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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 5 (March 1), 1999:
pp. 1707-1714
TEL/PDGF R Induces Hematologic Malignancies in Mice That
Respond to a Specific Tyrosine Kinase Inhibitor
By
Michael H. Tomasson,
Ifor R. Williams,
Robert Hasserjian,
Chirayu Udomsakdi,
Shannon M. McGrath,
Juerg Schwaller,
Brian Druker, and
D. Gary Gilliland
From the Division of Hematology, Brigham and Women's Hospital,
Boston; Howard Hughes Medical Institute, Harvard Medical School,
Boston, MA; Department of Pathology, Emory University, Atlanta, GA;
Hammersmith Hospital, London, UK; and Oregon Health Sciences
University, Portland, OR.
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ABSTRACT |
The TEL/PDGF R fusion protein is expressed as the consequence of a
recurring t(5;12) translocation associated with chronic myelomonocytic
leukemia (CMML). Unlike other activated protein tyrosine kinases
associated with hematopoietic malignancies, TEL/PDGFR is invariably
associated with a myeloid leukemia phenotype in humans. To test the
transforming properties of TEL/PDGFR in vivo, and to analyze the
basis for myeloid lineage specificity in humans, we constructed
transgenic mice with TEL/PDGFR expression driven by a
lymphoid-specific immunoglobulin enhancer-promoter cassette. These mice
developed lymphoblastic lymphomas of both T and B lineage, demonstrating that TEL/PDGFR is a transforming protein in vivo, and
that the transforming ability of this fusion is not inherently restricted to the myeloid lineage. Treatment of TEL/PDGFR transgenic animals with a protein tyrosine kinase inhibitor with in vitro activity
against PDGFR (CGP57148) resulted in suppression of disease and a
prolongation of survival. A therapeutic benefit was apparent both in
animals treated before the development of overt clonal disease and in
animals transplanted with clonal tumor cells. These results suggest
that small-molecule tyrosine kinase inhibitors may be effective
treatment for activated tyrosine kinase-mediated malignancies both
early in the course of disease and after the development of additional
transforming mutations.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
DESPITE SIGNIFICANT ADVANCES in the
therapy of human leukemia, most adults still die of their disease or
complications of therapy. Chronic myelomonocytic leukemia (CMML) is
exemplary of the problem: although indolent at presentation, CMML
progresses in most cases to acute myeloid leukemia (AML), which is
frequently a fatal complication of the disease.1 There are
currently no therapies for CMML or other of the myelodysplastic
syndromes that are known to prolong survival apart from bone marrow
transplant for selected patients.2,3 There is therefore a
need to develop more effective therapies for these diseases.
The TEL/PDGFR fusion protein is generated by t(5;12) (q33;p13), a
recurring cytogenetic abnormality associated exclusively with
CMML.4-9 Transformation of hematopoietic cells by
TEL/PDGFR is the consequence of oligomerization, mediated by the
pointed (PNT) domain of TEL, resulting in constitutive activation of
the kinase domain of PDGFR.10,11 However, the basis for
the myeloid lineage specificity of TEL/PDGFR in human leukemia is
not known. Certain leukemogenic fusion proteins have the ability to
mediate transformation of multiple lineages of hematopoietic cells in cell culture and in animal model systems,12,13 and are
associated with both myeloid and lymphoid disease in humans. For
example, the BCR/ABL fusion protein expressed as a result of the
t(9;22) translocation is associated with chronic myelogenous and acute lymphoblastic leukemias (ALLs) in humans, and causes myeloid and lymphoid malignancies in murine bone marrow transplant (BMT)
models.12 In contrast, certain translocations such as
t(7;9), leading to overexpression of TAN-1, the human homolog of
Drosophila Notch-1, are exclusively associated with a single
hematopoietic lineage.14 t(7;9) has been identified only in
T-cell leukemia/lymphoma in humans. The lineage specificity appears to
be intrinsic to TAN-1, because murine BMT experiments, in which
activated TAN-1 expression can be achieved in all lineages of
hematopoietic cells through retroviral infection, gives rise to only
T-lineage hematopoietic disease in mice. TEL/PDGFR is exclusively
associated with CMML in humans, but it is not known whether this
myeloid lineage specificity is due to intrinsic properties of
TEL/PDGFR. To test the ability of TEL/PDGFR to transform primary
hematopoietic cells, and to investigate the basis for lineage
specificity in humans, transgenic mice were prepared in which
TEL/PDGFR expression was directed to the lymphoid compartment by the
immunoglobulin heavy-chain enhancer/promoter (EµVHP).
These mice developed lymphoblastic lymphomas and were used to assess
the in vivo efficacy of CGP57148, a specific tyrosine kinase inhibitor
with known activity against TEL/PDGFR in vitro.15
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MATERIALS AND METHODS |
Construction of transgenic mice.
The backbone plasmid pBSVE6K containing EµVHP and the
-globin splice acceptor and poly A sequences was obtained as a gift from Dr Fred Alt, Harvard Medical School, Boston, MA. The TEL/PDGFR cDNA cloned by Golub et al4 was inserted 3' of the
EµVHP cassette (Fig 1).4,16
The prokaryotic plasmid sequences were removed from the above
constructs by restriction enzyme digestion with BssHII and gel
purification. Each purified construct was diluted to a concentration of
1.5 to 3.0 ng/µL and 1 fL was microinjected into the pronucleus of a
FVB strain murine oocyte. The injected eggs were implanted into
the oviduct of a pseudopregnant mouse in the Transgenic Core
Facility at Brigham and Women's Hospital. At 2 weeks of age, founder
mice were identified by Southern blot analysis of DNA isolated from
tail clippings. Progeny of these founder lines were generated by
crossing with normal FVB mice.
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| Fig 1.
EµVHP-TEL/PDGFR transgenic construct.
The immunoglobulin heavy-chain enhancer/promoter cassette contains the
682-bp EcoRI-Xba I fragment of the immunoglobulin-mu
enhancer (Eµ), ligated to the 330-bp HindII-Eco I
promoter fragment of the VH gene (VHP). The
3' end of the construct consists of the 1.6-kb
BamHI-Pst I fragment of the genomic human -globin
gene containing a small (19-bp) piece of exon 2, the entire 849-bp
intron 2, including splice donor and acceptor sites, all of exon 3, and
a fragment of nearly 500 bp of 3'UTR, including the
polyadenylation signal. Into the multicloning site of pBSVE6K,
between these 2 cassettes, the 2.2-kb cDNA for TEL/PDGFR fusion gene
was inserted. The stop codon in TEL/PDGFR prevents the -globin
exons from being translated, but the existence of these exons in the
transgene mRNA was exploited to assay for expression. Primers used in
RT-PCR are indicated by small arrows. Restriction sites: B2 = BssHII, B1 = BamHI, R1 = EcoRI, S2 = SacII, H = HindIII.
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Assessment of transgenic mice.
Animals were monitored three times per week for the development of
disease by inspection and palpation of the spleen and cervical, femoral, and axillary lymph nodes. Mice with clinically evident disease
were killed by CO2 asphyxiation followed by cervical
dislocation. Bone marrow cells were isolated by flushing femurs and
tibias with phosphate-buffered saline (PBS). Single-cell suspensions were prepared by passing spleen and lymph node tumor tissue through nylon mesh (Falcon, Lincoln Park, NJ) wetted with PBS.
Tumor cell transplantation.
A single-cell suspension from pathologically enlarged lymph nodes was
prepared as above. Cells were counted and diluted to a concentration of
2 × 104/mL. Recipient mice were subjected to a single
dose of 450 rad and 0.5 mL of the tumor cell suspension was
administered by tail vein injection.
Expression analysis.
Single-cell suspensions of tissues were prepared as above. Total
cellular RNA was isolated by resuspending 107 cells in 1 mL
of a monophase phenol/guanidinium thiocyanate solution (TRIzol;
GibcoBRL, Gaithersburg, MD) and processing according to the
manufacturer's instructions. A 4-µg quantity of total mRNA from each
sample was reverse-transcribed using AMV reverse transcriptase (GibcoBRL) for 1 hour at 42°C. One microliter of cDNA solution was
subjected to 35 cycles of the polymerase chain reaction (94°C for
60 seconds, 56°C for 60 seconds and 72°C for 60 seconds) using primers TPY3F, 5'-TAC AAA AAG TAC CAG CAG-3' and HBG1,
5'-GCG AGC TTA GTG ATA CTT GT-3'. Cells from representative
tissues were washed twice with PBS and lysed in 1% NP40, 150 mmol/L
NaCl, 20 mmol/L Tris pH 7.4, 10% glycerol containing 1 mmol/L
phenylmethylsulfonylfluoride, 20 µg/mL aprotinin, and 1 mmol/L sodium
orthovanadate at 5 × 107 cells/mL. Equal amounts of
lysates (100 µg) were analyzed by denaturing polyacrylamide gel
electrophoresis in the presence of sodium dodecyl sulfate (SDS) and
Western blotting with polyclonal rabbit anti-PDGFR tail (Pharmingen,
San Diego, CA) as described.17
Histology.
Murine tissues were fixed for 24 hours in 10% neutral buffered
formalin and embedded in paraffin. Femurs were subjected to an
additional decalcification step in RDO (Apex Engineering Products, Plainfield, IL) for 4 hours before processing. The 3-µm sections were
deparaffinized and stained with hematoxylin and eosin (H&E).
Flow cytometric analysis.
Single-cell suspensions of bone marrow, spleen, lymph nodes, blood, and
thymus were prepared. Red blood cells were lysed in ammonium chloride
solution (150 mmol/L NH4Cl, 10 mmol/L KHCO3, 0.1 mmol/L EDTA, pH 7.4) for 5 minutes at room temperature. The cells
were washed in PBS with 0.1% NaN3 and 0.1% bovine serum albumin (BSA; staining buffer). To block nonspecific Fc
receptor-mediated binding, the cells were preincubated with supernatant
from the 2.4G2 hybridoma line (anti-CD16/CD32; cell line obtained from American Type Culture Collection, Rockville, MD) for 20 minutes on ice.
Aliquots of 0.5 to 1.0 × 106 cells were then stained
for 20 minutes on ice with monoclonal antibodies specific for B220
(CD45R), CD24 (heat-stable antigen), CD117 (c-kit), IgM, CD3,
CD2,  TCR, CD25 (IL-2R chain), CD40, CD43, CD4, CD8, and
Ly-51 (BP-1) (Pharmingen) conjugated with fluorescein isothiocyanate
(FITC), phycoerythrin (PE), or biotin. Binding of biotinylated primary
antibodies was detected using PE-conjugated streptavidin (Immunotech,
Westbrook, ME) or FITC-conjugated avidin (Southern Biotechnology,
Birmingham, AL). Cells were washed once in staining buffer followed by
two-color flow cytometric analysis with a FACScan (Becton Dickinson,
San Jose, CA). A minimum of 10,000 events was acquired and the data
were analyzed using CellQuest software (Version 3.1; LCC International,
McLean, VA). The results are presented as contour plots showing FITC
and PE fluorescence signals of viable cells gated on the basis of
forward and side scatter signals.
Immunoglobulin gene rearrangement.
Genomic DNA was prepared from single-cell suspensions of lymph node
tumor cells, and control DNA was isolated from the tail of an
unaffected littermate as described.18,19 A 10-µg quantity of genomic DNA was digested with the appropriate restriction
endonucleases overnight, subjected to electrophoresis on a 1% agarose
gel, and transferred to nylon membranes (Hybond N+;
Amersham, Arlington Heights, IL) using alkaline transfer as described.20 A 1.5-kb Pst 1 IgH fragment, a 2.4-kb
HindIII-BamHI IgK fragment, and 2.0-kb EcoRI
TCR fragment were used as probes (a gift of Dr Benjamin Rich),
random-labeled using 32P dCTP (Boehringer Mannheim,
Indianapolis, IN). Hybridization was performed at 65°C for 16 hours
and membranes were washed with 2× SSC, 0.1% SDS for 20 minutes
at room temperature; 1× SSC, 0.1% SDS at 65° for 20 minutes;
and exposed to photographic film (BioMax; Eastman Kodak, Rochester, NY)
at  80°C overnight.
Tyrosine kinase inhibitor treatment.
CGP57148 was diluted in PBS to a concentration of 3 mg/mL; 50 mg/kg was
injected intraperitoneally (IP) every day for 30 days. Control mice
were injected IP with 0.5 mL of PBS alone at the same time. P
values for statistical significance were calculated by the log-rank
(Mantel-Cox) method. Statistical calculations and Kaplan-Meier survival
analyses were performed using the program Statview (SAS Institute,
Cary, NC).
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RESULTS |
Construction of Eµ-TEL/PDGFR transgenic
mice.
The ability of TEL/PDGFR to transform lymphoid cells in vivo was
tested using a transgenic mouse model in which the immunoglobulin heavy-chain enhancer/promoter EµVHP was used to direct
TEL/PDGFR expression to the lymphoid compartment.
EµVHP would be expected to result in expression
predominantly to the B-cell compartment, but expression in T cells has
also been well described.21,22 A total of 10 founder mice
were identified as having integrated the TEL/PDGFR
construct. Germline transmission allowed the establishment of eight transgenic lines. One founder failed to breed successfully, and another developed a mediastinal mass and died at 65 days old before
analysis or breeding could be completed. Two of the eight lines (I and
N lines) consistently developed lymphoid malignancies. The most
extensive analysis of tumors (40 separate tumors) was performed on mice
from the I line, all of which developed diffuse lymphadenopathy and
massive hepatosplenomegaly at a median age of 4 months
(Fig 2A). N line mice also
consistently developed tumors at the same median age (seven tumors
analyzed) that presented in several different patterns (see below).
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| Fig 2.
Gross pathology of Eµ-TEL/PDGFR lymphoblastic
lymphoma in the I line and microscopic histopathology of tumors and
affected tissues from Eµ-TEL/PDGFR mice. (A) Gross dissection of
an affected I-line mouse shows enlargement of the liver (L), spleen
(S), and thymus (T), as well as massive lymphadenopathy in the cervical
(C), axillary (A), and femoral (F) node groups. (B) Touch preparation
of the lymph node from an affected I-line mouse shows tumor composed of
intermediate-sized lymphoid cells with scant cytoplasm, finely
dispersed nuclear chromatin, and a brisk mitotic rate, features of
lymphoblastic lymphoma. (C) Clusters of lymphoma cells present in
hepatic sinusoids. (D) Lymphoma cells in N-line mouse with T-cell
mediastinal lymphoma phenotype show involvement of the lung in
peribronchial distribution. (E) Lymphoma cells from the same mouse as
in D infiltrating the spleen, with some residual megakaryocytes and
erythroid cells. (F) Bone marrow replacement by lymphoblasts in an
N-line mouse with ALL phenotype. (G) Same N-line mouse as in F showing
splenic involvement with lymphoblastic leukemia (H&E).
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Microscopic and flow cytometric analysis of the malignant phenotype.
Histopathologic examination of I line mice showed tumor with features
of lymphoblastic lymphoma, including intermediate-sized lymphoid cells
with scant cytoplasm, dispersed nuclear chromatin, and a brisk mitotic
rate. All lymph node groups were affected, as well as periportal tissue
in the liver, diffuse involvement of the spleen with effacement of
normal splenic architecture, and of bone marrow with lymphoblastic
lymphoma (Fig 2A through C, and data not shown). Flow cytometry of cell
suspensions from bone marrow, spleen, blood, and lymph nodes was
performed to determine cell lineage of the tumors. All I-line tissues
analyzed contained a dominant population of B220+,
CD3 , CD40, CD43+,
HSA+, and BP-1+ cells (Fig
3A), consistent with hardy fraction C
lymphoblastic lymphoma.23,24 This phenotype corresponds
most closely with a late pro-B- or early pre-B-cell lymphoma in
humans. However, while all mice of this line showed early B-cell
phenotype, other individual mice from the same line had variable
staining for CD43 and CD117 (c-kit). These different
immunophenotypes show some variability in the stage of differentiation
of these tumors, an observation that has been made in several other
mouse models of lymphoid malignancy.25-28
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| Fig 3.
Flow cytometric analysis and immunoglobulin gene
rearrangement studies of tumor cells from transgenic animals. (A) Flow
cytometric analysis of involved tissues from from affected mice. I-line
mice and N-line mice with lymphoma phenotype (I-836) show a large
population of early B cells (B220+, CD3,
CD43+, BP-1+, CD117+,
IgM) in lymph node tumors (shown), as well as in bone
marrow, spleen, and peripheral blood (data not shown). N-line animals
with leukemic phenotype (N-2051) do not have adenopathy, but show a
similar immunophenotype in the spleen (shown), marrow and blood (not
shown). Normal cells can also be seen in these samples. Cells from an
N-line animal with mediastinal tumor phenotype (N-2288) show tumor
cells that are CD3+, B220,
CD2+, aTCR+, and both
CD4+/CD8+, as well as
CD4+/CD8 and
CD4/CD8+. (B-D) Immunoglobulin and T-cell
receptor gene rearrangements in tumors from transgenic animals. (B)
EcoRI digests, µVJ probe; faint low-molecular-weight bands
in transgenic samples are due to nonspecific probe binding. (C)
EcoRI + BamHI digests, kC probe; (D) HindIII
digests, TCR probe. Southern blot analyses of genomic DNA from tumor
samples show clonal rearrangements of the immunoglobulin heavy-chain
locus in I-line mice. The N-line founder shows a clonal rearrangement
of the TCR locus. Conversely, tumors from I-line mice do not show
rearrangements of the kappa light-chain locus or TCR locus. Genomic
tail DNA from an unaffected littermate is used as a control and the
germline bands are indicated by an arrow.
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TEL/PDGFR lymphoid tumors are clonal.
Clonality of tumors was assessed in transgenic mice by Southern blot
analysis with probes for the immunoglobulin heavy-chain, kappa
light-chain, and TCR loci (Fig 3B through D). Clonal rearrangements were detected in I-line tumor tissue with probes for the immunoglobulin heavy-chain locus, but not for the kappa light-chain or TCR loci, consistent with the histopathologic and flow cytometry data.
Furthermore, these data show that in addition to expression of
TEL/PDGFR, tumorigenesis requires additional mutations to give rise
to the full malignant phenotype.
TEL/PDGFR is expressed in involved tissues from
transgenic mice.
Expression of TEL/PDGFR in tissues involved with lymphoma was
confirmed by both reverse-transcriptase polymerase chain reaction (RT-PCR) and by Western blot analysis in I-line (Fig
4) and N-line mice (data not shown). RT-PCR
used primers flanking an intron in the TEL/PDGFR construct (Fig 1)
so that amplimers resulting from contaminating genomic DNA could be
distinguished from those arising from cDNA on the basis of size.
TEL/PDGFR transcript was detected in marrow, spleen, and nodes of
transgenic animals, but not in marrow or spleen of controls (Fig 4A).
TEL/PDGFR protein was detected by Western blotting with
anti-PDGFR antibody in nodes and bone marrow, but not in kidney of
transgenic mice or marrow of control mice (Fig 4B).
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| Fig 4.
Assessment of TEL/PDGFR transgene expression: the
fusion protein is expressed in tumors and hematopoietic tissues from
transgenic mice, but not in tissues from normal controls. (A) PCR of
RNA isolated from transgenic and normal mouse tissues. The forward
primer (TPY3F, 5'-TAC AAA AAG TAC CAG CAG-3') binds the
3' end of the PDGFR, and the reverse primer (HBG1,
5'-GCG AGC TTA GTG ATA CTT GT-3') anneals to the antisense
strand of the human -globin exon 3 and were designed to span the
-globin intron in the transgenic construct (see Fig 2). Samples
contaminated with genomic DNA give a larger, 1.6-kb product (data not
shown). The 0.7-kb product expected from the amplification of spliced
mRNA is seen in samples isolated from transgenic mouse tumor, spleen,
and bone marrow, but not in normal spleen, bone marrow, or in reverse
transcriptase-negative controls. (B) Western blot of whole-cell
lysates from tumors (N), bone marrow (M), and kidney (K). The antibody
used was  tail PDGFR. The TEL/PDGFR protein runs as a doublet,
as reported previously,10 due to an alternate start site
for translation within the TEL gene.
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I-line Hardy C lymphoblastic lymphoma is transplantable.
To confirm the malignant phenotype of lymphoma cells, 104
tumor cells in single-cell suspensions derived from I-line lymph node
tumors were transplanted by intravenous injection from an affected
transgenic animal into sublethally irradiated syngeneic mice.
Tumors developed in these transplanted mice 3 to 4 weeks after
transplantation and affected liver, lymph node, marrow, and spleen
tissues. Histopathologic and flow cytometric analysis was consistent
with B-cell lymphoblastic lymphoma, demonstrating that the transplanted
tumors retained the same morphology and immunophenotype as the primary
B-cell lymphoblastic lymphoma (data not shown). Tumors also developed
in unirradiated recipient mice that received 105 lymph node
cells (data not shown).
Other transgenic lines of TEL/PDGFR mice also develop
lymphoblastic lymphoma.
The N-line founder developed a massive mediastinal mass with
involvement of pulmonary parenchyma and spleen. Histopathologic examination was consistent with lymphoblastic lymphoma (Fig 2 D and E).
Flow cytometric analysis of tumor cells (Fig 3A) showed CD3+, CD2+, TCR+, and
B220 cells consistent with T-cell lymphoblastic
lymphoma. Southern blot analysis confirmed a clonal rearrangement of
the TCR locus, but not of immunoglobulin heavy-chain or kappa chain
loci (Fig 3B through D). RT-PCR confirmed the presence of TEL/PDGFR
transcript (data not shown). Five F1 progeny of the N-line founder have
developed a distinct clinical phenotype consisting of massive
splenomegaly, weight loss, and eventually hind limb paralysis. The
spleen and bone marrow of these animals was involved with lymphoblastic
lymphoma (Fig 2F and G) with an identical immunophenotype to that seen in I-line animals, and there were tumor cells circulating in the blood
(data not shown). The phenotype of splenomegaly with bone marrow
involvement by Hardy C lymphoblastic lymphoma cells is most analogous
to human pre-B-cell ALL. Last, one mouse from the N line developed
massive lymphadenopathy that was histologically and
immunophenotypically identical to that seen in I-line mice.
Treatment with a specific tyrosine kinase inhibitor.
Transgenic I-line mice were treated with daily IP injections of
CGP57148 (50 mg/kg).29 CGP57148 is a specific inhibitor of
the PDGFR and ABL kinases at concentrations as low as 1 µmol/L,15 but has no effect on a broad spectrum of other
tyrosine and serine/threonine kinases at concentrations as high as 100 µmol/L. Mice were treated for 30 consecutive days, and did not
display any obvious toxicity from the treatment. Control mice were
treated with PBS for the same duration. There was a statistically
significant prolongation of survival in mice treated with CGP57148
compared with PBS controls (Fig 5A).
Latency was prolonged by approximately the same duration as CGP57148
therapy in this experiment, suggesting that CGP57148 may have inhibited
growth of these cells or prevent tumor progression, but did not
eradicate TEL/PDGFR-expressing cells at the concentration and dosing
schedule used in this experiment.
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| Fig 5.
Improved survival of Eµ-TEL/PDGFR transgenic mice
treated with the specific tyrosine kinase inhibitor CGP57148. (A)
Premalignant mice model: 6 I-line mice between 5 and 7 weeks old
without evidence of malignancy on examination were treated with daily
IP injections of CGP57148 for 30 days ( ). Concurrently, 9 similar
animals were treated with PBS as a control (). Time to development
of overt malignancy is delayed and survival is improved in animals
treated with CGP57148. (B) Tumor transplant model: tumor cells from an
I-line mouse with massive lymphadenopathy were put into single-cell
suspension, and 104 cells were injected into sublethally
irradiated syngeneic mice. Ten were treated with CGP57148 () and 9 were treated with PBS (-) for 21 days. Again, mice treated with
CGP57148 had a statistically significant improvement in survival. In
both figures, an open bar represents the duration and timing of
CGP57148 or PBS.
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In a separate experiment, the effect of CGP57148 was tested in a
transplant model. A total of 104 tumor cells derived from
affected lymph nodes of transgenic animals were injected intravenously
into 19 sublethally irradiated animals; 10 animals then received daily
IP CGP57148 as above and nine received PBS. Again, animals treated with
the specific kinase inhibitor had a statistically significant
inhibition of development of lymphoblastic lymphoma and survived longer
than control animals (P = .0040, Fig 5B). These data suggest
that even after acquisition of clonal disease through additional
mutations, CGP57148 is capable of inhibiting lymphoblastic lymphoma
cell growth. Prolongation of latency was approximately equivalent to
the duration of CGP57148 therapy.
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DISCUSSION |
CMML is a subtype of myelodysplastic syndrome and is characterized by
dysplastic monocytosis, variable bone marrow fibrosis, and progression
to AML. AML is frequently a fatal complication of CMML, but the
molecular genetic basis for progression of disease is poorly understood
and there are no known therapies to prevent progression.
t(5;12)(q31;p13), a recurring cytogenetic abnormality associated with
CMML, results in fusion of the amino terminus of TEL, containing the
PNT oligomerization domain, to the tyrosine kinase domain of PDGFR.
The transformation of Ba/F3 cells by TEL/PDGFR is dependent on
PDGFR tyrosine kinase activity. A kinase inactive point mutant of
TEL/PDGFR is not transforming,10 and the PDGFR kinase-specific inhibitor CGP57148 inhibits the growth of
TEL/PDGFR-transformed Ba/F3 cells.15 t(5;12)(q31;p13) is
exclusively associated with a myeloid leukemia phenotype; it is never
seen in association with lymphoproliferative disorders or lymphoid
malignancy. We have developed an animal model of TEL/PDGFR-mediated
malignancy to (1) characterize the basis for myeloid lineage
specificity of TEL/PDGFR in humans, (2) test the transforming
properties of the fusion protein in vivo, (3) develop a model system
for studying progression of disease, and (4) test therapeutic
interventions targeted at the PDGFR kinase.
Expression of TEL/PDGFR was directed to the lymphoid lineage using
the immunoglobulin heavy-chain enhancer-promoter, Eµ. TEL/PDGFR
was capable of transforming primary lymphoid lineage cells, as
evidenced by the development of B and T lymphoblastic lymphomas in
different transgenic lines of mice. Expression of TEL/PDGFR was
assayed in mice that developed lymphomas. Tumor, but not unaffected
tissues, expressed the TEL/PDGFR mRNA and protein. Eµ is known to
direct expression primarily to the B-cell compartment, but low levels
of expression have been documented in other cell types, including T
cells. In fact, Eµ-E2A/PBX transgenic mice developed T-cell neoplasms
exclusively.30 Because Eµ-TEL/PDGFR transgenic mice
develop lymphoid malignancy, the myeloid lineage specificity of
TEL/PDGFR observed in human leukemias is not due to an inherent
inability of the fusion protein to transform lymphoid lineage lymphoid
cells. These data contrast with transforming proteins such as TAN-1,
which has T-cell lineage specificity both in humans and in murine BMT
models, or BCR/ABL, which causes both myeloid and lymphoid malignancy
in humans and in murine models. The basis for myeloid lineage
specificity of TEL/PDGFR in humans may relate to events in myeloid
differentiation that favor acquisition of the TEL/PDGFR gene rearrangement.
Southern blot analysis of tumor tissue for immunoglobulin gene
rearrangement showed clonal tumors in both B and T lymphoblastic lymphomas. TEL/PDGFR is transmitted in the germ line, and would be
expected to generate polyclonal tumors if it were sufficient to cause
hematopoietic malignancy. The finding of clonal tumors shows
acquisition of additional mutations in disease pathogenesis in this
model, and recapitulates human CMML, in which acquisition of second
mutations is associated with progression of disease to AML. For
example, in the index case of CMML with t(5;12)(q31;p13), progression
to AML was associated with acquisition of t(8;21).4 However, in most cases, the nature of the second mutation is unknown. The transgenic mouse model of TEL/PDGFR-mediated hematologic malignancy provides a reagent for identifying additional mutations associated with disease progression.
Transformation of CMML to AML is frequently a fatal complication.
However, the clinical phenotype of CMML is relatively benign, and many
patients are asymptomatic at presentation. Specific tyrosine kinase
inhibitors of the 2-phenylaminopyrimidine class offer the opportunity
for intervention in disease progression. CGP57148 is a specific
inhibitor of the PDGFR and ABL kinases, with negligible effect on
other tyrosine and serine/threonine kinases at concentrations in the
0.1 to 1 µmol/L range.15,31,32 Furthermore, CGP57148 inhibits the growth of TEL/PDGFR-transformed Ba/F3 cells, and inhibition can be rescued by addition of IL-3, demonstrating that the
effect of CGP57148 is specific for PDGFR.15
To determine whether CGP57148 could prolong disease latency in the
TEL/PDGFR transgenic model, drug was administered by daily IP
injections for 30 days. Although the compound can inhibit both native
PDGFR and c-Abl kinases, there was no observed toxicity. Furthermore, there was statistically significant prolongation of
latency of disease of approximately the same duration as administration of drug. CGP57148 has previously been shown to be active in vivo against BCR/ABL.31 Our data suggest that CGP57148 may also
be useful clinically in prolonging disease latency in humans with TEL/PDGFR-mediated disease. Drug efficacy could potentially be improved by testing modulations in dose, schedule, or route of administration in this animal model. For example, the half-life of
CGP57148 in vivo measured in rats is approximately 4 hours (B. Druker,
unpublished observation); thus, single-day dosing might
not be expected to give maximal efficacy.
The effect of CGP57148 was then tested on tumor cells in a transplant
model in which tumor cells were introduced into a syngeneic recipient.
There was also a statistically significant prolongation of disease
latency in this context as well, demonstrating that CGP57148 is able to
inhibit the growth of tumor cells expressing TEL/PDGFR even after
the acquisition of additional mutations. However, tumor cells were
apparently not killed by the drug treatment. It is plausible that
CGP57148 inhibits the proliferation of cells mediated by TEL/PDGFR,
but that after cessation of therapy, tumor cells are again able to
proliferate. This hypothesis could be further explored by testing the
effect of CGP57148 ex vivo on tumor cells before transplantation.
The present study shows that TEL/PDGFR can cause hematopoietic
malignancy in a transgenic mouse model, and is capable of causing
lymphoid malignancy in this context, despite myeloid lineage specificity in humans. CGP57148, a PDGFR-specific tyrosine kinase inhibitor, is nontoxic in mice under the conditions used, and prolongs
disease latency both in transgenic mice and in transplanted tumor
cells. Although these pilot experiments used a small number of animals,
the results were statistically significant. Further analysis of
CGP57148 and other compounds in this animal model may identify novel
therapies to treat CMML in humans and prevent disease progression.
| |
ACKNOWLEDGMENT |
The authors thank M. Ryan for assistance in preparing the manuscript
and K. Weilbaecher, M. Carroll, D. Sternberg, and T. Ross for helpful
discussions and critical reading of the manuscript.
| |
FOOTNOTES |
Submitted July 29, 1998; accepted October 22, 1998.
Supported in part by the Lauri Strauss Leukemia Foundation and the
Leukemia Society of America (LSA) Grant No. 5461-98 (M.H.T.), National
Institutes of Health Grant No. P01CA66996-01, and the Lawrence Family
Foundation. D.G.G. is an investigator in the Howard Hughes Medical
Institute and is the Stephen Birnbaum Scholar of the LSA.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to D. Gary Gilliland, MD, PhD,
Division of Hematology, Brigham and Women's Hosptal, Harvard
Institutes of Medicine, Room 420, Boston, MA 02115.
| |
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Abstract
Introduction