLT3 provided a useful model of leukemia.11 These mice typically sicken and die, with large spleens full of leukemic cells, within 2 to 3 weeks following injection. Mice were injected with BaF3/ITD cells on day 1, and then were given a single subcutaneous dose of CEP-701 on day 12 (Figure 8). Two mice were killed at each time point following drug treatment. The spleens were harvested and the leukocytes within isolated for analysis of in vivo inhibition of FLT3 phosphorylation. In vivo inhibition of FLT3 occurred, with the effect lasting up to 8 hours after the single dose of CEP-701 (Figure 8).
The next experiment was to see if CEP-701, administered on a daily
basis, could prolong the survival of Balb/c mice injected with the
BaF3/ITD leukemia cells. Based on the results from the single-dose
studies shown in Figure 8, dosing intervals of 4 times per day would be
optimal to maintain continuous FLT3 inhibition. However, practical
considerations limited the dosing to a maximum of every 8 hours. Figure
9 shows the survival curves of mice
treated either with vehicle control or with increasingly intense dosing regimens. Survival was significantly prolonged (P < .001)
in all 3 treated groups, with a clear dose-response effect. In a
similar experiment, using EOL-1 cells injected into NOD-RAG mice,
CEP-701 significantly (P < .001) prolonged the survival
of treated mice (data not shown).
In this report, we have described the characterization of CEP-701 as a FLT3 inhibitor. We have demonstrated its potency and relative selectivity for inhibiting wild-type and constitutively activated FLT3 in BaF3/ITD model cells and in myeloid leukemia-derived cell lines. CEP-701 is cytotoxic to some, but not all, FLT3-expressing cell lines. In BaF3/ITD, EOL-1, and MV4-11 cell lines, the drug was cytotoxic in a dose-responsive fashion that paralleled its inhibition of FLT3 phosphorylation. The cytotoxic effects began at approximately 5 nM and were maximal by 50 nM CEP-701. It is formally possible that the cytotoxicity observed could be mediated through inhibition of vascular endothelial growth factor receptor (VEGFR) 1 or 2, which are known to be up-regulated in AML and CML. However, the IC50 values of CEP-701 for these related receptor tyrosine kinases are 77 and 65 nM, respectively.38 Therefore, it is unlikely that the cytotoxic effects are mediated through inhibition of these VEGFRs. In BV173 cells, FLT3 phosphorylation was inhibited by CEP-701, but no cytotoxicity was observed, suggesting that these cells do not rely on FLT3 signaling for survival. The variation in cytotoxic response to CEP-701 was also seen in the primary blasts from AML patient samples. CEP-701 inhibited the phosphorylation of wild-type, ITD mutant, and Asp835 mutant FLT3 in these primary AML blasts, and a cytotoxic response was seen in some samples with wild-type FLT3 and in most samples with FLT3/ITD mutations. We have now tested a total of 14 FLT3/ITD AML samples with FLT3 inhibitors (4 in this study, 8 in our previously published study using the tyrphostin AG1295,23 and 2 unpublished experiments, March 2001). Twelve of the 14 FLT3/ITD samples displayed a cytotoxic response, whereas a much smaller proportion of nonmutant samples responded. The variable cytotoxic response brought about by CEP-701 raises several important issues regarding the use of FLT3 inhibitors in leukemia. It implies that FLT3 is often not the only signaling mechanism that must be inhibited to achieve a cytotoxic response. There are clearly other important signals in BV173 cells (presumably BCR/ABL) and in the nonresponsive blast samples that are unaffected by CEP-701. The inhibition of FLT3 phosphorylation probably results in the withdrawal of a strong antiapoptotic signal. However, it probably does not by itself introduce a proapoptotic stimulus. A combination of FLT3 inhibition and DNA damaging agents might well prove to be synergistic in killing leukemia cells. The results from our animal experiments represent an important step in the preclinical development of CEP-701. In mice with FLT3/ITD leukemia, a single dose of the drug completely inhibited FLT3 phosphorylation in vivo for several hours, and dosing 2 to 3 times per day significantly prolonged survival. The lack of a complete cure of the mice by CEP-701 could be due to several factors. The in vivo phosphorylation study showed that FLT3 inhibition after a single dose only lasts up to 8 hours. Maintaining inhibition continuously (ie, 24 h/d) may be required to maximize efficacy. In addition, a number of mechanisms of resistance to tyrosine kinase inhibitors are known to exist (as has been seen with imatinib mesylate), any one of which may be operative in these experiments.39,40 Further studies will be necessary to determine which of these mechanisms might affect the efficacy of CEP-701. CEP-701 is a derivative of the indolocarbazole K252, a microbial-derived compound.41 Other than FLT3, the only known tyrosine kinases inhibited by CEP-701 with similar potency are members of the TRK family (high-affinity nerve growth factor receptors).38 Preliminary dose finding (phase I) studies of orally administered CEP-701 have been completed (results in preparation). The drug was well tolerated by patients and there were no serious or irreversible toxicities. The pharmacokinetic data from these studies, coupled with the encouraging results demonstrating in vivo FLT3 inhibition in mice, suggest that a safe and tolerable oral dose of CEP-701 can achieve FLT3-inhibitory drug levels in the plasma of patients. The cytotoxicity displayed by leukemia cell lines and primary AML blasts in response to FLT3 inhibition lead to the prediction that CEP-701 will be an effective therapy in a significant number of patients with AML. The activity of imatinib mesylate in both the blast crisis phase of CML and Philadelphia chromosome-positive acute lymphocytic leukemia has demonstrated that a tyrosine kinase inhibitor can be an effective therapy in an acute leukemia.42 A clinical trial of CEP-701 in patients with AML is now underway.
Submitted November 14, 2001; accepted January 25, 2002.
Supported by grants from the National Cancer Institute (CA70970 to D.S., and K23 CA81262 to B.D.S.), Leukemia and Lymphoma Society (to D.S.), Children's Cancer Foundation (to D.S.), Cephalon, Inc. (to D.S.), and National Institutes of Health training grant 5T32CA09071 (to M.L.). Partial funding for the studies described in this report was provided by Cephalon, Inc.
D.S. is a paid consultant to Cephalon, Inc. The terms of this arrangement are being managed by the Johns Hopkins University in accordance with its conflict of interest policies.
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: Donald Small, Johns Hopkins University School of Medicine, Bunting Blaustein Cancer Research Bldg, Rm 251, 1650 Orleans St, Baltimore, MD 21231-1000; e-mail: donsmall{at}jhmi.edu.
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B. D. Smith, M. Levis, M. Beran, F. Giles, H. Kantarjian, K. Berg, K. M. Murphy, T. Dauses, J. Allebach, and D. Small Single-agent CEP-701, a novel FLT3 inhibitor, shows biologic and clinical activity in patients with relapsed or refractory acute myeloid leukemia Blood, May 15, 2004; 103(10): 3669 - 3676. [Abstract] [Full Text] [PDF]
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S. Trudel, S. Ely, Y. Farooqi, M. Affer, D. F. Robbiani, M. Chesi, and P. L. Bergsagel Inhibition of fibroblast growth factor receptor 3 induces differentiation and apoptosis in t(4;14) myeloma Blood, May 1, 2004; 103(9): 3521 - 3528. [Abstract] [Full Text] [PDF]
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S. A. Armstrong, M. E. Mabon, L. B. Silverman, A. Li, J. G. Gribben, E. A. Fox, S. E. Sallan, and S. J. Korsmeyer FLT3 mutations in childhood acute lymphoblastic leukemia Blood, May 1, 2004; 103(9): 3544 - 3546. [Abstract] [Full Text] [PDF]
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M. G. Mohi, C. Boulton, T.-L. Gu, D. W. Sternberg, D. Neuberg, J. D. Griffin, D. G. Gilliland, and B. G. Neel Combination of rapamycin and protein tyrosine kinase (PTK) inhibitors for the treatment of leukemias caused by oncogenic PTKs PNAS, March 2, 2004; 101(9): 3130 - 3135. [Abstract] [Full Text] [PDF]
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R. Zheng, A. D. Friedman, M. Levis, L. Li, E. G. Weir, and D. Small Internal tandem duplication mutation of FLT3 blocks myeloid differentiation through suppression of C/EBP{alpha} expression Blood, March 1, 2004; 103(5): 1883 - 1890. [Abstract] [Full Text] [PDF]
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D. G. Gilliland, C. T. Jordan, and C. A. Felix The Molecular Basis of Leukemia Hematology, January 1, 2004; 2004(1): 80 - 97. [Abstract] [Full Text] [PDF]
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R. Zheng, M. Levis, O. Piloto, P. Brown, B. R. Baldwin, N. C. Gorin, M. Beran, Z. Zhu, D. Ludwig, D. Hicklin, et al. FLT3 ligand causes autocrine signaling in acute myeloid leukemia cells Blood, January 1, 2004; 103(1): 267 - 274. [Abstract] [Full Text] [PDF]
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A.-M. O'Farrell, J. M. Foran, W. Fiedler, H. Serve, R. L. Paquette, M. A. Cooper, H. A. Yuen, S. G. Louie, H. Kim, S. Nicholas, et al. An Innovative Phase I Clinical Study Demonstrates Inhibition of FLT3 Phosphorylation by SU11248 in Acute Myeloid Leukemia Patients Clin. Cancer Res., November 15, 2003; 9(15): 5465 - 5476. [Abstract] [Full Text] [PDF]
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Y. Minami, K. Yamamoto, H. Kiyoi, R. Ueda, H. Saito, and T. Naoe Different antiapoptotic pathways between wild-type and mutated FLT3: insights into therapeutic targets in leukemia Blood, October 15, 2003; 102(8): 2969 - 2975. [Abstract] [Full Text] [PDF]
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Q. Yao, R. Nishiuchi, Q. Li, A. R. Kumar, W. A. Hudson, and J. H. Kersey FLT3 Expressing Leukemias Are Selectively Sensitive to Inhibitors of the Molecular Chaperone Heat Shock Protein 90 through Destabilization of Signal Transduction-Associated Kinases Clin. Cancer Res., October 1, 2003; 9(12): 4483 - 4493. [Abstract] [Full Text] [PDF]
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C. M. Zwaan, S. Meshinchi, J. P. Radich, A. J. P. Veerman, D. R. Huismans, L. Munske, M. Podleschny, K. Hahlen, R. Pieters, M. Zimmermann, et al. FLT3 internal tandem duplication in 234 children with acute myeloid leukemia: prognostic significance and relation to cellular drug resistance Blood, October 1, 2003; 102(7): 2387 - 2394. [Abstract] [Full Text] [PDF]
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C. J. Strock, J.-I. Park, M. Rosen, C. Dionne, B. Ruggeri, S. Jones-Bolin, S. R. Denmeade, D. W. Ball, and B. D. Nelkin CEP-701 and CEP-751 Inhibit Constitutively Activated RET Tyrosine Kinase Activity and Block Medullary Thyroid Carcinoma Cell Growth Cancer Res., September 1, 2003; 63(17): 5559 - 5563. [Abstract] [Full Text] [PDF]
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K. Murata, H. Kumagai, T. Kawashima, K. Tamitsu, M. Irie, H. Nakajima, S. Suzu, M. Shibuya, S. Kamihira, T. Nosaka, et al. Selective Cytotoxic Mechanism of GTP-14564, a Novel Tyrosine Kinase Inhibitor in Leukemia Cells Expressing a Constitutively Active Fms-like Tyrosine Kinase 3 (FLT3) J. Biol. Chem., August 29, 2003; 278(35): 32892 - 32898. [Abstract] [Full Text] [PDF]
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K. Spiekermann, R. J. Dirschinger, R. Schwab, K. Bagrintseva, F. Faber, C. Buske, S. Schnittger, L. M. Kelly, D. G. Gilliland, and W. Hiddemann The protein tyrosine kinase inhibitor SU5614 inhibits FLT3 and induces growth arrest and apoptosis in AML-derived cell lines expressing a constitutively activated FLT3 Blood, February 15, 2003; 101(4): 1494 - 1504. [Abstract] [Full Text] [PDF]
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F. Ravandi, M. Talpaz, and Z. Estrov Modulation of Cellular Signaling Pathways: Prospects for Targeted Therapy in Hematological Malignancies Clin. Cancer Res., February 1, 2003; 9(2): 535 - 550. [Abstract] [Full Text] [PDF]
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M. L. Guzman, C. F. Swiderski, D. S. Howard, B. A. Grimes, R. M. Rossi, S. J. Szilvassy, and C. T. Jordan Preferential induction of apoptosis for primary human leukemic stem cells PNAS, December 10, 2002; 99(25): 16220 - 16225. [Abstract] [Full Text] [PDF]
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K. W. H. Yee, A. M. O'Farrell, B. D. Smolich, J. M. Cherrington, G. McMahon, C. L. Wait, L. S. McGreevey, D. J. Griffith, and M. C. Heinrich SU5416 and SU5614 inhibit kinase activity of wild-type and mutant FLT3 receptor tyrosine kinase Blood, September 26, 2002; 100(8): 2941 - 2949. [Abstract] [Full Text] [PDF]
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D. G. Gilliland and J. D. Griffin The roles of FLT3 in hematopoiesis and leukemia Blood, August 13, 2002; 100(5): 1532 - 1542. [Abstract] [Full Text] [PDF]
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F. J. Giles, A. Keating, A. H. Goldstone, I. Avivi, C. L. Willman, and H. M. Kantarjian Acute Myeloid Leukemia Hematology, January 1, 2002; 2002(1): 73 - 110. [Abstract] [Full Text]
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Top
Abstract
Introduction
cells destined to
become dendritic cells.
, PI3-kinase, STAT5, and ERK1/2 are important signaling proteins
linked to activation of FLT3.
20°C, was diluted into medium just before use. For every
experiment described in this report, each sample within the experiment
contained identical amounts of DMSO (< 0.25%). For in vivo mouse
experiments, CEP-701 was formulated in a vehicle of 40% polyethylene
glycol 1000, 10% povidone C30, and 2% benzyl alcohol in sterile
water. All antibodies used were obtained from Santa Cruz Biotechnology
(Santa Cruz, CA) except for antiphospho-STAT5 and antiphospho-STAT3
(Cell Signaling, Beverly, MA), and 4G10 (Upstate Biotechnology, Lake
Placid, NY). Interleukin-3 (IL-3), FL, PDGF, stem cell factor (SCF),
and KIT ligand (KL) were from Peprotech (Rocky Hill, NJ). All other
reagents were from Sigma (St Louis, MO). Restriction enzymes were from
Invitrogen (Rockville, MD).
