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NEOPLASIA
From the Division of Hematology/Oncology, Department of
Pathology, Brigham and Women's Hospital, and the Howard Hughes Medical
Institute, Harvard Medical School, Boston, MA; and Department of
Pathology, Emory University, Atlanta, GA.
FLT3 receptor tyrosine kinase is expressed on lymphoid and myeloid
progenitors in the hematopoietic system. Activating mutations in FLT3
have been identified in approximately 30% of patients with acute
myelogenous leukemia, making it one of the most common mutations
observed in this disease. Frequently, the mutation is an in-frame
internal tandem duplication (ITD) in the juxtamembrane region that
results in constitutive activation of FLT3, and confers interleukin-3
(IL-3)-independent growth to Ba/F3 and 32D cells. FLT3-ITD mutants
were cloned from primary human leukemia samples and assayed for
transformation of primary hematopoietic cells using a murine bone
marrow transplantation assay. FLT3-ITDs induced an oligoclonal
myeloproliferative disorder in mice, characterized by splenomegaly and
leukocytosis. The myeloproliferative phenotype, which was associated
with extramedullary hematopoiesis in the spleen and liver, was
confirmed by histopathologic and flow cytometric analysis. The disease
latency of 40 to 60 days with FLT3-ITDs contrasted with wild-type FLT3
and enhanced green fluorescent protein (EGFP) controls, which did not
develop hematologic disease (> 200 days). These results demonstrate
that FLT3-ITD mutant proteins are sufficient to induce a
myeloproliferative disorder, but are insufficient to recapitulate the
AML phenotype observed in humans. Additional mutations that impair
hematopoietic differentiation may be required for the development of
FLT3-ITD-associated acute myeloid leukemias. This model system should
be useful to assess the contribution of additional cooperating
mutations and to evaluate specific FLT3 inhibitors in vivo.
(Blood. 2002;99:310-318) FLT3 (FMS-like receptor tyrosine kinase) also
referred to as fetal liver kinase 2 (FLK-2) or stem cell tyrosine
kinase 1 (STK-1) was originally described as a tyrosine kinase receptor
with strong sequence similarity to FMS, KIT, and platelet-derived
growth factor receptor (PDGFR).1,2 These proteins are all
members of the receptor tyrosine kinase type (RTK) III subfamily based
on a common domain structure including an interrupted kinase domain
(for review, see Rosnet and Birnbaum3). FLT3 is expressed
by cells found in the hematopoietic stem cell compartment
(Sca1+, Lin It is therefore interesting that activating mutations in FLT3 have been
found in approximately 25% to 30% of patients with acute myelogenous
leukemia (AML).9,10 The majority of FLT3 mutations are
internal tandem duplications (ITDs) in the juxtamembrane domain encoded
by exon 11, and were first reported in patients with AML in
1996.11 This mutation appears to confer a poor prognosis in several studies reported to date.12-14 All ITD repeats
were in-frame, but varied significantly in length. No such ITD
abnormalities were detected in remission samples, indicating that these
were acquired mutations.11 Subsequent studies have
confirmed the presence of the juxtamembrane ITDs in human leukemias,
with an overall incidence of approximately 20% in AML. In addition,
substitution mutations in the FLT3 kinase domain at Asp835 have
recently been reported in approximately 7% of patients with
AML.10 Both the FLT3-ITD and FLT3-Asp835 mutations
result in constitutive activation of the receptor and are associated
with FLT3 autophosphorylation and phosphorylation of downstream targets
such as STAT5 and mitogen-activated protein (MAP)
kinase.15-18 FLT3-ITD mutations are also found but at much
lower frequencies in related diseases such as myelodysplastic syndrome
(< 5%)9,10,19 and have rarely been identified in acute
lymphocytic leukemia (ALL) unless they are biphenotypic.20
Constitutive activation of RTKs is not unprecedented in leukemias;
however, the vast proportion of activated RTKs studied thus far are
associated with chronic myelogenous leukemia (CML) phenotypes and occur
as a result of a chromosomal translocation. Examples include the
BCR/ABL, TEL/PDGF Activating mutations in FLT3 differ from tyrosine kinase fusions
associated with the CML phenotypes. FLT3-ITD and activating loop
mutants are localized to the plasma membrane as opposed to the
cytoplasmic localization of tyrosine kinase fusions. The residual tyrosine kinase allele in the tyrosine kinase fusions
associated with chromosomal translocations is typically either
expressed at low levels or is localized to a different subcellular
compartment than the tyrosine kinase fusion. In contrast, both the
wild-type and FLT3-ITD alleles are expressed at comparable levels and
are localized to the plasma membrane where they may interact. Perhaps most importantly, activating mutations in FLT3 are most frequently associated with AML rather than CML phenotypes.9
Furthermore, FLT3-ITDs are most often observed in AML patients negative
for other cytogenetic abnormalities.14 Taken together,
these data suggest that FLT3-ITDs may play a critical role in the
development of AML.
We have investigated the transforming properties of the FLT3-ITD in
primary hematopoietic cells using a murine BMT assay. We report that
the FLT3-ITDs cloned from primary human myeloid leukemias confer
factor-independent growth to Ba/F3 cells and induce a
myeloproliferative phenotype similar to that observed with the
translocation-associated tyrosine kinase fusions. These data have
important implications for the contribution of the FLT3-ITD to the
pathogenesis of human AML. Furthermore, this model should provide a
useful system for analysis of cooperativity of FLT3 with other
mutations and gene rearrangements associated with human leukemia and
for testing of FLT3-specific low molecular weight inhibitors.
Cloning and vector construction
All FLT3 mutations were confirmed by sequencing and the entire cDNA was
subcloned from pGEM to MSCV-EB neo42 or MSCV2.2 GFP
(kindly provided by W. Pear, University of Pennsylvania)43 into the HpaI site in each case.
Cell culture and virus production
Mouse strains and BMT BALB/c mice were purchased from Taconic (Germantown, NY). BMT assays were carried out as described previously.32,44 Briefly, 4- to 6-week-old male donor mice were primed with intraperitoneal injection of 5'-fluorouracil (150 mg/kg, Sigma, St Louis, MO) and subsequently killed after 6 days by CO2 asphyxiation. Bone marrow was flushed from femurs and tibias, and red blood cells were lysed (Red Blood Cell Lysis, RBCL buffer, Sigma). Cells were cultured overnight with IL-3 (6 ng/mL, R & D Systems), IL-6 (10 ng/mL, R & D systems), and stem cell factor (10 ng/mL, Peprotech, Rocky Hill, NJ) in RPMI with 10% FCS (transplant medium). Cells were transduced by 2 rounds of spin-infection, at 24 hours and 48 hours after harvesting. Centrifugation of 1 mL viral supernatant and 4 × 106 cells in 3 mL transplant media containing 5 µg/mL Polybrene and 7.5 mM HEPES buffer was carried out for 90 minutes at 1800g. Cells were washed in PBS, resuspended in Hanks balanced salt solution (Life Technologies) and injected (5 × 105 cells/0.5 mL) into the lateral tail vein of lethally irradiated (2 × 450 cGy) female recipient mice. Mice were housed in microisolator cages with autoclaved chow and acidified water.Histopathology Murine tissues were fixed for at least 72 hours in 10% neutral buffered formalin (Sigma), dehydrated in alcohol, cleared in xylene, and infiltrated with paraffin on an automated processor (Leica, Bannockburn, IL). The tissue sections (4 µm thick) from paraffin-embedded tissue blocks were placed on charged slides and deparaffinized in xylene, rehydrated through graded alcohol solutions, and stained with hematoxylin and eosin. To visualize the reticulin fibers, rehydrated sections were sequentially treated with potassium permanganate (0.5%), potassium metabisulfite (2%), ferric ammonium sulfate (2%), ammonical silver solution, formalin (10%), gold chloride (0.2%), potassium metasulfite (2%), sodium thiosulfate (2%), and counterstained with methyl green dye according to the published method.45Flow cytometric immunophenotyping and fluorescence-activated cell sorting Single-cell suspensions of spleen, bone marrow, and blood were prepared as described previously.32,44 Briefly, red blood cells were lysed in RBCL buffer (Sigma) for 5 minutes at room temperature and frozen in 90% fetal bovine serum and 10% dimethyl sulfoxide. Prior to analysis, cells were washed in PBS/0.1% NaN3/0.1% bovine serum albumin (BSA) and preincubated for 20 minutes on ice with supernatant from the 2.4G2 hybridoma cell line (anti-CD16/CD32; American Type Culture Collection, Rockville, MD) to block nonspecific Fc receptor-mediated binding. Cells were stained for 20 minutes on ice with monoclonal antibodies, washed in staining buffer, and stained with secondary antibodies where necessary. Antibodies used were allophycocyanin (APC)-conjugated Gr-1 and CD4; phycoerythrin (PE)-conjugated Mac 1, Thy 1.2, and CD8; biotin-conjugated CD19; and APC-conjugated streptavidin. All antibodies were purchased from Pharmingen, San Diego, CA apart from APC-CD4 and APC streptavidin, which were purchased from Caltag, Burlingame, CA. Flow cytometric analysis was carried out using a 4-color FACSort cytometer (Becton Dickinson, Mountain View, CA) and analyzed using CellQuest software.Fluorescence-activated cell sorting (FACS) was performed on a Coulter
Epics Altra at approximately 15 000 cells/s. A single cell suspension
from the spleen was resuspended in 1% BSA in PBS at 107
cells/mL and sorted into EGFP+ and EGFP Southern analysis DNA was prepared using a PUREGENE DNA isolation kit according to the manufacturer's protocol (Gentra Systems, Minneapolis, MN). After enzymatic digestion of 20 µg DNA with either EcoRI or XhoI and electrophoretic separation, the genomic DNA was blotted to Hybond-N+ nylon membranes (Amersham, Arlington Heights, IL) by capillary transfer.46 The green fluorescent protein (GFP) probe was a 750-bp fragment isolated using a NcoI/SalI digest from MSCV-EGFP. The 500-bp granzyme A probe was a kind gift from J. Pollock and T. Ley, Washington University. Probes were labeled with 32P by random priming using Radprime (Life Technologies), and Southern hybridization was performed as described.32Western analysis The Ba/F3 cells were washed 3 times in PBS and lysed in 10 mM Tris pH 7.5, 130 mM NaCl, 1% Triton X-100, 10 mM NaF, 10 mM sodium phosphate, 10 mM sodium pyrophosphate, 10 mM EDTA, 1 mM sodium vanadate, and protease inhibitor cocktail tablets (Roche-Boehringer, Indianapolis, IN). Total cell lysate (150 µg) was separated on a 7.5% gel and Western analysis was performed as described previously44 using a 1:200 dilution of anti-FLT3 SC-479 (Santa Cruz, Santa Cruz, CA) as the primary antibody, followed by horseradish peroxidase-conjugated secondary antibody (Amersham Life Science) and visualization by enhanced chemoluminescence (Amersham Life Science).
Cloning of FLT3-ITD mutations from primary human AML cells The DNA samples from peripheral blood of 12 patients with AML were screened by PCR (see "Materials and methods") for length mutations in the juxtamembrane region, encoded by exon 11. Four independent FLT3-ITD mutants were cloned, which had a duplicated sequence of between 7 and 25 amino acids (Figure 1). The length and position of the duplication varied between samples and differed from other published FLT3-ITD mutants.9,11,13,15 No abnormalities were observed in samples from 54 healthy individuals, consistent with published data.9 Each FLT3-ITD mutant, including a previously reported FLT3-ITD mutant (Npos is Mut117) positive control, and the wild-type FLT3 gene were subcloned into a MSCV murine ecotropic retrovirus that expressed either the neomycin selectable marker or the EGFP under the control of an internal ribosome entry site (IRES).
Expression and transforming properties of FLT3 and FLT3 mutants in Ba/F3 cells To confirm that the FLT3-ITD mutants we cloned had similar properties to those previously reported, Ba/F3 cells were stably transduced with MSCV-neo retrovirus containing either the wild-type FLT3 or FLT-ITD cDNAs. Pools of transduced cells were maintained in IL-3 and selected with G418 for 2 weeks prior to analysis of factor-independent growth. Each of the FLT3-ITD mutants including the previously reported positive control Npos,17 conferred IL-3 factor-independent growth to Ba/F3 cells. Parental cells or cells stably transduced with the wild-type FLT3 died after IL-3 deprivation (Figure 2A). There were no significant differences in growth rates in replicate experiments over the time course of this assay. A point mutation in the activation loop that inhibits kinase activity of wild-type FLT347 abrogated transforming activity of FLT3-ITD when mutated in the context of W51 (data not shown). Western blot analysis confirmed that the cell pools expressed comparable levels of FLT3 and each FLT3-ITD mutant at the expected molecular weights of approximately 130 and 155 kd (Figure 2B), and that parental Ba/F3 cells did not express FLT3 (Figure 2B). The FLT3 wild-type protein was identified by immunoprecipitation analysis as 2 distinct bands of approximately 130 and 155 kd, respectively. It has been previously demonstrated by expression in COS-1 and COS-7 cells that these 2 bands are generated from a predicted 110-kd protein via posttranslational glycosylation.15,47
Two independent FLT3-ITD mutants, but not wild-type FLT3, induce a lethal myeloproliferative disease in a murine BMT assay Donor mice were treated with 5-fluorouracil to increase the number of cycling stem cells for retroviral transduction, as described in "Materials and methods." Equivalent titers of FLT3-ITD or FLT3 were then transduced into primary mouse bone marrow cells using the MSCV-EGFP retrovirus. Transduced bone marrow for each construct was also analyzed by flow cytometry to confirm EGFP expression in a comparable fraction of the transplanted cells. Transduced bone marrow cells were transferred by injection into the lateral tail vein of lethally irradiated syngeneic recipient mice. Mice receiving transplants of cells transduced with retrovirus containing the wild-type FLT3 or the MSCV vector expressing EGFP engrafted normally and had a normal survival with a follow-up of more than 200 days (Figure 3). In contrast, animals transduced with cells containing either the FLT3-ITD/51 or FLT3-ITD/78 developed a lethal hematopoietic disease with a median latency of approximately 40 to 50 days (Figure 3 and Table 1).
Animals receiving transplants of FLT3-ITD developed marked
leukocytosis, with a white blood cell (WBC) count ranging from 28 to
145 × 103/µL (Table 1). Differential counts analyzed
by the scatter property of peripheral blood of several animals
indicated that the increase in WBCs was due almost entirely to
neutrophils, which were increased from between 13.7% to 21.8% in 3 controls to 75.7% to 87% in 3 FLT3-ITD-diseased animals analyzed.
Animals with FLT3-ITD-induced disease appeared to have a higher
proportion of mature neutrophils with fewer immature myeloid cells
observed than in animals with myeloproliferative disease associated
with other tyrosine kinase fusions.28 The absolute numbers
of other blood cells were unchanged and no abnormality in erythrocyte
count or composition was observed. Wright-Giemsa stains of peripheral
blood demonstrated leukocytosis with myeloid lineage cells in all
stages of maturation, predominantly terminally differentiated
neutrophils (Figure 4A).
Gross pathologic examination demonstrated emaciation, ruffled fur, and
marked splenomegaly, with spleen weights ranging from 300 to 1000 mg in
FLT3-ITD/51 and 250 to 900 mg in FLT3-ITD/78 compared to 100 to 171 mg
in wild-type FLT3 or EGFP controls (Table 1). The slight increase in
spleen weight in the controls was typical for animals after irradiation
and BM transplantation. Bone marrow stained with hematoxylin-eosin
demonstrated hypercellularity with a marked predominance of maturing
myeloid lineage cells, consistent with a myeloproliferative disease
(Figure 4B). In addition, the bone marrow, as in other mouse models of
myeloproliferative disease,48 displayed a significant
degree of reticulin fibrosis as demonstrated by silver stain when
compared to control FLT3 mice (Figure 4E,F). A reduced yield of cells
was obtained from the marrow of FLT3-ITD animals compared to EGFP and
wild-type FLT3 controls. Splenic architecture was effaced by a marked
expansion of red pulp comprised of maturing myeloid cells (Figure 4C)
and scattered admixed megakaryocytes (not shown). Analysis of the liver
demonstrated a perivascular parenchymal infiltration comprised predominantly of maturing myeloid elements, with admixed erythroid and
megakaryocytic elements similar to that seen in the spleen (Figure 4D).
The lungs showed evidence of pulmonary hemorrhage, as observed in BMT
assays with other tyrosine kinase fusions such as with
TEL/PDGF Flow cytometric analysis of blood and spleen cells from FLT3-ITD mice
further confirmed the myeloproliferative phenotype. Several FLT3-ITD
animals were examined in detail showing 57% to 70% of cells in the
spleen (n = 3) and 70% to 90% of cells in the blood (n = 5) were
positive for the late myeloid markers Gr-1 (Ly 6-G) and Mac-1,
indicative of mature neutrophils, compared to 5% to 9% and 16% to
24% Gr-1, Mac-1 double-positive cells in spleen and blood of controls,
respectively. A representative set of samples of one EGFP control, one
wild-type FLT3, and one FLT3-ITD are presented in Figure
5. Gr-1+ cells were also
EGFP+, consistent with proviral integration in these cells.
In addition, significant proportions of Gr-1+ cells were
EGFP
No abnormalities were observed in T-cell (Figure 5A) or B-cell (Figure 5A, B) populations. Comparable results were obtained from immunophenotypic analysis of cells from the bone marrow (n = 4), with an increase in Gr-1+ cells from 54% on average in controls to 87% average in FLT3-ITD mice. Southern blot analysis of spleen, bone marrow, and blood
demonstrated proviral integration in all affected tissues. Data from the spleen are shown in Figure 6, where
the DNA was digested to excise the proviral DNA as a single band (3 kb
for MSCV-EGFP or 6 kb for the FLT3 series in MSCV-EGFP) or cleaved once
within the construct and randomly in the flanking DNA, according to the insertion position, which demonstrated the clonality. DNA integrity and
equal loading was demonstrated by reprobing the blot for an endogenous
gene, granzyme A (Figure 6, lower panel). Southern blot analysis of
control mice sacrificed at the same time confirmed transduction of bone
marrow cells with FLT3-ITD and MSCV-EGFP, although these animals did
not develop disease. Restriction digests designed to determine
clonality indicated that the myeloproliferative disease induced by the
FLT3-ITD was oligoclonal (Figure 6, upper panel).
This is the first demonstration that expression of FLT3-ITD, but not wild-type FLT3, has transforming properties in primary bone marrow cells. FLT3-ITD expression causes a myeloproliferative phenotype characterized by leukocytosis and comprised mainly of mature neutrophils. Splenomegaly with extramedullary hematopoiesis in the spleen and the liver are also consistently observed. Myeloproliferative disease induced by FLT3-ITD has a latency of 40 to 60 days and is oligoclonal. These findings demonstrate that FLT3-ITD is sufficient to induce a
myeloproliferative disease. However, although the FLT3-ITD is primarily
associated with AML in humans, it is not sufficient to induce an AML
phenotype in the murine BMT assay. These data suggest that FLT3-ITDs
may require additional cooperating mutations to generate the AML
phenotype. Evidence supporting this hypothesis includes clinical
observations that FLT3-ITD may coexist with virtually any known
chromosomal translocation associated with AML, including the t(8;21),
inv(16), and t(15;17) associated with the AML1/ETO,
CBF Initial analysis suggests that FLT3 and FLT3-ITD use the same signal
transduction pathways. On stimulation with FL, FLT3 activates signal
transduction pathways through engagement of SH2-containing proteins,
including PLC The mechanism by which the ITD activates the FLT3 tyrosine kinase is not understood. However, insertion of the duplicated residues may disrupt a kinase inhibitory domain, resulting in kinase activation. Several lines of evidence support this hypothesis. First, FLT3-ITD in-frame mutations in patients with AML may range in size from several to more than 40 amino acids and vary in position within the juxtamembrane domain. In addition, it has recently been demonstrated that deletion of several amino acid residues in the juxtamembrane domain of FLT3 also results in constitutive activation of FLT3.58 The observation that a spectrum of insertions or deletions in the JM domain serve to activate the kinase is most consistent with loss of function of a kinase inhibitory domain through disruption of the tertiary structure. Second, in-frame deletions in the juxtamembrane domain of c-KIT, a related type III receptor tyrosine kinase, results in constitutive activation of c-KIT in gastrointestinal stromal cell tumors and mastocytoses.63,64 Third, regulation of the FLT1 receptor tyrosine kinase through an autoinhibitory function of the JM domain has recently been reported.65 Collectively, these data support the hypothesis that disruption of an autoinhibitory motif in the JM domain, either by deletion or insertion, results constitutive activation of the FLT3 kinase. Structural analysis of FLT3 and related mutants will be required to confirm this hypothesis. An additional question regarding the mechanism of FLT3-ITD transformation relates to the involvement of the wild-type FLT3 in modulating the signal transduction by the mutant. It is plausible that wild-type FLT3 could potentiate or impair signal transduction by FLT3-ITD. The data from the FLT3-ITD murine BMT assay are consistent with previously reported data on the role of FL and FLT3 receptor in hematopoiesis. FL synergizes with other growth factors in colony assays to stimulate proliferation of hematopoietic progenitors and myeloid precursors but not erythroid precursors.66,67 Furthermore, mice that do not express the FLT3 receptor have reduced B lymphopoiesis.68 On transplantation, reduced T lymphopoiesis and myelopoiesis are also observed.68 Mice that lack FL have reduced numbers of myeloid and B-lymphoid progenitors and a deficiency in natural killer and dendritic cells.69 In addition, mice treated with exogenous FL accumulate progenitor cells in the peripheral blood.70 Overexpression of FL in a BMT assay induces a proliferative phenotype71 and can predispose animals to leukemia after a 5-month latency period where the malignant cells were all shown to express FLT3.72 Taken together these data suggest that the FLT3-FL signaling pathway has a unique role in expansion of stem and progenitor cells and sustained activation of FLT3 can lead to hyperproliferation of cells expressing the FLT3 receptor. Our findings confirm the role of FLT3 in cellular proliferation and support the hypothesis that FLT3-ITD mutations contribute to the pathogenesis of human leukemias. Each independent FLT3-ITD mutant tested was sufficient to induce a myeloproliferative disease in the murine BMT assay, a phenotype similar to that generated by the activated tyrosine kinases that result from chromosomal translocations.21-38,40,41 These data suggest that it may be worthwhile to investigate other myeloproliferative syndromes, such as myeloid metaplasia with myelofibrosis, polycythemia vera, and essential thrombocythemia, for the presence of activating mutations in FLT3. Finally, the recently reported successes in the therapy of chronic myelogenous leukemias associated with the BCR/ABL gene rearrangement with the ABL-specific kinase inhibitor STI57173,74 suggest that small molecule inhibitors of FLT3 may have therapeutic efficacy in AML.75 This murine model should also allow pharmacologic and therapeutic properties of small molecule inhibitors to be tested in vivo.
We thank L. Seaton for administrative assistance. We thank J. Daley and S. Lazo for expert cell sorting, and D. Cain and S. Amaral for technical support. We also thank E. Anastasiadou, A. Dash, D. Sternberg, and other members of the Gilliland and Tenen laboratories for valuable discussions.
Submitted May 7, 2001; accepted September 4, 2001.
Supported in part by National Institutes of Health grants CA66996 and DK50654. L.M.K. is an associate and D.G.G. is an associate investigator of the Howard Hughes Medical Institute.
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: D. Gary Gilliland, Harvard Institutes of Medicine, 4 Blackfan Cir, Rm 418, Boston, MA 02115; e-mail: gilliland{at}calvin.bwh.harvard.edu.
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Top
Abstract
Introduction
)
R, TEL/JAK2, HIP1/PDGF
80°C. Viral titers were estimated by transduction of Ba/F3 cells in
the presence of Polybrene (10 µg/mL) as described
previously.
fusion proteins,
respectively
, RAS-GTPase activating protein, the p85 subunit of
PI3K, SHC, GRB2, STAT5, VAV, FYN, and SRC, among
others.