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NEOPLASIA
From the Department of Medicine/Hematology and
Oncology, and the Department of Neurology, University of Münster,
Germany and the Department of Hematology and Oncology, Osaka University
Medical School, Japan.
Somatic mutations of the receptor tyrosine kinase Flt3 consisting
of internal tandem duplications (ITD) occur in 20% of patients with
acute myeloid leukemia. They are associated with a poor prognosis of
the disease. In this study, we characterized the oncogenic potential
and signaling properties of Flt3 mutations. We constructed chimeric
molecules that consisted of the murine Flt3 backbone and a 510-base
pair human Flt3 fragment, which contained either 4 different ITD
mutants or the wild-type coding sequence. Flt3 isoforms containing ITD
mutations (Flt3-ITD) induced factor-independent growth and resistance
to radiation-induced apoptosis in 32D cells. Cells containing
Flt3-ITD, but not those containing wild-type Flt3 (Flt3-WT), formed
colonies in methylcellulose. Injection of 32D/Flt3-ITD induced rapid
development of a leukemia-type disease in syngeneic mice. Flt3-ITD
mutations exhibited constitutive autophosphorylation of the immature
form of the Flt3 receptor. Analysis of the involved signal transduction
pathways revealed that Flt3-ITD only slightly activated the MAP kinases
Erk1 and 2 and the protein kinase B (Akt) in the absence of ligand and
retained ligand-induced activation of these enzymes. However, Flt3-ITD
led to strong factor-independent activation of STAT5. The relative
importance of the STAT5 and Ras pathways for ITD-induced colony
formation was assessed by transfection of dominant negative (dn)
forms of these proteins: transfection of dnSTAT5 inhibited colony
formation by 50%. Despite its weak constitutive activation by
Flt3-ITD, dnRas also strongly inhibited Flt3-ITD-mediated colony
formation. Taken together, Flt3-ITD mutations induce factor-independent
growth and leukemogenesis of 32D cells that are mediated by the Ras and
STAT5 pathways.
(Blood. 2000;96:3907-3914) The receptor tyrosine kinase Flt31-3
and its ligand FL4-6 play an important role in survival and
self-renewal of early hematopoietic progenitors and monocytic
precursors and in early lymphoid development.7-9 The
expression of Flt3 on leukemic cell lines and samples from patients
with acute leukemia has been extensively studied. Flt3 is expressed on
most acute leukemias of myeloid and B-lymphoid origin.10,11 Incubation of leukemic blasts with FL results in enhanced DNA synthesis in some, but not all cases of acute myeloid
leukemia (AML)10-15 and in a reduced rate of spontaneous apoptosis of AML blasts.16 Chronic overexpression of Flt3
ligand in hematopoietic progenitors has been shown to induce
hematologic malignancies after a long latency period.17
Somatic mutations involving a set of in-frame internal tandem
duplications (ITDs) occur at the end of exon 11 of the Flt3 gene. These
result in the duplication of a stretch of several amino acids in the
juxtamembrane region of the receptor.18 ITD mutations are
detectable in about 20% of AML samples18 but not in normal
hematopoiesis,19 and they are associated with high
leukemic cell numbers in patients with acute promyelocytic
leukemia.20 The presence of ITD mutations of Flt3 on AML
blasts indicates a very poor prognosis.21-23 So far,
little is known about the functional consequences of ITD mutations.
Expression of ITD-containing Flt3 in COS-7 cells resulted in its
constitutive autophosphorylation.20 Our own results
indicated that constitutive autophosphorylation of Flt3 is detectable
in about 10% of AML cases.24 However, autophosphorylation
did not correlate with ITD mutations. The growth of AML blasts with ITD mutations in stroma cell cultures was inhibited compared with blasts
not containing ITD mutations.25
In this study, we show that ITD mutations of Flt3 (Flt3-ITD) have
oncogenic potential. ITD mutations confer factor-independent growth and
radiation resistance onto 32D cells. Also, they induce colony growth in
semisolid media, which is not supported by Flt3 ligand in 32D cells
transfected with wild-type Flt3 (Flt3-WT). When injected into syngeneic
hosts, Flt3-ITD, but not Flt3-WT, leads to the rapid development of a
disease resembling leukemia. Analysis of the signaling properties of
Flt3-ITD reveals weak constitutive activation of Ras and Akt with
ligand-dependent augmentation and strong constitutive activation of
STAT5, a novel feature of Flt3 signaling. STAT5 is not activated by a
ligand-bound wild-type receptor. Analyses by dominant negative (dn)
constructs show an important role of Ras and STAT5 for ITD-induced
colony formation. Taken together, ITD mutations of Flt3 have powerful
transforming potential in myeloid cells mediated by Ras and aberrant
STAT5 activation.
Reagents
Cell lines
Construction of expression plasmids and transfection into 32Dcl3 cells ITD mutations were detected in patients with AML as described previously.24 The membrane proximal region of 4 mutated Flt3 receptor sequences, as well as wild-type human Flt3 derived from the Oci-AML5 cell line, were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) using Pfu DNA polymerase. The sense primer was 5'-GCA CAT CTT GTG AGA CGA TCC-3' and the antisense primer was 5'-CAC CAT AGC AAC AAT ATT CAA AAA TC-3', yielding a product corresponding to nucleotides 1562 to 2092 of the published sequence of human Flt3.3 One of 2 SspI sites of the PCR products, which is unique to the human Flt3 sequence, was removed by site-directed mutagenesis without alteration of the resulting amino acid code. A BsmBI/SspI fragment of these PCR products spanning nt1567 to 2077 was substitutionally introduced into the sequence of murine Flt3 (provided by Dr I. Lemischka, Princeton, NJ). The sequences of all mutants were confirmed by DNA sequencing. These constructs were cloned into an expression vector (pAL) under the control of the 5'LTR of the Moloney murine sarcoma virus (MoMSV), derived from pLXSN (Clontech, Palo Alto, CA). Ten micrograms of plasmid DNA of either plasmid and 1 µg pMAM/BSD (Kaken Pharmaceutical, Japan) were cotransfected into 32Dcl3 cells by electroporation. Cells were selected with 15 µg/mL blasticidin (Invitrogen, Groningen, The Netherlands) in IL-3 supplemented culture. Polyclonal cell lines were used for further experiments. The ovine STAT5/Y694F mutant (dnSTAT5)26 was subcloned into an expression vector under the control of the 5'LTR of the MoMSV (pOPALI), derived from pOPI3CAT (Stratagene, La Jolla, CA), which contained a neomycin resistance marker.Lac inducible system The 32Dcl3 were first transfected with pLAM-LacR, which was derived from pMAM and expresses the Lac repressor. The 32D/pLAM-LacR were further transfected with pOPRSVI (Stratagene) containing RasS17N (dnRas), the expression of which is suppressed by the Lac repressor in the uninduced state, and induced by the addition of isopropyl thiogalactose (IPTG). Highly inducible dnRas-expressing clones were selected by limiting dilution. One of the clones was cotransfected with the pAL/Flt3 constructs and pPur (Clontech) as a selection marker, and puromycin-selected polyclonal lines were used for further experiments. The expression of dnRas was induced by incubating the cell lines with 0.5 mmol/L IPTG for 24 hours, and the expression was verified by Western blotting with anti-ras Ab (Upstate Biotechnology).Flow cytometry Cells were preincubated for 15 minutes at 4°C with mouse IgG and subsequently stained for 15 minutes with the indicated antibody. The cells were washed twice in phosphate-buffered saline (PBS)/0.1% bovine serum albumin (BSA) and analyzed with a FACSCalibur (Becton Dickinson, Heidelberg, Germany).Immunoprecipitation and Western blot analysis The 32Dcl3 cells transfected with Flt3 constructs were starved from IL-3 and serum for 12 hours in 0.5% FCS. Subsequently, cells were resuspended in 1 mL medium for 10 minutes at 37°C with or without 100 ng/mL FL, washed once with ice-cold PBS, and lysed with buffer containing 50 mmol/L HEPES (pH 7.4), 10% glycerol, 150 mmol/L NaCl, 1% Triton X-100, 1 mmol/L EDTA, 1 mmol/L EGTA, 50 µmol/L ZnCl, 25 mmol/L NaF, proteinase inhibitors (Complete; Boehringer, Mannheim, Germany), 1 µmol/L pepstatin, and 1 mmol/L sodium orthovanadate. Cell lysates were clarified at 20 000g for 20 minutes. For immunoprecipitation, cell lysates were incubated with rabbit polyclonal antibody to murine Flt3 (Upstate Biotechnology) and with Protein A/G-Plus-Sepharose (Santa Cruz Biotechnology). The immunoprecipitates were washed 3 times with lysis buffer. Immunoprecipitates as well as total lysates were resuspended in SDS sample buffer, heated, and separated by SDS PAGE. Gels were blotted on Immobilon P membrane (Millipore, Bedford, MA) and stained with the indicated antibody. Antibody binding was detected by incubation with an horseradish peroxidase (HRP)-labeled secondary antibody, followed by chemiluminescence detection (ECL-Plus, Amersham Pharmacia Biotech, Uppsala, Sweden).3H thymidine incorporation A total of 1 × 104 32D cells was IL-3 and serum starved (0.5% FCS) for 12 hours. Subsequently, cells were placed in 200 µL medium supplemented with the indicated concentrations of FL. After a 24-hour incubation at 37°C, 0.037 MBq (1 µCi) 3H thymidine was added to each well, and cells were incubated for an additional 8 hours. Cells were harvested on glass fiber filters, and -emission of the bound DNA was analyzed with a
scintillation counter. Experiments were repeated at least 3 times. Each
data point represents the mean ± standard deviation of 4 wells.
Radiation-induced apoptosis A total of 2.5 × 105 cells was starved from IL-3 and serum for 3 hours, placed in 24-well plates, and exposed to 5 Gy -irradiation. Immediately after irradiation, cells were supplemented
with FL (100 ng/mL), IL-3 (1 ng/mL), or no factor. Cell viability was analyzed using the Annexin-V assay. Cells staining negative for both
Annexin-V and propidium iodide were counted as viable cells.
Electrophoretic mobility shift assay The 32D cells exposed to the indicated cytokines were washed with ice-cold PBS containing 0.5 mmol/L PMSF and 0.2 mmol/L NaVO3 and lysed in 500 µL cold buffer I (20 mmol/L HEPES, pH 7.9, 0.2% [vol/vol] Nonidet P-40, 10% [vol/vol] glycerol, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L DTT, 0.2 mmol/L NaVO3, 0.5 mmol/L PMSF, 10 µg/mL leupeptin, and 10 µg/mL aprotinin). After incubation on ice for 10 minutes, cell lysates were centrifuged at 14 000 rpm for 10 minutes at 2°C. The pellets were resuspended in 100 µL buffer II, containing 20 mmol/L HEPES (pH 7.9), 20% (vol/vol) glycerol, 420 mmol/L NaCl, 2 mmol/L EDTA, 1.5 mmol/L MgCl2, 0.5 mmol/L DTT, 2 mmol/L NaVO3, 0.5 mmol/L PMSF, 10 µg/mL leupeptin, and 10 µg/mL aprotinin, and incubated for 30 minutes at 4°C. After centrifugation at 14 000 rpm for 20 minutes at 2°C, the supernatant was dialyzed against buffer III (20 mmol/L HEPES [pH 7.9], 20% [vol/vol] glycerol, 100 mmol/L NaCl, 2 mmol/L EDTA, 0.5 mmol/L DTT, 2 mmol/L NaVO3, 0.5 mmol/L PMSF) for 4 hours at 4°C. The DNA mobility shift assay was performed by incubating 10 µg nuclear extract with 1 µg poly(dI-dC) and 20 000 cpm of the end-labeled, double-stranded probe in binding buffer (10 mmol/L Tris [pH 7.5], 20% [vol/vol] glycerol, 50 mmol/L NaCl, 1 mmol/L DTT, and 1 mmol/L EDTA) in a final volume of 20 µL. The binding reaction was incubated for 20 minutes at 25°C. The double-stranded oligonucleotide was 5'-AGA TTT CTA GGA ATT CAA TCC-3', harboring the consensus binding site for STAT5.27 DNA-protein complexes were run on a 4% nondenaturing polyacrylamide gel carried out in 0.5x Tris-Borate-EDTA buffer. The gel was dried and exposed to film. DNA supershift assays were performed by incubation of the nuclear proteins with a specific antibody directed against STAT5a/b (Santa Cruz Biotechnology, 2 µg each per assay) for 15 minutes at 25°C before the addition of the probe.Clonal growth in methylcellulose To analyze clonal growth, 1 mL of a culture mixture containing Iscove modified Dulbecco medium (IMDM; Life Technologies, Grand Island, NY), 1% methylcellulose, and 10% FCS was plated on a 35-mm culture dish in the presence of FL (20 to 200 ng/mL), IL-3 (1 ng/mL), or no cytokine. Stably transfected 32Dcl3 cells were seeded at a concentration of 3 × 102 or 3 × 103 cells per dish. The assays were plated as quadruplicates, and colonies were counted on day 6. The numbers given show representative results of one of at least 3 independent experiments per construct. To analyze the effect of the expression of dnSTAT5 on the colony growth, 32Dcl3/Flt3-ITD cells were electroporated with either 20 µg pOPALI/STAT5Y694F or the same amount of pOPALI/CAT. One day after the electroporation, cells were seeded at a concentration of 1 × 105 cells per dish in 1 mL of a culture mix containing IMDM, 1% methylcellulose, 20% FCS, and 0.6 mg/dL G418. The colonies were counted on day 8.In vivo tumorigenesis assay Eight- to 10-week-old female C3H/HeJ mice, which were syngeneic with 32Dcl3 cells, were used for the in vivo tumorigenesis experiments. The 1 × 106 cells were inoculated by intravenous injection. Moribund animals were killed. Bone marrow cells were extracted from the femur and stained with Pappenheim stain. The experimental protocols had been reviewed and approved by the local Animal Experimentation Committee.
Construction and expression of the chimeric mutant Flt3 receptors We have previously identified Flt3-ITD mutations from patients with AML.24 To examine the role of ITD mutations in leukemogenesis, we constructed mouse Flt3 complementary DNA (cDNA) mutants containing the mutated human Flt3 sequences (Figure 1A). Four different ITD mutations were cloned into the mouse backbone. These mutations were chosen because of their variable involvement of tyrosine residues and differences in the lengths of the duplication. The wild-type human Flt3 cDNA of this region was amplified from OCI-AML5. All constructs were introduced into the IL-3-dependent myeloid progenitor cell line 32D under the control of the MoMSV 5'LTR. After selection of the bulk culture for 14 days in the presence of IL-3, stable expression of murine Flt3 was established as shown in Figure 1B. All Flt3-ITD mutant receptors were expressed at comparable levels with the wild-type human/murine chimera Flt3 (Flt3-WT) or with wild-type murine Flt3 (data not shown). The parental or mock transfected 32D lines did not express Flt3 (Figure 1B).
Factor-independent DNA synthesis of 32D/Flt3-ITD We analyzed the DNA-synthesis of the different mutants by 3H thymidine incorporation. The parental 32Dcl3 line did not respond to FL, whereas the Flt3-WT transfectant (32D/Flt3-WT) grew dependent on either IL-3 or FL. However, all cell lines containing Flt3-ITD (32D/Flt3-ITD) proliferated independently of exogenously added IL-3 or FL (Figure 2). In addition, FL did not further increase the rate of DNA synthesis in 32D/Flt3-ITD. Thymidine incorporation of 32D/Flt3-ITD was much higher compared with Flt3-WT cells stimulated with maximal levels of FL. As shown in Figure 2A, DNA synthesis of ITD-containing 32D cells without exogenously added growth factors approximately equaled their synthesis rate under the influence of maximal IL-3 stimulation (10 ng/mL).
Resistance to radiation-induced apoptosis of 32D/Flt3-ITD It had been reported that FL mediates survival of early hematopoietic precursors as well as AML cells.16,28 Therefore, we examined the role of ITD mutants in DNA damage-induced apoptosis. As shown in Figure 3, 32D/Flt3-WT were highly sensitive to-irradiation, with less than
10% viable cells 24 hours after irradiation. On FL exposure, up to
60% of 32D/Flt3-WT were rescued from apoptosis. Interestingly, cells
transfected with Flt3-ITD were entirely resistant to
irradiation-induced apoptosis. The resistance was independent of FL
presence or absence.
Clonal growth of 32D/Flt3-ITD in methylcellulose To further analyze the effect of Flt3-ITD mutants on proliferation and survival, we examined the ability of 32D/Flt3-ITD cells to form colonies. The 32D cells transfected with the empty vector produced colonies in the presence of IL-3, but not in the presence of FL (Figure 4). Surprisingly, Flt3-WT could not support clonal growth of the 32D cells even in the presence of up to 200 ng/mL FL (Figure 4A). To confirm these findings, we repeated the experiment with 10 times the number of cells per dish. In addition, appropriate expression of Flt3 on the surface of the cells was checked immediately before plating. We still did not detect any colonies, evidencing that Flt3-WT did not allow clonal growth of 32D cells in methylcellulose (Figure 4B). Results obtained with wild-type mouse Flt3 were identical (data not shown). In stark contrast, Flt3-ITD led to colony formation of 32D cells, even in the absence of IL-3 and FL (Figure 4). Taken together, these results indicate that signaling of Flt3-ITD may elicit a signal quality different from the wild-type isoforms of Flt3.
Flt3-ITD induce leukemogenesis of 32D cells in vivo Previously, we have shown that a mutation in the catalytic domain of Flt3 leads to factor-independent receptor autophosphorylation, proliferation, and survival of 32D cells.24 However, when we injected cells carrying these mutations into syngeneic mice, they developed obvious disease very late, with a latency period of several months (M.M., R.S., H.S., unpublished data). Therefore, we investigated whether Flt3-ITD mutants enhance leukemogenesis of 32D cells in vivo. The 32D/Flt3-ITD1, 32D/Flt3-ITD2, 32D/Flt3-WT, and 32D/mock cells were intravenously injected into syngeneic mice. In all but one of the mice injected with 32D/Flt3-ITD1 or 32D/Flt3-ITD2, a disease resembling leukemia developed, with massive enlargement of spleen and liver and infiltration of the bone marrow by blastlike cells (Figure 5). Most animals injected with these cells died within 5 weeks after injection (Figure 6). Mice injected with 32D/mock or 32D/Flt3-WT did not develop obvious disease up to 3 months after injection. After this time, 2 of 10 animals injected with 32D/Flt3-WT developed a similar disease, as did the animals injected with the ITD-containing cells, and died.
Constitutive activation of the Flt3 receptor and ligand-dependent activation of MAPK and Akt in 32D/Flt3-ITD To analyze the signaling properties of Flt3-ITD in comparison to Flt3-WT, we first studied the autophosphorylation status of the Flt3 mutants in transfected 32D cells. The cells were IL-3 and serum starved for 12 hours and FL stimulated in the absence of serum for 10 minutes. From the cell lysates, Flt3 was immunoprecipitated and immunoblotted with an antiphosphotyrosine moAb or an anti-Flt3 polyclonal antibody. Interestingly, we detected aberrant and constitutive autophosphorylation of the lower band of the mutant Flt3 isoforms, which corresponds to its immature, possibly intracellular form (Figure 7).29 In contrast, the mature form seems to retain its ligand-specific autophosphorylation pattern, at least in some cases. Also, the balance between the immature and the mature forms of the receptor was disturbed, because only weak expression of the mature form of the receptor was seen on Flt3 immunoblots.
To examine downstream signals, total cell lysates of all 4 ITD mutant cell lines were subjected to immunoblotting with antibodies specific to the activation status of signal transduction molecules. The loading conditions of the gels were verified by antibodies detecting the molecules independent of their activation state. To detect MAPK activation, we used an anti-pErk antibody, which is specific for Erk1/2 phosphorylated on tyrosine 204. To detect the activation of PI3K-dependent pathways, we analyzed the activation of Akt, a serine/threonine protein kinase downstream of PI3K, which has been implicated in cell survival. The anti-pAkt-specific antibody detects Akt, which is phosphorylated at serine 473. As shown in Figure 7, Erk1/2 and Akt were activated ligand dependently in 32D/Flt3-ITD cells, with slightly increased basal levels of phosphorylation. Only one ITD construct, ITD2, led to significant constitutive activation of the Erk proteins. Taken together, the ITD mutations induced factor-independent autophosphorylation of the immature form of Flt3. In the absence of ligand, they did not induce strong activation of Erk proteins or of Akt. Constitutive activation of STAT5 in 32D/Flt3-ITD To analyze STAT5 activation, we first used an antibody specific for STAT5a/b phosphorylated on tyrosine 694. Flt3-WT induced very weak phosphorylation of STAT5 after FL stimulation (Figure 8A) However, the ITD mutants induced strong constitutive phosphorylation of STAT5. To confirm the activation of STAT5 in a different assay system, we performed electrophoretic mobility shift assays using a STAT5-specific probe (Figure 8B). On stimulation with IL-3, both WT and ITD mutants showed similar DNA binding activity of STAT5. Only 32D/Flt3-ITD cells showed constitutive DNA binding activity, and even FL stimulation could not induce STAT5 DNA binding in 32D/Flt3-WT. The DNA binding activity of 32D/Flt3-ITD was confirmed to be STAT5 by supershift analyses with an anti-STAT5 antibody.
Effects of dominant negative Ras and STAT5 on ITD-induced colony formation To examine the consequences of STAT5 activation in 32D/Flt3-ITD, we analyzed the effects of dnSTAT5 on the colony formation of 32D/Flt3-ITD. After transfection of STAT5Y694F (dnSTAT5), the colony formation of 32D/Flt3-ITD1 was reduced by approximately 50%, compared with the transfection of a control vector (Figure 9A).
As shown in Figure 7, we observed weak MAPK activation by some of the ITD mutations in the absence of exogenously added Flt3 ligand. Also, inactivation of STAT5 did not abrogate the Flt3-ITD-induced colony growth completely. Therefore, we were interested in examining the relative role of the Ras/MAPK pathway for the function of the ITD mutations. We used the Lac-inducible system, in which the expression of dnRas (Ras S17N) was repressed by the Lac repressor. Repression was relieved by IPTG, leading to highly inducible expression of the dominant negative construct, as shown in Figure 9B. The colony formation induced by the ITD forms of Flt3 was strongly inhibited by dnRas induction, as shown in Figure 9B for ITD1. Similar results were obtained with ITD2 (data not shown), which had caused considerable constitutive activation of MAPK in the pErk blot (Figure 7). IPTG alone did not show any effect on cell lines transfected with the empty control vector.
In this report, we demonstrate that internal tandem repeat mutations of Flt3, which are readily detectable in about 20% of cases with AML18,30 and which indicate a poor prognosis of the disease,21-23 elicit transforming ability in the factor-dependent murine myeloid cell line 32D. Our analyses of the autophosphorylation pattern of the Flt3 mutations indicate that ITD mutations cause constitutive autophosphorylation of Flt3. We also demonstrate that the observed activation of Flt3 is sufficient to mediate proliferation and survival in the absence of ligand, even if the mature form of the receptor is only marginally autophosphorylated. Furthermore, the mutant receptors alter cellular behavior in a way not seen with ligand-activated wild-type Flt3. The mechanism of Flt3 activation by mutation in the juxtamembrane domain remains elusive. Constitutive activation of the close relative Kit by point mutations and small deletions in the juxtamembrane region has been studied extensively,31,32 showing that ligand-independent dimerization occurs in the cellular membrane. Mutations in the kinase domain of Kit cause intracellular dimerization and activation of the receptor.33 Recently, it has been reported that in the Kit juxtamembrane domain, methionine 552 to isoleucine 563 form a putative alpha-helical conformation, and that substitution of any of 4 different amino acids sitting along the hydrophobic side of the alpha-helix leads to receptor activation.34 We also were interested in juxtamembrane deletion mutants of Flt3 and constructed a deletion mutant, in which all 4 potential tyrosine autophosphorylation sites were eliminated. This mutant also showed ligand-independent receptor autophosphorylation, proliferation, and apoptosis resistance, suggesting that the conformational change of the juxtamembrane domain, due to either addition or deletion, leads to receptor activation and similar biologic effects (M.M., R.F., H.S., manuscript in preparation). Taken together, ITD mutations in the Flt3 juxtamembrane region seem to disturb the conformation of a tertiary structure, which normally exerts inhibitory control over unoccupied receptor tyrosine kinases. The ITD mutations significantly shorten the latency period, which is required for Flt3 to transform 32D cells into a fully oncogenic cell line in vivo. The low, but detectable transforming ability of induced wild-type Flt3 expression is in accordance with the ability of c-kit to induce leukemogenesis in 32D cells, with a long latency period.35 The additional effect of the ITD mutations most likely depends on the ligand-independent activation of Flt3. We found that all types of Flt3-ITD do not only confer constitutive activation of the major biologic functions of Flt3, but also induce additional functions in 32D cells. Of particular interest is the ability of ITD mutations to support clonal growth. The surprising difference between activated wild-type Flt3 and the ITD mutations could be attributable to a complementary signal necessary for the Flt3 response in semisolid media, but not in suspension culture. The ITD mutations obviously bypass the necessity for this. They seem to provide an intracellular signal different from activated wild-type receptors. We analyzed intermediates of several signaling pathways and could indeed find qualitative differences in the signals provided by wild-type and ITD receptors. Our results show that Flt3 activates Akt, which has been shown to be an enzyme regulated by phosphoinositide products of PI3 kinase. The importance of PI3 kinase signaling for Flt3-mediated proliferation and survival is controversial, because the binding of the enzyme to Flt3 has not been demonstrated with certainty. On the other hand, it has been recently shown that Flt3 activates PI3 kinase by alternative mechanisms.36,37 Thus, our results showing the activation of Akt by wild-type Flt3 are in line with the latter experiments. We and others have shown that the Ras/Raf/MAP kinase pathways are involved in proliferation and survival induced by ligand-activated wild-type Flt3.38-40 Although the ITD mutants induced autonomous proliferation and survival of 32D cells, constitutive activation of MAP kinase and Akt was only weak in most of the ITD-containing cell lines. In contrast, STAT5 is strongly and constitutively activated by all ITD mutations tested, whereas STAT5 phosphorylation by wild-type Flt3 is very weak and does not translate into its enhanced DNA binding after Flt3 stimulation. After the initial submission of this paper, the constitutive activation of STAT5 by ITD mutations and the lack of STAT5 activation by wild-type Flt3 has been independently described by others,41 confirming our results of differential signaling between wild-type and ITD-mutated Flt3. Our results provide the first evidence for the relative importance of
the different signaling pathways for Flt3/ITD function: both Ras and
STAT5 are important for Flt3/ITD-induced colony formation of 32D cells.
Transfection of dominant negative STAT5 does not inhibit colony growth
completely. One explanation could be that other signaling pathways,
possibly other STAT proteins, can substitute for STAT5. The relative
importance of Ras-dependent signaling pathways is unexpected, given the
low constitutive activation of MAPK by the Flt3 mutations. On the other
hand, wild-type Flt3 strongly activates the Ras/MAPK pathway, but does
not induce colony formation (Figures 4 and 7). Obviously, basic
activity of the Ras/MAPK pathway is a necessary, but not sufficient,
condition for 32D cells to grow in colonies. In recent reports, it was
shown that STAT proteins are coactivated by Ras-dependent signals:
expression of dominant negative Ras in a T-cell line inhibited
IL-2-induced STAT5 activity on a In conclusion, we provide evidence that frequently occurring Flt3 mutations from patients with AML are leukemogenic in 32D cells. The leukemogenic effect is based on several intracellular signals of these receptor mutants, which involve the basal Ras activation and the constitutive activation of STAT5. Future work will have to elucidate these signaling pathways in still more detail. This will hopefully result in a more sophisticated picture of how proliferation and survival of leukemic blasts are regulated and how they may be subjected to specific therapeutic interventions.
We thank Dr I. Lemischka for providing the murine Flt3 cDNA, and Dr F. Rosenthal for providing the 32D cells. We thank Astrid Kolkmeyer, Corinna Dördelmann, and Steffi Wetzlich for their excellent technical assistance.
Submitted December 28, 1999; accepted August 1, 2000.
Supported by grant Se 600/2-2 from the Deutsche Forschungsgemeinschaft and Se 119804 and KR 229838 from the IMF Münster.
This paper represents partial fulfillment of the requirements for the PhD program of R.F. and for the MD program of R.S. and C.S. at the University of Münster.
M.M. and R.F. contributed equally to this 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: Hubert Serve or Wolfgang E. Berdel, Department of Medicine/ Hematology and Oncology, Medizinische Universitätsklinik Münster, Albert-Schweitzer-Strasse 33, 48129 Münster, Germany; e-mail: serve{at}uni-muenster.de.
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C. Reindl, K. Bagrintseva, S. Vempati, S. Schnittger, J. W. Ellwart, K. Wenig, K.-P. Hopfner, W. Hiddemann, and K. Spiekermann Point mutations in the juxtamembrane domain of FLT3 define a new class of activating mutations in AML Blood, May 1, 2006; 107(9): 3700 - 3707. [Abstract] [Full Text] [PDF]
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B. F. Goemans, C. M. Zwaan, A. Harlow, A. H. Loonen, B. E. S. Gibson, K. Hahlen, D. Reinhardt, U. Creutzig, M. C. Heinrich, and G. J. L. Kaspers In vitro profiling of the sensitivity of pediatric leukemia cells to tipifarnib: identification of T-cell ALL and FAB M5 AML as the most sensitive subsets Blood, November 15, 2005; 106(10): 3532 - 3537. [Abstract] [Full Text] [PDF]
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C. Choudhary, J. Schwable, C. Brandts, L. Tickenbrock, B. Sargin, T. Kindler, T. Fischer, W. E. Berdel, C. Muller-Tidow, and H. Serve AML-associated Flt3 kinase domain mutations show signal transduction differences compared with Flt3 ITD mutations Blood, July 1, 2005; 106(1): 265 - 273. [Abstract] [Full Text] [PDF]
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R. Grundler, C. Miething, C. Thiede, C. Peschel, and J. Duyster FLT3-ITD and tyrosine kinase domain mutants induce 2 distinct phenotypes in a murine bone marrow transplantation model Blood, June 15, 2005; 105(12): 4792 - 4799. [Abstract] [Full Text] [PDF]
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D.-E. Schmidt-Arras, A. Bohmer, B. Markova, C. Choudhary, H. Serve, and F.-D. Bohmer Tyrosine Phosphorylation Regulates Maturation of Receptor Tyrosine Kinases Mol. Cell. Biol., May 1, 2005; 25(9): 3690 - 3703. [Abstract] [Full Text] [PDF]
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T. Illmer, C. Thiede, A. Fredersdorf, S. Stadler, A. Neubauer, G. Ehninger, and M. Schaich Activation of the RAS Pathway Is Predictive for a Chemosensitive Phenotype of Acute Myelogenous Leukemia Blasts Clin. Cancer Res., May 1, 2005; 11(9): 3217 - 3224. [Abstract] [Full Text] [PDF]
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K. Bagrintseva, S. Geisenhof, R. Kern, S. Eichenlaub, C. Reindl, J. W. Ellwart, W. Hiddemann, and K. Spiekermann FLT3-ITD-TKD dual mutants associated with AML confer resistance to FLT3 PTK inhibitors and cytotoxic agents by overexpression of Bcl-x(L) Blood, May 1, 2005; 105(9): 3679 - 3685. [Abstract] [Full Text] [PDF]
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L. Tickenbrock, J. Schwable, M. Wiedehage, B. Steffen, B. Sargin, C. Choudhary, C. Brandts, W. E. Berdel, C. Muller-Tidow, and H. Serve Flt3 tandem duplication mutations cooperate with Wnt signaling in leukemic signal transduction Blood, May 1, 2005; 105(9): 3699 - 3706. [Abstract] [Full Text] [PDF]
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S. Fukuda, H. E. Broxmeyer, and L. M. Pelus Flt3 ligand and the Flt3 receptor regulate hematopoietic cell migration by modulating the SDF-1{alpha}(CXCL12)/CXCR4 axis Blood, April 15, 2005; 105(8): 3117 - 3126. [Abstract] [Full Text] [PDF]
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J. Schwable, C. Choudhary, C. Thiede, L. Tickenbrock, B. Sargin, C. Steur, M. Rehage, A. Rudat, C. Brandts, W. E. Berdel, et al. RGS2 is an important target gene of Flt3-ITD mutations in AML and functions in myeloid differentiation and leukemic transformation Blood, March 1, 2005; 105(5): 2107 - 2114. [Abstract] [Full Text] [PDF]
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 P. Chen, M. Levis, P. Brown, K.-T. Kim, J. Allebach, and D. Small FLT3/ITD Mutation Signaling Includes Suppression of SHP-1 J. Biol. Chem., February 18, 2005; 280(7): 5361 - 5369. [Abstract] [Full Text] [PDF]
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K.-T. Kim, K. Baird, J.-Y. Ahn, P. Meltzer, M. Lilly, M. Levis, and D. Small Pim-1 is up-regulated by constitutively activated FLT3 and plays a role in FLT3-mediated cell survival Blood, February 15, 2005; 105(4): 1759 - 1767. [Abstract] [Full Text] [PDF]
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W. Fiedler, H. Serve, H. Dohner, M. Schwittay, O. G. Ottmann, A.-M. O'Farrell, C. L. Bello, R. Allred, W. C. Manning, J. M. Cherrington, et al. A phase 1 study of SU11248 in the treatment of patients with refractory or resistant acute myeloid leukemia (AML) or not amenable to conventional therapy for the disease Blood, February 1, 2005; 105(3): 986 - 993. [Abstract] [Full Text] [PDF]
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P. Brown, M. Levis, S. Shurtleff, D. Campana, J. Downing, and D. Small FLT3 inhibition selectively kills childhood acute lymphoblastic leukemia cells with high levels of FLT3 expression Blood, January 15, 2005; 105(2): 812 - 820. [Abstract] [Full Text] [PDF]
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M. Wadleigh, D. J. DeAngelo, J. D. Griffin, and R. M. Stone After chronic myelogenous leukemia: tyrosine kinase inhibitors in other hematologic malignancies Blood, January 1, 2005; 105(1): 22 - 30. [Abstract] [Full Text] [PDF]
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K. Y. Chung, G. Morrone, J. J. Schuringa, B. Wong, D. C. Dorn, and M. A. S. Moore Enforced expression of an Flt3 internal tandem duplication in human CD34+ cells confers properties of self-renewal and enhanced erythropoiesis Blood, January 1, 2005; 105(1): 77 - 84. [Abstract] [Full Text] [PDF]
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T. Kindler, F. Breitenbuecher, S. Kasper, E. Estey, F. Giles, E. Feldman, G. Ehninger, G. Schiller, V. Klimek, S. D. Nimer, et al. Identification of a novel activating mutation (Y842C) within the activation loop of FLT3 in patients with acute myeloid leukemia (AML) Blood, January 1, 2005; 105(1): 335 - 340. [Abstract] [Full Text] [PDF]
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K. W. H. Yee, M. Schittenhelm, A.-M. O'Farrell, A. R. Town, L. McGreevey, T. Bainbridge, J. M. Cherrington, and M. C. Heinrich Synergistic effect of SU11248 with cytarabine or daunorubicin on FLT3 ITD-positive leukemic cells Blood, December 15, 2004; 104(13): 4202 - 4209. [Abstract] [Full Text] [PDF]
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N. J. Lacayo, S. Meshinchi, P. Kinnunen, R. Yu, Y. Wang, C. M. Stuber, L. Douglas, R. Wahab, D. L. Becton, H. Weinstein, et al. Gene expression profiles at diagnosis in de novo childhood AML patients identify FLT3 mutations with good clinical outcomes Blood, November 1, 2004; 104(9): 2646 - 2654. [Abstract] [Full Text] [PDF]
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J. J. Clark, J. Cools, D. P. Curley, J.-C. Yu, N. A. Lokker, N. A. Giese, and D. G. Gilliland Variable sensitivity of FLT3 activation loop mutations to the small molecule tyrosine kinase inhibitor MLN518 Blood, November 1, 2004; 104(9): 2867 - 2872. [Abstract] [Full Text] [PDF]
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I. J. Griswold, L. J. Shen, P. La Rosee, S. Demehri, M. C. Heinrich, R. M. Braziel, L. McGreevey, A. D. Haley, N. Giese, B. J. Druker, et al. Effects of MLN518, a dual FLT3 and KIT inhibitor, on normal and malignant hematopoiesis Blood, November 1, 2004; 104(9): 2912 - 2918. [Abstract] [Full Text] [PDF]
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Y. Li, H. Li, M.-N. Wang, D. Lu, R. Bassi, Y. Wu, H. Zhang, P. Balderes, D. L. Ludwig, B. Pytowski, et al. Suppression of leukemia expressing wild-type or ITD-mutant FLT3 receptor by a fully human anti-FLT3 neutralizing antibody Blood, August 15, 2004; 104(4): 1137 - 1144. [Abstract] [Full Text] [PDF]
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S. Takahashi, M. J. McConnell, H. Harigae, M. Kaku, T. Sasaki, A. M. Melnick, and J. D. Licht The Flt3 internal tandem duplication mutant inhibits the function of transcriptional repressors by blocking interactions with SMRT Blood, June 15, 2004; 103(12): 4650 - 4658. [Abstract] [Full Text] [PDF]
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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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C. Muller-Tidow, B. Steffen, T. Cauvet, L. Tickenbrock, P. Ji, S. Diederichs, B. Sargin, G. Kohler, M. Stelljes, E. Puccetti, et al. Translocation Products in Acute Myeloid Leukemia Activate the Wnt Signaling Pathway in Hematopoietic Cells Mol. Cell. Biol., April 1, 2004; 24(7): 2890 - 2904. [Abstract] [Full Text] [PDF]
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K. Bagrintseva, R. Schwab, T. M. Kohl, S. Schnittger, S. Eichenlaub, J. W. Ellwart, W. Hiddemann, and K. Spiekermann Mutations in the tyrosine kinase domain of FLT3 define a new molecular mechanism of acquired drug resistance to PTK inhibitors in FLT3-ITD-transformed hematopoietic cells Blood, March 15, 2004; 103(6): 2266 - 2275. [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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L.-Y. Shih, C.-F. Huang, J.-H. Wu, P.-N. Wang, T.-L. Lin, P. Dunn, M.-C. Chou, M.-C. Kuo, and C.-C. Tang Heterogeneous Patterns of FLT3 Asp835 Mutations in Relapsed de Novo Acute Myeloid Leukemia: A Comparative Analysis of 120 Paired Diagnostic and Relapse Bone Marrow Samples Clin. Cancer Res., February 15, 2004; 10(4): 1326 - 1332. [Abstract] [Full Text] [PDF]
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T. Taketani, T. Taki, K. Sugita, Y. Furuichi, E. Ishii, R. Hanada, M. Tsuchida, K. Sugita, K. Ida, and Y. Hayashi FLT3 mutations in the activation loop of tyrosine kinase domain are frequently found in infant ALL with MLL rearrangements and pediatric ALL with hyperdiploidy Blood, February 1, 2004; 103(3): 1085 - 1088. [Abstract] [Full Text] [PDF]
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 D. W. Sternberg and D. G. Gilliland The Role of Signal Transducer and Activator of Transcription Factors in Leukemogenesis J. Clin. Oncol., January 15, 2004; 22(2): 361 - 371. [Abstract] [Full Text] [PDF]
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C.-K. So, Y. Nie, Y. Song, G.-Y. Yang, S. Chen, C. Wei, L.-D. Wang, N. A. Doggett, and C. S. Yang Loss of Heterozygosity and Internal Tandem Duplication Mutations of the CBP Gene Are Frequent Events in Human Esophageal Squamous Cell Carcinoma Clin. Cancer Res., January 1, 2004; 10(1): 19 - 27. [Abstract] [Full Text] [PDF]
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J. E. Lancet and J. E. Karp Farnesyltransferase inhibitors in hematologic malignancies: new horizons in therapy Blood, December 1, 2003; 102(12): 3880 - 3889. [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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W. Fiedler, R. Mesters, H. Tinnefeld, S. Loges, P. Staib, U. Duhrsen, M. Flasshove, O. G. Ottmann, W. Jung, F. Cavalli, et al. A phase 2 clinical study of SU5416 in patients with refractory acute myeloid leukemia Blood, October 15, 2003; 102(8): 2763 - 2767. [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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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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S. Meshinchi, D. L. Stirewalt, T. A. Alonzo, Q. Zhang, D. A. Sweetser, W. G. Woods, I. D. Bernstein, R. J. Arceci, and J. P. Radich Activating mutations of RTK/ras signal transduction pathway in pediatric acute myeloid leukemia Blood, August 15, 2003; 102(4): 1474 - 1479. [Abstract] [Full Text] [PDF]
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C. R. Antonescu, G. Sommer, L. Sarran, S. J. Tschernyavsky, E. Riedel, J. M. Woodruff, M. Robson, R. Maki, M. F. Brennan, M. Ladanyi, et al. Association of KIT Exon 9 Mutations with Nongastric Primary Site and Aggressive Behavior: KIT Mutation Analysis and Clinical Correlates of 120 Gastrointestinal Stromal Tumors Clin. Cancer Res., August 1, 2003; 9(9): 3329 - 3337. [Abstract] [Full Text] [PDF]
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F. J. Giles, A. T. Stopeck, L. R. Silverman, J. E. Lancet, M. A. Cooper, A. L. Hannah, J. M. Cherrington, A.-M. O'Farrell, H. A. Yuen, S. G. Louie, et al. SU5416, a small molecule tyrosine kinase receptor inhibitor, has biologic activity in patients with refractory acute myeloid leukemia or myelodysplastic syndromes Blood, August 1, 2003; 102(3): 795 - 801. [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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K. Spiekermann, K. Bagrintseva, R. Schwab, K. Schmieja, and W. Hiddemann Overexpression and Constitutive Activation of FLT3 Induces STAT5 Activation in Primary Acute Myeloid Leukemia Blast Cells Clin. Cancer Res., June 1, 2003; 9(6): 2140 - 2150. [Abstract] [Full Text] [PDF]
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P. M. Chan, S. Ilangumaran, J. La Rose, A. Chakrabartty, and R. Rottapel Autoinhibition of the Kit Receptor Tyrosine Kinase by the Cytosolic Juxtamembrane Region Mol. Cell. Biol., May 1, 2003; 23(9): 3067 - 3078. [Abstract] [Full Text] [PDF]
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A.-M. O'Farrell, T. J. Abrams, H. A. Yuen, T. J. Ngai, S. G. Louie, K. W. H. Yee, L. M. Wong, W. Hong, L. B. Lee, A. Town, et al. SU11248 is a novel FLT3 tyrosine kinase inhibitor with potent activity in vitro and in vivo Blood, May 1, 2003; 101(9): 3597 - 3605. [Abstract] [Full Text] [PDF]
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M. Benekli, M. R. Baer, H. Baumann, and M. Wetzler Signal transducer and activator of transcription proteins in leukemias Blood, April 15, 2003; 101(8): 2940 - 2954. [Abstract] [Full Text] [PDF]
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M. Mizuki, J. Schwable, C. Steur, C. Choudhary, S. Agrawal, B. Sargin, B. Steffen, I. Matsumura, Y. Kanakura, F. D. Bohmer, et al. Suppression of myeloid transcription factors and induction of STAT response genes by AML-specific Flt3 mutations Blood, April 15, 2003; 101(8): 3164 - 3173. [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. D. Bohmer, L. Karagyozov, A. Uecker, H. Serve, A. Botzki, S. Mahboobi, and S. Dove A Single Amino Acid Exchange Inverts Susceptibility of Related Receptor Tyrosine Kinases for the ATP Site Inhibitor STI-571 J. Biol. Chem., February 7, 2003; 278(7): 5148 - 5155. [Abstract] [Full Text] [PDF]
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S. Frohling, R. F. Schlenk, J. Breitruck, A. Benner, S. Kreitmeier, K. Tobis, H. Dohner, and K. Dohner Prognostic significance of activating FLT3 mutations in younger adults (16 to 60 years) with acute myeloid leukemia and normal cytogenetics: a study of the AML Study Group Ulm Blood, December 15, 2002; 100(13): 4372 - 4380. [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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R. Zheng, A. D. Friedman, and D. Small Targeted inhibition of FLT3 overcomes the block to myeloid differentiation in 32Dcl3 cells caused by expression of FLT3/ITD mutations Blood, December 1, 2002; 100(12): 4154 - 4161. [Abstract] [Full Text] [PDF]
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R. L. Darley, L. Pearn, N. Omidvar, M. Sweeney, J. Fisher, S. Phillips, T. Hoy, and A. K. Burnett Protein kinase C mediates mutant N-Ras-induced developmental abnormalities in normal human erythroid cells Blood, December 1, 2002; 100(12): 4185 - 4192. [Abstract] [Full Text] [PDF]
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K. Spiekermann, K. Bagrintseva, C. Schoch, T. Haferlach, W. Hiddemann, and S. Schnittger A new and recurrent activating length mutation in exon 20 of the FLT3 gene in acute myeloid leukemia Blood, October 16, 2002; 100(9): 3423 - 3425. [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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L.-Y. Shih, C.-F. Huang, J.-H. Wu, T.-L. Lin, P. Dunn, P.-N. Wang, M.-C. Kuo, C.-L. Lai, and H.-C. Hsu Internal tandem duplication of FLT3 in relapsed acute myeloid leukemia: a comparative analysis of bone marrow samples from 108 adult patients at diagnosis and relapse Blood, September 18, 2002; 100(7): 2387 - 2392. [Abstract] [Full Text] [PDF]
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G. W. Reuther, Q. T. Lambert, J. F. Rebhun, M. A. Caligiuri, L. A. Quilliam, and C. J. Der RasGRP4 Is a Novel Ras Activator Isolated from Acute Myeloid Leukemia J. Biol. Chem., August 16, 2002; 277(34): 30508 - 30514. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
