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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 96 No. 2 (July 15), 2000:
pp. 664-670
NEOPLASIA
The Grb2 binding site is required for the induction of chronic
myeloid leukemia-like disease in mice by the Bcr/Abl tyrosine kinase
Ryan P. Million and
Richard A. Van Etten
From The Center for Blood Research, Department of Genetics, Harvard
Medical School, Boston, MA.
 |
Abstract |
The BCR/ABL oncogene results from a balanced translocation
between chromosomes 9 and 22 and is found in patients with chronic myeloid leukemia (CML) and in some patients with acute B-lymphoid leukemia. The Bcr/Abl fusion protein is a constitutively active tyrosine kinase that stimulates several intracellular signaling pathways, including activation of Ras through direct binding of the
SH2-containing adapter protein Grb2 to Bcr tyrosine 177. A tyrosine-to-phenylalanine mutation (Y177F) at this site blocks the
co-association of Bcr/Abl and Grb2 in vivo and impairs focus formation
by Bcr/Abl in fibroblasts. However, the Bcr/Abl Y177F mutant can
transform hematopoietic cell lines and primary bone marrow cells in
vitro, so the importance of the Bcr/Abl-Grb2 interaction to myeloid
and lymphoid leukemogenesis in vivo is unclear. We have recently
demonstrated the efficient induction of CML-like myeloproliferative
disease by BCR/ABL in a murine bone marrow transduction/transplantation model system. The Y177F mutation greatly
attenuates the myeloproliferative disease induced by BCR/ABL, with mice developing B- and T-lymphoid leukemias of longer latency. In
addition, the v-abl oncogene of Abelson murine leukemia virus, whose protein product lacks interaction with Grb2, is completely defective for the induction of CML-like disease. These results suggest
that direct binding of Grb2 is required for the efficient induction of
CML-like myeloproliferative disease by oncogenic Abl proteins.
(Blood. 2000;96:664-670)
© 2000 by The American Society of Hematology.
| |
Introduction |
The product of the t(9;22) Philadelphia chromosome
translocation, the BCR/ABL oncogene, is found in virtually all
patients with the myeloproliferative syndrome, chronic myeloid leukemia (CML), and in approximately 20% of patients with acute B-lymphoblastic leukemia.1 The Bcr/Abl fusion protein is a constitutively
active tyrosine kinase that can transform fibroblasts,2
factor-dependent hematopoietic cells,3 and primary bone
marrow B-cell progenitors4 in vitro. Expression of Bcr/Abl
stimulates a diverse array of cell signaling pathways,5
including the activation of Ras, phosphatidylinositol 3-kinase, STATs,
SAPK/JNK, and c-Myc. Studies with dominant negative mutants have
demonstrated that Ras activation is required for the transformation of
fibroblasts6 and hematopoietic cells6,7 by
Bcr/Abl.
One documented mechanism of Ras activation by Bcr/Abl is through
interaction with the Grb2/Sos protein complex.8,9 The SH2
domain of the Grb2 adapter protein binds directly to
phosphorylated tyrosine 177 in the N-terminal Bcr portion of the
Bcr/Abl fusion protein. A tyrosine-to-phenylalanine mutation (Y177F)
at this site blocks co-association of Bcr/Abl and Grb2 in vivo,
impairs Ras activation, and decreases focus formation by Bcr/Abl in
fibroblasts.8 However, the Bcr/Abl Y177F mutant is still
able to activate Ras and transform cytokine-dependent hematopoietic
cell lines to become cytokine independent for survival and
growth.10,11 Further, Bcr/Abl Y177F is capable of
transforming primary bone marrow-derived B-lymphoid progenitors in
vitro.11 These conflicting in vitro observations call into
question the relevance of the Bcr/Abl-Grb2 interaction to myeloid and
lymphoid leukemogenesis in vivo.
A complete understanding of the pathogenesis of human
Philadelphia-positive leukemia requires expression of the
BCR/ABL oncogene in the hematopoietic system. Currently, the
only model in which BCR/ABL induces a form of leukemia similar
to CML is the murine bone marrow transduction/transplantation
system12 (for review see Van Etten13).
Recently, we14 and others15,16 demonstrated the
induction of a myeloproliferative disease closely resembling human CML
in 100% of mice transplanted with marrow transduced with the p210
BCR/ABL oncogene. That system14 allows a direct comparison of the leukemogenic activity of different forms and mutants
of activated ABL oncogenes after the transduction of an identical pool of primary hematopoietic cells. In this study, we
compared p210 BCR/ABL and the p210 BCR/ABL Y177F mutant
for their ability to induce leukemia in the murine bone marrow
transduction/transplantation assay. The p210 Y177F mutant was found to
be defective for the induction of CML-like myeloproliferative disease
in mice. In addition, we demonstrated that p160 v-Abl, a Gag/Abl fusion
protein produced by Abelson murine leukemia virus, does not bind to
Grb2 and completely lacks the ability to cause CML-like disease.
| |
Materials and methods |
Cell culture and transfection
293T cells were grown in Dulbecco modified Eagle's medium
supplemented with 10% heat-inactivated fetal calf serum,
penicillin/streptomycin, 2 mmol/L L-glutamine, and
nonessential amino acids. For transient transfection, cDNA encoding the
p210 form of Bcr/Abl, the p210 Y177F point mutant,8 the
p160 form of v-Abl, or the p145 murine-type IV c-Abl were introduced
into the expression vector pcDNA3 (Invitrogen, Carlsbad, CA). Abl
proteins were expressed in 293T cells by modified calcium phosphate
transfection as previously described.17
Far Western and immunoprecipitation Western blotting
Transfected 293T cells were lysed in RIPA buffer as previously
described,18 and whole cell lysates were either
fractionated directly by 5% to 20% gradient sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or
immunoprecipitated with polyclonal anti-Abl (anti-GEX4)19
or anti-Grb2 (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies
before electrophoresis. After SDS-PAGE, proteins were electroblotted to
nitrocellulose membranes. For Far Western analysis, GST-Grb2(SH2)
fusion protein20 and parental GST protein were purified
from Escherichia coli by affinity chromatography on glutathione
agarose (Molecular Probes, Eugene, OR) and hybridized to membranes
essentially as described.21 Bound GST proteins were
detected by the hybridization of membranes with affinity-purified polyclonal rabbit-anti-GST antibodies22 and enhanced
chemiluminescence (Amersham, Arlington Heights, IL). Immunoprecipitated
proteins were detected by blotting with monoclonal anti-Abl (8E9;
Pharmingen, San Diego, CA) or polyclonal anti-Grb2 antibodies and
enhanced chemiluminescence.
Bone marrow transduction/transplantation
ABL oncogenes were introduced into the retroviral vector
MSCVneo,23 and high-titer, helper-free retroviral
stocks were prepared by transient transfection of 293T cells using the
kat ecotropic packaging system.24 Viral stocks were
titered by the transduction of NIH 3T3 cells with serial dilutions of
stock, followed by the selection for neomycin resistance. Each viral
stock had a titer of 3 to 5 × 106 neomycin-resistant
colony-forming units per milliliter and lacked detectable
replication-competent helper virus, as assessed by a sensitive provirus
mobilization assay.25 Titers within this range gave an
equivalent proviral copy number in transduced NIH 3T3 cells
(approximately 2 proviral copies per diploid genome). Retroviral
transduction of bone marrow, followed by transplantation into lethally
irradiated syngeneic Balb/c recipient mice, was performed exactly as
previously described.14 In all cases, donors were
pretreated with 5-fluorouracil (200 mg/kg intravenously) 4 days before bone marrow harvest. In some experiments, genomic DNA was
isolated from samples of transduced primary bone marrow just before
transplantation. Southern blot analysis indicated that P210
Y177F-transduced bone marrow had a proviral copy number that was equal
to or greater than that of P210 wild-type-transduced marrow (data not shown).
Analysis of diseased mice
Premoribund mice were killed by CO2 asphyxiation, and
hematopoietic organs and tissues were harvested and analyzed by
cytospin, histopathology, and Southern blotting of genomic DNA as
previously described.14 Mast cell tumors were identified by
basophilic Wright-Giemsa stain. For flow cytometric analysis, tumor
cell populations were incubated with antibodies to murine CD90
(Thy1.2), CD8a, CD24, CD43, CD45RA (B220), and CD11b (Mac1), all from
Pharmingen, stained with fluorescein isothiocyanate-conjugated donkey
anti-rat IgG (Jackson Immunoresearch, West Grove, PA), and analyzed on a FACScan with CellQuest software (Becton Dickinson, Mountain View, CA).
| |
Results |
Bcr/Abl Y177F and v-Abl proteins do not bind Grb2
We detected a direct interaction between p210 Bcr/Abl and Grb2 by
Far Western blotting, using the Grb2 SH2 domain as a probe (Figure
1A), and we found the 2 proteins to
co-immunoprecipitate in vivo (Figure 1B). Both interactions were
completely abolished by the Y177F point mutation, in agreement with
previous reports.8,9 In addition, no binding of Grb2 to the
p160 form of v-Abl was detected in either assay (Figure 1).
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| Fig 1.
Lack of association of Grb2 with the Bcr/Abl Y177F mutant
and with v-Abl.
(A) Far Western blot. The indicated Abl proteins were expressed by
transient transfection in 293T cells, whole cell extracts fractionated
by SDS-PAGE and transferred to nitrocellulose filters, and hybridized
with a GST-Grb2 (SH2) fusion protein (left pair) or GST alone (right
pair). Bound GST protein was detected by anti-GST antibodies and
enhanced chemiluminescence. Filters were subsequently stripped and
rehybridized with anti-Abl antibody (right panel of each pair).
Molecular weight standards are at left, and the positions of p210
Bcr/Abl and c-Abl proteins are indicated by arrowheads at the right.
(B) Co-immunoprecipitation. The indicated Abl proteins were expressed
by transient transfection of 293T cells, immunoprecipitated with
anti-Grb2 (mock-transfected cells only, left lane) or anti-Abl
antibodies (all other samples), fractionated by SDS-PAGE, transferred
to nitrocellulose, and hybridized with anti-Abl (top panel) or
anti-Grb2 (bottom panel) antibodies. Molecular weight standards are at
the right, and the position of Grb2 protein is indicated by the
arrowhead.
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p210 Y177F mutant preferentially induces B- and T-lymphoid leukemia
in vivo
The leukemogenic activity of p210 BCR/ABL, p210 Y177F, and
p160 v-abl were assessed in the mouse model system by
retroviral transduction of bone marrow from 5-fluorouracil-treated
donors. p210 BCR/ABL induced CML-like disease in all recipients
within 3 to 4 weeks of transplantation (Figure
2). These animals demonstrated distinctive
pathologic features (Table 1), including
high peripheral blood leukocyte counts (100-400 × 103/µL), massive splenomegaly (0.7-1.0 g), and
infiltration of spleen, liver, and lungs by maturing neutrophils, as
previously described.12,14
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| Fig 2.
The p210 BCR/ABL Y177F and v-abl
oncogenes are defective for the induction of CML-like disease in mice.
Kaplan-Meier-style survival curve for recipients of bone marrow from
5-FU-treated donors transduced with the indicated ABL
oncogene. The symbols in each curve designate individual mice (n = 15
for P210 WT, n = 10 for P210 Y177F, and n = 10 for v-abl).
Solid black symbols, CML-like disease; open symbols, B-lymphoid
leukemia; light gray symbols, macrophage disease; dark gray symbols,
mast cell disease; hatched symbols, T-lymphoid leukemia. Animals
diagnosed with 2 or more disease processes simultaneously, based on
histopathologic and molecular analysis (see text), are indicated by
multi-shaded symbols. The difference in survival between recipients of
p210 WT-transduced marrow and either p210 Y177F- or
v-abl-transduced marrow was highly significant
(P < .0001, Mantel-Cox test). One recipient of
v-abl-transduced marrow died 285 days after transplantation of
nonleukemic causes. Asterisks indicate mice with increased peripheral
blood neutrophils (see text).
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Mice transplanted with p210 Y177F-transduced bone marrow had strikingly
different fates. The survival of these animals was extremely prolonged
compared to recipients of wild-type p210-transduced marrow, ranging
from 10 to 22 weeks (Figure 2). Further, the Y177F animals succumbed to
distinct leukemias (Table 1). Several mice developed B-lymphoid
leukemia/lymphoma within 10 to 14 weeks, characterized by peripheral
lymphadenopathy, moderate splenomegaly, and a malignant hemorrhagic
pleural effusion that appeared to be the cause of death. The tumor
cells were positive for B220 (CD45R), CD43, 6C3/BP1, and CD24 cell
surface antigens and demonstrated largely germline configuration of the
immunoglobulin heavy chain locus (data not shown), consistent with a
late pro-B cell phenotype.26 Southern blot analysis of
genomic DNA from these tumors demonstrated monoclonal or oligoclonal
proviral integration (data not shown). These B-lymphoid leukemias were
identical to B-cell malignancies frequently observed in recipients of
marrow from non-5-FU-treated donors transduced with wild-type
BCR/ABL.14
Most recipients of p210 Y177F-transduced marrow developed a novel form
of T-cell leukemia/lymphoma (Figure 2, Table 1), not previously
observed by us in primary recipients of BCR/ABL- transduced marrow. These T-cell lymphomas were characterized by a massive abdominal mesenteric tumor, an extremely enlarged thymus, or both (Figure 3A). Spleen weights (0.1-0.2 g) and
peripheral white blood cell counts (5-10 × 103/µL)
were only moderately elevated. Abdominal ascites was also observed in
several animals. Tumor cells obtained from abdominal tumors and thymic
masses expressed the T-lymphoid cell surface antigens Thy-1 (CD90),
CD8, CD43, and CD24 (Figure 3B) but were negative for B-cell antigens
B220 and 6C3/BP-1 and the myeloid antigen CD11b (Mac-1). DNA from these
tumors contained 1 or 2 distinct proviral integrations (Figure 3C),
confirming the presence of BCR/ABL in the malignant cells. In
addition, these tumors exhibited germline configuration of the
immunoglobulin heavy chain genes (data not shown) but clonal
rearrangements of the T-cell receptor  locus (Figure 3C), verifying
their T-lymphoid origin. Assay of tumor cell-conditioned medium for
replication-competent retrovirus was negative (data not shown),
excluding the possibility that the T-lymphoma was induced by Moloney
murine leukemia virus contaminating the BCR/ABL virus stock.
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| Fig 3.
The Bcr/Abl Y177F mutant preferentially induces
T-lymphoid leukemia.
(A) Photomicrograph of section of abdominal tumor that
developed 105 days after transplantation with p210 Y177F-transduced
marrow (mouse 4, Table 1), hematoxylin-eosin stain, magnification
400×. Note the population of cells with large nuclei, prominent
nucleoli, and moderate cytoplasm and the frequent mitotic and apoptotic
figures (arrowheads). (B) Flow cytometric analysis of tumor cells from
A, demonstrating uniform expression of Thy1.2, CD43, and CD24, variable
expression of CD8, but lack of expression of the B-lymphoid marker B220
and myeloid marker Mac1. In each panel, expression of the indicated
antigen is shown by the gray plot, whereas staining by an isotype
control antibody is shown by the transparent plot. (C) Southern blot
analysis of T-lymphoid tumor DNA. Left panel: genomic DNA from
abdominal tumor (tum) and thymus (thy) from p210 Y177F mouse 4 demonstrates a single proviral integrant when hybridized with
a radioactive probe from the retroviral neo gene, whereas
thymus DNA from mouse 8 exhibits 2 different proviral clones. Control
DNA (C) demonstrates the intensity of 1 proviral copy per diploid
genome. Right panel: tumor and thymus DNA from both these mice show
loss of the germline band of the T-cell receptor chain locus
(indicated by the GL arrowhead in the control sample) and clonal
rearrangements of both alleles when hybridized with a TCR probe.
Note the thymic lymphoma from mouse 8 shows 4 new bands,
corresponding to distinct biallelic rearrangements in each of
the 2 clones. Positions of DNA size markers (in kb) are indicated on
the left.
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Although all recipients of p210 Y177F-transduced marrow died
of lymphoid leukemia, a few animals had some features of CML-like disease (Figure 2, asterisks; Table 1), including moderately increased
peripheral blood and bone marrow neutrophils and infiltration of
maturing myeloid cells into the spleen and, in some cases, the lungs.
In all such mice, analysis of provirus integration patterns showed a
single proviral integrant in tissues containing the myeloid cells that
was different from those found in the lymphoma cells (Figure
4A). This indicates that the excess myeloid
cells in these mice were derived from the transduction of a bone marrow target cell that was distinct from the target for induction of the T
lymphoma.
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| Fig 4.
Distinct target cells for different leukemias
induced by p210 BCR/ABL Y177F and v-abl.
(A) Unique proviral integrants in myeloid cells from mice with T
lymphoma and increased neutrophils. Genomic DNA from thymus (thy),
abdominal tumor (tum), ascites (asc), spleen (spl), liver (liv), and
peripheral blood (pb) of p210 Y177F mouse 7, and tumor, ascites,
pleural effusion (eff), peripheral blood, and bone marrow (bm) of mouse
8 (Figure 2, Table 1) were analyzed by Southern blot for provirus
integration pattern using a neo probe. In mouse 7 the thymus,
tumor, and ascites were composed exclusively of malignant T-lymphoid
cells, the liver was a mixture of T-lymphoma and myeloid cells, and the
spleen and peripheral blood were exclusively maturing neutrophils
(peripheral blood leukocyte count = 356 000/µL). A single provirus
is present in the T-lymphoma cells with a distinct single proviral
clone in neutrophils. In mouse 8, the tumor was composed exclusively of
T-lymphoma cells, the ascites and pleural effusion were mostly lymphoma
cells with a small amount of myeloid cells, the peripheral blood was
30% lymphoblasts and 70% neutrophils (peripheral blood leukocyte
count = 48 000/µL), and the bone marrow was 50% lymphoblasts and
50% myeloid cells. Again, there is a single provirus in the T-lymphoma
cells and a different single proviral clone in the myeloid cells. DNA
from a control cell line (con) indicates 1 proviral copy per diploid
genome. Positions of DNA size markers (in kb) are indicated on the
left. (B) Unique proviral integrants in spleen from mice with
v-abl-induced B-lymphoid leukemia and simultaneous macrophage
or mast cell disease. Genomic DNA from spleen (spl) and lymph node (LN)
from 2 representative mice (animals 1 and 8 in Figure 2, Table 2) with
v-abl-induced B-lymphoid leukemia and coexisting macrophage
and mast cell tumors (1) or mast cell tumors (8) were analyzed as in A. Lymph nodes from both mice were composed exclusively of malignant
lymphoblasts, whereas spleen contained a mixture of lymphoblasts and
infiltrating malignant macrophages or mast cells. B lymphoblasts from
mouse 1 contained 2 proviral clones, and 4 additional integrants were
present in spleen DNA. Similarly, the B-lymphoid disease was biclonal
in mouse 8, with 2 additional integrants detected in spleen. DNA from a
control cell line (con) indicates 1 proviral copy per diploid genome.
Positions of DNA size markers (in kb) are indicated on the left.
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p160 v-abl is completely defective for induction of
CML-like disease in mice
Animals reconstituted with marrow transduced with v-abl also
showed differences from the wild-type p210 BCR/ABL transplant recipients. Survival of these animals was prolonged, though not to the
extent of p210 Y177F animals (Figure 2). None of the v-abl animals developed CML-like disease; rather, these mice developed 3 distinct malignancies in varying combinations (Figure 2, Table 2). Eight of 10 mice developed B-lymphoid
leukemia that was identical to that observed in p210 Y177F mice. Six of
10 exhibited mast cell disease, characterized by tumors of malignant
mast cells in liver, spleen, bone marrow, and occasionally lungs. The
histopathology of mast cell disease was similar to that observed
previously in recipients of v-abl-transduced bone
marrow.27 Finally, 2 of 10 mice developed malignancies of
monocytes/macrophages, with tumors predominantly occurring in
the liver. As was B-lymphoma, this disease was observed previously in
recipients of wild-type p210-transduced marrow when donors were not
pretreated with 5-FU.14
Several recipients of v-abl-transduced bone marrow
developed 2 leukemias simultaneously, whereas 1 mouse had evidence of
all 3 diseases (Figure 2). Identification of such mice was possible because of the unique clinicopathologic features of each form of
leukemia. The distinct origin of the different leukemias was confirmed
by the presence of unique proviral integrants in DNA from the various
tumors (Figure 4B). Although some neutrophils were present in bone
marrow and, to a lesser extent, in spleen of some v-abl mice,
the total lack of increased peripheral blood neutrophils and of lung
infiltration indicated that v-abl was completely defective for
the induction of CML-like disease under these conditions.
| |
Discussion |
Previous work has demonstrated that mice transplanted with
p210 BCR/ABL-transduced bone marrow from 5-FU-treated donors
develop exclusively CML-like disease.14-16 In contrast,
when bone marrow from non-5-FU-treated donors is used14 or
when viral stocks of lower titer are used,12 recipients
instead develop a mixture of CML-like disease, B-lymphoid leukemia, and
monocyte/macrophage tumors. The time of development of these distinct
hematologic malignancies differs, with CML-like disease occurring
within 3 to 6 weeks of transplantation, B-lymphoid leukemia within 4 to 12 weeks, and macrophage disease within 8 to 20 weeks. Analysis of
provirus integration patterns suggests that the bone marrow target cells for induction of the 3 diseases after retroviral transduction are distinct.12,14 The target cell for the
CML-like disease exhibits a multilineage repopulating ability
consistent with an early multipotential progenitor/stem cell, whereas
the target cells for B-lymphoid leukemia and macrophage disease are lineage-restricted and resemble committed B-lymphoid and monocyte progenitors, respectively.14
In the current study, we found the p210 Y177F mutant to be
severely compromised in its ability to induce CML-like disease in mice,
and we found p160 v-abl to be completely defective. Instead, recipients of bone marrow transduced with these oncogenes developed fatal hematologic malignancies of other lineages, including B- and
T-lymphoid leukemia, macrophage disease, and malignant mast cell
disease. Similar results for p210 Y177F have been reported by
others.28 Although the retroviral target cells for the
T-lymphoid leukemia/lymphoma and mast cell tumors have not been
characterized, it is plausible that they are committed
T-lymphoid29 and mast cell27
progenitors as well. Together, these observations suggest a model for
understanding the patterns of leukemia induced by p210 Y177F and
v-abl after the retroviral transduction of murine bone marrow.
If a given form or mutant of an ABL oncogene is defective for
the induction of CML-like disease in stem cells but is competent to
induce leukemia on transduction of another target cell, transplanted animals will not develop CML-like disease early after transplantation but will succumb later to leukemia induced by the transduction of a
sensitive target cell. Our results suggest that the p210 Y177F mutant
is greatly deficient in its ability to induce the overproduction of
maturing myeloid cells characteristic of the CML-like disease and that,
rather than dying from vastly increased levels of neutrophils and the
accompanying hepatic and pulmonary dysfunction, the mice survive and
later develop lymphoid leukemia. The moderate increase in neutrophils
observed in some p210 Y177F recipients suggests that this mutant has
some residual capacity for the stimulation of myelopoiesis, and
it demonstrates that this model system is sensitive enough to detect
partial defects in leukemogenesis in different hematopoietic lineages.
The appearance of a unique proviral clone in myeloid cells from these
mice supports the hypothesis that the CML-like disease results from the
transduction of a target cell distinct from that for the lymphoid
leukemias. In related experiments, we have observed that the Bcr/Abl
SH2 domain contributes to the efficient induction of CML-like disease in mice but is completely dispensable for the induction of B-lymphoid leukemia.30 Because mice transplanted with p210 SH2
mutant-transduced 5-FU-treated marrow die from B-lymphoid leukemia
within 6 to 10 weeks after transplantation (data not shown) whereas
most recipients of p210 Y177F-transduced marrow survive longer than
this, we infer that the p210 Y177F mutant is also somewhat compromised
in its ability to induce B-lymphoid leukemia after marrow transduction. In support of this, when marrow from non-5-FU-treated donors was transduced with p210 Y177F, no recipient developed CML-like disease or
B-lymphoid leukemia by 12 weeks after transplantation (data not shown),
whereas all recipients of non-5-FU marrow transduced with P210
wild-type died from CML or B-lymphoid leukemia within this
period.14 A partial defect in B-lymphoid leukemogenesis by
p210 Y177F may explain the conflicting reports of the ability of this
mutant to transform bone marrow B-cell progenitors in vitro.8,11 Verification of this model of differential
leukemogenesis will require purification and transduction of the
individual target cells from bone marrow before transplantation.
Tyrosine 177 was initially identified as a potential Grb2 binding site
because the immediate C-terminal amino acid sequence (Y177VNV) contained an asparagine residue at position +2,
which is strongly preferred by the Grb2 SH2 domain.31,32
The p210 BCR/ABL Y177F mutant lacks direct binding to Grb2 in
vitro and fails to co-precipitate with Grb2 in vivo (Figure
2).8,9 Because Grb2 is linked to the activation of Ras
through binding of the Sos guanine nucleotide exchange
protein33 and because both Grb234 and
Ras6,7 contribute to Bcr/Abl transformation, it is likely
that direct binding of Grb2 to pY177 in Bcr/Abl is required for the
pathogenesis of CML. However, there are several other SH2-containing
signaling proteins that also prefer asparagine at position +2 to the
tyrosine, including the adapter protein 3BP2 and c-Abl
itself.32,35 Establishing the identity of the critical
effector molecule binding to phosphorylated Y177 in Bcr/Abl will
require further study. Assuming direct binding of Grb2 is required for
the induction of CML by Bcr/Abl, a mechanistic explanation of this
requirement is lacking. Although the Bcr/Abl Y177F mutant does not
induce transcription of a Ras-responsive reporter gene in
fibroblasts,8 subsequent studies demonstrated that Bcr/Abl Y177F can still stimulate guanosine triphosphate loading of Ras in
hematopoietic cells,10 possibly through the activation of pathways such as Shc.11 There are at least 2 models that
might explain a requirement for the direct binding of Grb2 by Bcr/Abl for the induction of CML. Perhaps very high levels of Ras activation, mediated by the interaction of Grb2/Sos with Bcr/Abl, are necessary for
the stimulation of myelopoiesis by Bcr/Abl. Alternatively, by analogy
to Ras activation by transmembrane tyrosine kinases such as epithelial
growth factor-receptor,36 it may be the stimulation of Ras
in the immediate vicinity of Bcr/Abl that is critical for the
pathogenesis of CML. These alternative models of Bcr/Abl signaling can
now be experimentally tested.
We found that the product of the transforming gene of Abelson murine
leukemia virus, v-Abl, lacked binding to Grb2 in vitro and in
vivo and was completely unable to cause CML-like disease under
conditions in which p210 Bcr/Abl induced this leukemia with 100%
efficiency. These observations strengthen the correlation between
binding of Grb2 and induction of CML-like disease by activated forms of
Abl. In an early version of the marrow transduction/transplantation model system that used replication-competent helper virus to increase infection efficiency, it was reported that v-Abl did induce CML-like disease, with moderate elevations of neutrophils in peripheral blood
and spleen.37 However, the neutrophils in such mice lack the v-abl provirus38 and probably represent a
reactive process to cytokines produced by some Abl-induced
tumors,39 not a true myeloproliferative disease. Others
have described a chronic myeloproliferative disease induced by the
transduction of bone marrow with v-abl,40 but
careful analysis of the description and histopathology of the disease
process suggests that these animals developed a mixture of B-lymphoid
leukemia, mast cell tumors, and macrophage disease, similar to those
observed in this study. We conclude that the v-Abl tyrosine kinase is
unable to induce CML-like disease in this model system. In addition to
failing to bind Grb2, the p160 Gag/Abl fusion protein differs from
Bcr/Abl both in the nature of the polypeptide fused to Abl and in the
site of fusion within Abl, and it lacks the Abl SH3 domain. The SH3
domain of p210 Bcr/Abl is not required for the induction of CML-like
disease in mice,41 but, because Bcr motifs other than the
Grb2 binding site42 may contribute to Bcr/Abl
transformation, further experiments will be necessary to determine
whether lack of Grb2 binding is the sole explanation for the inability
of v-Abl to induce CML- like disease.
Although other oncogenic tyrosine kinases such as the Tel-Jak2 fusion
protein43 induce a myeloproliferative-like disease after
the transduction of murine bone marrow, it is clearly not the case that
any activated tyrosine kinase will induce CML-like disease in mice.
This suggests that the activation of specific signaling pathways, such
as that mediated by the Grb2-Bcr/Abl interaction, is necessary for the
pathogenesis of CML-like myeloproliferative disease. Careful comparison
of the signaling induced in hematopoietic cells by different tyrosine
kinases should help identify these mechanisms. This study also
validates the interaction between Grb2 and tyrosine 177 of Bcr as a
target for rational drug design. Small molecules that specifically
disrupt binding of the Grb2 SH2 domain to tyrosine-phosphorylated
ligands may have clinical usefulness in the treatment of chronic-phase
CML. In conclusion, our study provides strong support for the use of
animal models to study human leukemia. In this instance, the bone
marrow transduction/transplantation model system has resolved an
important unanswered question about the pathogenesis of CML. Our
results also illustrate the complexity of analyzing leukemogenesis in
vivo. Careful and creative application of this and other animal models
should continue to provide novel insights into the molecular
pathophysiology of human leukemia.
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Acknowledgments |
We thank Dr Ann-Marie Pendergast for providing the Bcr/Abl Y177F
mutant, Dr Wojciech Swat for the gift of the TCR probe, Dr Jim Griffin
for the GST-Grb2(SH2) construct, and Dr Warren Pear for helpful
discussions and for communicating data before publication.
| |
Footnotes |
Supported in part by National Institutes of Health grants CA09595
(R.P.M.) and CA57593 (R.A.V.E.).
R.A.V.E. is a Scholar of the Leukemia Society of America and the Carl
and Margaret Walter Scholar in Blood Research at Harvard Medical School.
Reprints: Richard A. Van Etten, Center for Blood Research,
Department of Genetics, Harvard Medical School, 200 Longwood Avenue,
Boston MA 02115; e-mail: vanetten{at}cbr.med.harvard.edu.
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.
| |
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Abstract
Introduction