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PLENARY PAPER
From the Center for Blood Research, Department of
Genetics, Harvard Medical School, Boston, MA; Department of Biomedical
Research, St Elizabeth's Hospital, Tufts University School of
Medicine, Boston, MA; and Hamon Center for Therapeutic Oncology
Research, University of Texas Southwestern Medical Center, Dallas, TX.
The effect of mutations in the Src homology 2 (SH2) domain of the
BCR/ABL oncogene on leukemogenesis was tested in a
quantitative murine bone marrow transduction/transplantation
assay that accurately models human Philadelphia-positive B-lymphoid
leukemia and chronic myeloid leukemia (CML). The SH2 domain was
not required for induction of B-lymphoid leukemia in mice by
BCR/ABL. Under conditions where the p190 and p210 forms of
BCR/ABL induce fatal CML-like myeloproliferative disease
within 4 weeks, p210 SH2 mutants induced CML-like disease in some mice
only after a significant delay, with other recipients succumbing to
B-lymphoid leukemia instead. In contrast, p190 BCR/ABL SH2
point and deletion mutants rapidly induced CML-like disease. These
results provide the first direct evidence of significant differences in
cell signaling by the Bcr/Abl tyrosine kinase between these distinct
leukemias. Contrary to previous observations, high levels of
phosphatidylinositol 3-kinase (PI 3-kinase) activity in primary
malignant lymphoblasts and myeloid cells from recipients of marrow
transduced with the BCR/ABL SH2 mutants were found. Hence,
the decreased induction of CML-like disease by the p210 BCR/ABL SH2 mutants is not due to impaired activation of PI
3-kinase.
(Blood. 2001;97:4-13) The Bcr/Abl fusion protein is the product of the
t(9;22) Philadelphia chromosome translocation found in the human
leukemias chronic myeloid leukemia (CML) and acute lymphoblastic
leukemia.1 Bcr/Abl exists in 2 major forms, p190 and p210,
generated by distinct breakpoints within the BCR gene on
chromosome 22. Relative to c-Abl, the Bcr/Abl proteins exhibit elevated
tyrosine kinase activity2,3 and have gained the ability to
transform fibroblasts,2 factor-dependent hematopoietic
cells,3 and primary bone marrow-derived B-lymphoid cells4 in vitro. In a retroviral bone marrow
transduction/transplantation model, p190 and p210 induce an identical
fatal CML-like myeloproliferative disease in recipients of transduced
marrow when donors are pretreated with 5-fluorouracil
(5-FU).5 Mice with the CML-like illness succumb within 4 weeks after transplantation and exhibit massive expansion of maturing
myeloid cells, principally neutrophils, with involvement of peripheral
blood, bone marrow, spleen, liver, and lungs. Analysis of proviral
integration shows the CML-like disease to be polyclonal and involve
multiple myeloid and B-lymphoid lineages, implicating a target cell
that is an early multipotential progenitor or stem cell.5
In contrast, when donors are not treated with 5-FU, recipients of p190-
and p210-transduced marrow develop a mixture of CML-like disease,
B-lymphoid leukemia, and macrophage tumors.5 p190 is
more potent than p210 for induction of B-lymphoid leukemia, a result
anticipated by in vitro studies.4 Bcr/Abl-induced
B-lymphoid leukemia develops between 4 and 12 weeks after transplant,
is monoclonal or oligoclonal by proviral integration and involves only
the B-lymphoid lineage,5 suggesting a lineage-restricted
target cell similar to that characterized for Abelson murine leukemia
virus,6 which requires additional events in addition to
BCR/ABL transduction for full malignant transformation.
Thus, the phenotype of the leukemia induced by BCR/ABL in
primary bone marrow depends critically on the type of cell that is
transduced, and it is plausible that the leukemogenic process in these
distinct cells may be quite different.
Bcr/Abl constitutively activates several cell signaling pathways that
contribute to transformation, including activation of Myc,7 Ras,8 c-Raf-1,9
MAPK/ERK,10 SAPK/JNK,11
STAT,12,13 NF- The role of the Src homology 2 (SH2) domain of Bcr/Abl in
transformation has been extensively studied. Mutation of the highly conserved FLVRES motif in the phosphotyrosine binding site blocks binding of the Abl SH2 domain to tyrosine phosphorylated proteins in
vitro,23 and significantly impairs transformation of
fibroblasts by SH3-deleted c-Abl23 and by p190 Bcr/Abl,
which is restored by overexpression of c-Myc.24 SH2 is not
required for transformation of cytokine-dependent hematopoietic cell
lines3,25,26 or primary bone marrow B-lymphoid cells by
Bcr/Abl in vitro,27 but the latter exhibit decreased and
delayed tumor formation in SCID mice. A recent study reported that p210
Bcr/Abl SH2 mutants were completely defective in activation of PI
3-kinase and the downstream serine/threonine kinase Akt in 32D
cells,28 and induced less hepatic and splenic myeloid
infiltration in SCID mouse recipients of transduced bone marrow,
relative to p210 wild-type. Cotransduction of marrow with retroviruses
expressing a p210 SH2 deletion mutant and a constitutively active
mutant of Akt increased spleen size and liver involvement by myeloid
cells in recipients. However, it was not clear whether the myeloid
infiltration represented acute myeloid leukemia or a CML-like process,
whether the disorder was fatal or transplantable, and whether the
myeloid cells contained the BCR/ABL gene or were a reactive
process. The role of the Bcr/Abl SH2 domain in lymphoid and myeloid
leukemogenesis is therefore unclear. In this study, we have tested the
leukemogenic activity of Bcr/Abl SH2 mutants in a bone marrow
transduction/transplantation system under conditions that accurately
model either Philadelphia-positive B-lymphoid leukemia or CML.
Cell lysates and PI 3-kinase assays
Western blotting
Bone marrow transduction/transplantation Generation and titering of retroviral stocks was as previously described5; all stocks had titers of 3 to 5 × 106 neomycin resistant colony-forming units per milliliters and gave equivalent proviral copy number, assessed by Southern blotting on transduction of NIH 3T3 cells or primary bone marrow. Balb/c mice (Taconic Farms, Germantown, NY) were used for all experiments. For induction of CML-like disease with BCR/ABL, marrow was harvested from male donors 4 days after treatment with 200 mg/kg 5-fluorouracil, prestimulated in vitro in medium containing 5% WEHI-3B cell-conditioned medium, 6 ng/mL IL-3, 10 ng/mL IL-6, and 50 ng/mL SCF (all from Peprotech, Rocky Hill, NJ). Subsequently, marrow was subjected to 2 rounds of cosedimentation with retroviral stocks in the same medium containing 2 µg/mL polybrene. Recipient female mice were prepared by 2 doses of 450 cGy gamma irradiation, and transduced marrow transplanted by intravenous injection of 0.5 × 106 cells per animal.For induction of B-lymphoid leukemia by BCR/ABL, marrow from donors that had not been 5-FU-treated was used, the prestimulation step was omitted, and cells subjected to a single round of cosedimentation with retroviral stock in medium containing 5% WEHI-3B-conditioned medium and 10 ng/mL IL-7. Immediately after transduction, cells were transplanted into lethally irradiated (2 × 450 cGy) syngeneic female recipient mice (1 × 106 cells each) or plated for Whitlock/Witte culture in RPMI 1640 medium supplemented with 10% fetal calf serum, 200 µmol/L L-glutamine, 50 µmol/L 2-mercaptoethanol, and penicillin/streptomycin. For negative selection of B220-expressing cells from p210 R1053K-transduced bone marrow, erythrocytes were lysed by addition of NH4Cl solution, leukocytes were washed and incubated with monoclonal antibody 6B2 (unconjugated, from Pharmingen, San Diego, CA) at 1.0 µg/106 cells. Antibody was removed by pelleting cells through a cushion of fetal calf serum, cell pellets resuspended and goat-antirat microbeads (Miltenyi Biotec) added, and incubated at 10°C for 15 minutes. Cells were then pelleted, washed, and subjected to 2 rounds of negative selection on a MicroMACS column with flow resistor, according to the manufacturer's instructions. Approximately 2 × 105 purified B220-negative transduced marrow cells were transplanted per animal into sublethally irradiated (450 cGy) female Balb/c mice.
SH2 mutation decreases the in vitro kinase activity of Bcr/Abl Previous studies showed that mutation of the Abl SH2 domain reduced the tyrosine kinase activity of p210 Bcr/Abl in vitro,3 and demonstrated decreased tyrosine phosphorylation of cellular proteins in fibroblasts24 and hematopoietic cell lines3,25,26 expressing Bcr/Abl SH2 mutants. To determine the relative effects of mutation of SH2 on the tyrosine kinase activities of p190 and p210 Bcr/Abl, we performed immune complex kinase assays on wild-type and SH2-mutated p190 and p210 (Figure 1). In agreement with previous reports,2,3,5 we found the in vitro kinase activity of wild-type p190 to be higher than wild-type p210, with both Bcr/Abl proteins significantly more active than c-Abl. Point mutation in the FLVRES motif decreased the kinase activity of both p190 and p210 Bcr/Abl by about 50%, whereas complete deletion of SH2 had a more dramatic effect on Bcr/Abl kinase activity, reducing p190 activity to near the level of c-Abl and p210 to significantly below that of c-Abl (Figure 1).
The Bcr/Abl SH2 domain is not required for activation of PI 3-kinase in factor-dependent myeloid and lymphoid cell lines The SH2 domain of Bcr/Abl is not required for transformation of IL-3-dependent B-lymphoid (Ba/F3) and myeloid (FDCP-1, 32D cl3) cell lines to become independent of IL-3 for survival and growth.3,25,26 In antiphosphotyrosine immunoprecipitates, extracts from each of the 3 cell lines transformed with wild-type p210 exhibited an 8- to 11-fold increase in PI 3-kinase activity relative to parental cells (Figure 2). In all 3 cell types, extracts from cells early after transformation by the p210 R1053K and p210 SH2 mutants showed prominent PI 3-kinase activity,
equal to or greater than that detected in wild-type p210-transformed
cells (Figure 2). Similar results were obtained when PI 3-kinase
activity was measured in whole cell lysates (data not shown). These
results demonstrate that the SH2 domain of Bcr/Abl is not required for activation of PI 3-kinase in hematopoietic cell lines.
The Bcr/Abl SH2 domain is not required for transformation of primary bone marrow B-lymphoid progenitors in vitro or for induction of B-lymphoid leukemia in mice To develop a model in which BCR/ABL induces exclusively B-lymphoid leukemia, we transduced bone marrow from non-5-FU-treated donor mice without prestimulation or prolonged incubation in cytokines (described in "Materials and methods"). Transduced cells were then either immediately cultured under conditions (Whitlock-Witte cultures) favoring propagation of B-lymphoid cells or transplanted into lethally irradiated syngeneic recipient mice. The p190-transduced marrow cultures reached maximal density (more than 1 × 106 cells/mL) of B220+ lymphoblasts within 7 days, with p210-transduced cultures delayed by 2 to 3 days, in agreement with previous observations4; the decrease in time required to reach confluence in our experiments is likely a consequence of increased transduction efficiency due to higher virus titers. We observed no decrease in the efficiency or kinetics of transformation by the p190 and p210 SH2 point and deletion mutants relative to the respective wild-type oncogenes, and no significant difference in the growth rates of the different populations (data not shown). These data confirm that the SH2 domain is dispensable for in vitro transformation of primary B-lymphoid cells by BCR/ABL.27Mice transplanted with marrow transduced by wild-type p190
BCR/ABL under these conditions developed B-lymphoid leukemia
with 100% incidence within 4 weeks after transplantation (Figure
3A), characterized by circulating
malignant B220+ lymphoblasts, lymphadenopathy, moderate
splenomegaly (average weight 0.25 g), normal thymus, and a
hemorrhagic malignant pleural effusion that was the cause of death. The
clinicopathologic features of the disease were identical to those
previously observed in this model system.5 Recipients of
marrow transduced with p190 Bcr/Abl containing an FLVRES point mutation
(R552K or R552L) developed identical leukemias with the same latency,
whereas p190 with a complete deletion of SH2 (
The Bcr/Abl SH2 mutants exhibit decreased kinase activity in vivo but no defect in PI 3-kinase activation in malignant lymphoblasts In primary malignant lymphoblasts isolated from mice transduced with p190 BCR/ABL and the SH2 mutants, we observed a fairly consistent global decrease in the tyrosine phosphorylation of cellular proteins in cells transformed by the p190 SH2 point mutants, and a further decrease with the SH2 deletion mutant (Figure 5). As previously observed,3,24 there was a prominent decrease in the tyrosine phosphorylation of proteins of molecular mass around 110 to 120 kd and 62 kd, but relatively similar levels of tyrosine phosphorylation of Bcr/Abl itself. We did not observe any novel tyrosine phosphorylated species in cells transduced with the p190 SH2 mutants.
To determine whether the BCR/ABL SH2 mutants were defective
in activation of PI 3-kinase activity in lymphoid cells, we prepared extracts from primary bone marrow B-lymphoid cells immediately after in
vitro transformation by BCR/ABL wild-type and the SH2 mutants. We observed prominent activation of PI 3-kinase in cells transformed by wild-type p190 and p210 BCR/ABL and no
significant or consistent decrease in PI 3-kinase activity with the SH2
point and deletion mutants (Figure 6A).
We also examined PI 3-kinase activation in malignant lymphoblasts
isolated from lymph nodes or pleural effusion from mice transduced with
p190 BCR/ABL wild-type and the SH2 mutants. As a control, we
determined PI 3-kinase activity in extracts of B220+
splenocytes isolated from mice 8 weeks after transplantation with
untransduced bone marrow. In antiphosphotyrosine immunoprecipitates, we
found high levels of PI 3-kinase activity in lymphoblasts from wild-type p190-transduced mice relative to control B220+
splenocytes, and similar levels of activated PI 3-kinase with the p190
R552K, R552L, and
The phosphotyrosine-binding function of the Abl SH2 domain is required for efficient induction of CML-like myeloproliferative disease in mice by p210 BCR/ABL, but not p190 We next tested the leukemogenicity of the Bcr/Abl SH2 mutants under conditions (described in "Materials and methods") in which wild-type p210 and p190 Bcr/Abl induce CML-like myeloproliferative disease in 100% of transplant recipients within 4 weeks (Figure 7A,B).5 Recipients of marrow transduced with the p210SH2 mutant exhibited
prolonged survival (median 8.5 weeks; Figure 7B). Some recipients
developed typical CML-like illness5 between 6 and 8 weeks
after transplant, with greatly increased peripheral blood neutrophils,
hepatosplenomegaly with myeloid cell infiltration, and pulmonary
parenchymal infiltration and hemorrhage, with the pulmonary disease the
immediate cause of death. Other mice developed fatal B-lymphoid
leukemia between 7 and 11 weeks after transplant, characterized by
peripheral lymphadenopathy, a hemorrhagic malignant pleural effusion
and infiltration of spleen and marrow with malignant B220+
lymphoblasts. This leukemia was histopathologically identical to that
observed with the mice in Figure 3, with the immediate cause of death
being the pleural effusion. A third of the recipient mice developed
both diseases simultaneously (Figure 7B); because of the distinctive
clinicopathologic features of the 2 malignancies, such animals were
easily recognized. Analysis of genomic DNA from malignant myeloid cells
by Southern blotting demonstrated the presence of 4 to 14 independent
proviral clones (average 7) contributing to the CML-like disease (data
not shown); in animals with both diseases, different sets of proviral
clones were detected in lymphoblasts and myeloid cells (Figure
8), in accordance with previous
observations that the target cells for these diseases are
distinct.5 These results demonstrate that the Abl SH2
domain is not absolutely required for induction of CML-like disease by
p210 BCR/ABL, but the efficiency of disease induction is
lower in the absence of SH2.
Surprisingly, all but 2 of the mice transplanted with marrow transduced
by the p210 SH2 point mutant (R1053K) died from B-lymphoid leukemia
around 6 to 7 weeks after transplant (Figure 7B), with little or no
gross evidence of CML-like disease at autopsy. About half of these
recipients also had small macrophage tumors confined to the liver that
did not contribute to cause of death (data not shown). The remaining 2 mice developed mixed CML-like disease and B-lymphoid leukemia. At first
glance, these results suggest that the p210 SH2 FLVRES point mutant is
more defective for myeloid leukemogenesis than p210 with complete
deletion of SH2. However, analysis of recipients of p210
R1053K-transduced marrow between 3 and 6 weeks after transplant (Table
1) revealed that most exhibited palpable
splenomegaly and peripheral blood leukocytosis (70 to 200 × 103/µL) with excess neutrophils preceding the
development of B-lymphoid leukemia. This suggests that the R1053K
mutant is also able to inefficiently induce myeloproliferative disease,
but the subsequent rapid emergence of B-lymphoid leukemia obscures the
CML-like disease by the time of death. To demonstrate this, potential
target cells for BCR/ABL-induced B-lymphoid leukemia were
removed from p210 R1053K-transduced bone marrow by negative selection
of cells expressing B220 antigen. Recipients of B220-negative
p210 R1053K-transduced marrow developed predominantly CML-like disease
with latency similar to that of the B-lymphoid leukemia (Figure 7C),
confirming that the p210 R1053K mutant is capable of inducing delayed
CML-like disease if lymphoid leukemogenesis by this mutant is
attenuated. Interestingly, although recipients with CML-like disease in
Figure 7C lacked the lymphadenopathy and pleural effusion
characteristic of B-lymphoid leukemia, all exhibited minor
populations of B-lymphoid blasts in peripheral blood and spleen (data
not shown), suggesting that not all target cells for
BCR/ABL-induced B-lymphoid leukemia express B220.
In contrast to the p210 SH2 mutants, there was no defect in induction
of CML-like disease by the corresponding p190 SH2 point mutant R552L,
and only a slight delay for induction of CML-like disease by p190
The Bcr/Abl SH2 domain is not required for activation of PI 3-kinase in primary myeloid cells from mice with CML-like disease We assayed PI 3-kinase activity in extracts from spleens and peripheral blood leukocytes from mice with CML-like disease induced by p210 Bcr/Abl wild-type and p210SH2, populations that consisted predominantly of maturing myeloid cells. Relative to control bone marrow from normal Balb/c mice, we found elevated levels of PI 3-kinase
activity in antiphosphotyrosine immunoprecipitates from recipients of
p210 wild-type-transduced marrow and equivalent activity in extracts
from recipients of p210 SH2-transduced marrow (Figure
9). Similar results were obtained with
assay of whole cell lysates (data not shown). These results demonstrate
that the SH2 domain is not absolutely required for activation of PI 3-kinase in primary myeloid cells by Bcr/Abl.
There is no qualitative defect in activation of several signaling pathways in myeloid cells by p210 SH2 To determine whether there were defects in activation of specific signaling pathways in myeloid cells from mice with p210SH2-induced
CML-like disease, we performed Western blotting with phosphorylation-specific antibodies for STAT5, Akt, ERK, and JNK on
extracts from myeloid cells from mice with CML-like disease induced by
p210 wild-type and p210 SH2 (Figure
10). We found moderate but equal
tyrosine phosphorylation of STAT5 in both p210 wild-type and
SH2-expressing cells. There was some variability in the level of
phosphorylation of Akt, ERK, and JNK between individual samples, but no
consistent and significant decrease in activation of these kinases in
p210 SH2-expressing cells. Similarly, levels of c-Myc were somewhat
variable in different samples, but were not consistently lower in p210
SH2-transformed cells.
The Philadelphia chromosome is predominantly found in 2 distinct forms of human leukemia: the chronic myeloproliferative disease CML and the acute disease B-lymphoid or lymphoblastic leukemia. These leukemias are very different in their effect on the hematopoietic system, cell of origin, and response to therapy.1 CML is typified by overproduction of maturing myeloid cells with essentially normal differentiation, arises in a hematopoietic stem cell, and in chronic phase is very responsive to low-dose myelosuppressive agents like hydroxyurea and busulfan, and to interferon-alpha. In contrast, Philadelphia-positive B-lymphoblastic leukemia is characterized by a profound arrest in lymphoid differentiation, at least some cases appear to arise in a lymphoid progenitor, and the disease is only responsive to high-dose combinations of cytotoxic chemotherapeutic agents. Although both leukemias are directly caused by Bcr/Abl, the profound difference in the biology of these malignancies suggests that the critical signaling pathways for leukemogenesis by Bcr/Abl in the 2 disorders may differ as well. Our results provide the first direct evidence of a difference in Bcr/Abl signaling between these 2 diseases in a mouse model of Philadelphia-positive leukemogenesis. We found that the phosphotyrosine-binding function of the Abl SH2 domain is dispensable for induction of B-lymphoid leukemia by Bcr/Abl, but is required for efficient induction of CML-like disease by p210 Bcr/Abl. This difference is of potential clinical relevance, as small molecules designed to disrupt the interaction of the Abl SH2 domain with phosphotyrosine-containing ligands might have therapeutic activity in chronic phase CML, but would be predicted to be ineffective in treatment of Philadelphia-positive B-lymphoid leukemia. We found that mutation of the Abl SH2 domain impairs the intrinsic
tyrosine kinase activity of Bcr/Abl proteins. The R to K mutation in
the FLVRES motif decreases kinase activity of p190 and p210 Bcr/Abl by
about half, whereas complete deletion of SH2 profoundly lowers the
activity of p190 and p210 to a level near or below that of c-Abl,
respectively. A physical interaction between the Abl SH2 domain and Bcr
first exon sequences has been postulated to contribute to activation of
Abl kinase activity,30 and it is possible that the SH2
mutations interfere with this. Despite the negative effect on in vitro
and in vivo Bcr/Abl tyrosine kinase activity, elimination of Abl SH2
domain binding to phosphotyrosine has no discernible impact on the
ability of Bcr/Abl to transform primary bone marrow B-lymphoid
progenitors in vitro and to induce B-lymphoid leukemia in vivo.
Previous studies also found no defect in transformation of B-lymphoid
cells in vitro by the p190 R552L mutant, but when these cells were
adoptively transferred by subcutaneous injection into unirradiated SCID
mice, there was a 75% decrease in efficiency of leukemia induction
relative to p190 wild-type and an increase in disease latency from 2 weeks to 10 to12 weeks.27 The reason for the difference
with our results is not known, but might reflect decreased survival or
altered vascular migration of p190 R552L-transformed lymphoid cells
after subcutaneous injection in SCID mice. In our studies, a moderate
increase in disease latency was observed for p190 with a complete
deletion of the SH2 domain. The increased latency with this mutant
appears to reflect increased time required to develop the leukemia
rather than the aggressiveness of the established disease, because the
survival (4 to 7 weeks) of mice inoculated with p190 In contrast to lymphoid leukemogenesis, our results suggest a
significant role for phosphotyrosine-binding by SH2 in the induction of
CML-like myeloproliferative disease by p210 BCR/ABL. With a p210 mutant lacking SH2, we observed a decrease in the efficiency of
induction of CML-like illness, with a delay in development of disease
in some recipients and development of B-lymphoid leukemia in others.
The demonstration of a qualitative and quantitative defect in
leukemogenesis by the p210 We found that the SH2 domain was not required for efficient induction
of CML-like disease by the p190 form of Bcr/Abl: p190 R552L was
indistinguishable from wild-type p190, whereas p190 It is somewhat surprising that the defect in induction of CML-like
disease by the p210 We also found no consistent or qualitative defects in activation of
ERK, JNK, Akt, or STAT5 in p210 SH2-transduced myeloid cells. Further,
although the Abl SH2 domain is required for transcriptional induction
of the c-MYC gene after transient expression of
v-abl and BCR/ABL in fibroblasts,34
we observed similar levels of c-Myc protein in myeloid cells expressing
the Bcr/Abl In summary, we have shown here that the structural requirements for the Bcr/Abl fusion protein to induce B-lymphoid leukemia or CML are different. The phosphotyrosine-binding function of the Abl SH2 domain is not required for B-lymphoid leukemogenesis by Bcr/Abl but is necessary for efficient induction of CML-like myeloproliferative disease by p210 Bcr/Abl. Our results demonstrate the complexity of studying leukemogenesis in animal models by oncogenes like BCR/ABL, which can transform multiple distinct hematopoietic target cells with different consequences. It is obvious that any model system of BCR/ABL leukemogenesis must be very well characterized to allow useful insights into the pathophysiology of human Philadelphia-positive leukemia. Further application of this bone marrow transduction/transplantation model system should illuminate the critical signaling pathways used by Bcr/Abl to stimulate myelopoiesis, and aid in directing drug development for treatment of CML.
We thank Dr Ann-Marie Pendergast for the p190 R552L cDNA, Dr Tomasz Skorski for the gift of 32D cells, and Dr David Frank for the generous gift of antiphospho-STAT5 antibodies. R.A.V. is a Scholar of the Leukemia and Lymphoma Society of America and the Carl and Margaret Walter Scholar in Blood Research at Harvard Medical School.
Submitted June 12, 2000; accepted September 1, 2000.
Supported by NIH grants HL03310 (R.L.I.), CA94536 (L.V.), and CA57593 (R.A.V.).
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: Richard A. Van Etten, Center for Blood Research, 200 Longwood Ave, Boston, MA 02115; email: vanetten{at}cbr.med.harvard.edu.
1. Van Etten RA. The molecular pathogenesis of the Philadelphia-positive leukemias: implications for diagnosis and therapy. In: Freireich EJ,Kantarjian H, eds. Leukemia: Advances in Treatment and Research. Vol 64. Cancer Treatment and Research. Norwood, MA: Kluwer Academic Publishers; 1993:294-325.
2.
Lugo TG, Pendergast A, Muller AJ, Witte ON.
Tyrosine kinase activity and transformation potency of bcr-abl oncogene products.
Science.
1990;247:1079-1082
3.
Ilaria RL, Van Etten RA.
The SH2 domain of P210BCR/ABL is not required for transformation of hematopoietic factor-dependent cells.
Blood.
1995;86:3897-3904
4.
McLaughlin J, Chianese E, Witte ON.
Alternative forms of the bcr-abl oncogene have quantitatively different potencies for stimulation of immature lymphoid cells.
Mol Cell Biol.
1989;9:1866-1874
5.
Li S, Ilaria RL, Million RP, Daley GQ, Van Etten RA.
The P190, P210, and P230 forms of the BCR/ABL oncogene induce a similar chronic myeloid leukemia-like syndrome in mice but have different lymphoid leukemogenic activity.
J Exp Med.
1999;189:1399-1412
6.
Tidmarsh GF, Heimfeld S, Whitlock CA, Weissman IL, Muller-Sieburg CE.
Identification of a novel bone marrow-derived B-cell progenitor population that coexpresses B220 and Thy-1 and is highly enriched for abelson leukemia virus targets.
Mol Cell Biol.
1989;9:2665-2671 7. Sawyers CL, Callahan W, Witte ON. Dominant negative myc blocks transformation by ABL oncogenes. Cell. 1992;70:901-910[CrossRef][Medline] [Order article via Infotrieve].
8.
Sawyers CL, McLaughlin J, Witte ON.
Genetic requirement for Ras in the transformation of fibroblasts and hematopoietic cells by the Bcr-Abl oncogene.
J Exp Med.
1995;181:307-313
9.
Skorski T, Nieborowska-Skorska M, Szczylik C, et al.
c-Raf-1 serine/threonine kinase is required in BCR/ABL-dependent and normal hematopoiesis.
Cancer Res.
1995;55:2275-2278 10. Cortez D, Reuther G, Pendergast AM. The Bcr-Abl tyrosine kinase activates mitogenic signaling pathways and stimulates G1-to-S phase transition in hematopoietic cells. Oncogene. 1997;15:2333-2342[CrossRef][Medline] [Order article via Infotrieve].
11.
Raitano AB, Halpern JR, Hambuch TM, Sawyers CL.
The Bcr-Abl leukemia oncogene activates Jun kinase and requires Jun for transformation.
Proc Natl Acad Sci U S A.
1995;92:11746-11750
12.
Ilaria RL, Van Etten RA.
P210 and P190BCR/ABL induce the tyrosine phosphorylation and DNA binding activity of multiple specific STAT family members.
J Biol Chem.
1996;271:31704-31710 13. Shuai K, Halpern J, ten Hoeve J, Rao X, Sawyers CL. Constitutive activation of STAT5 by the BCR-ABL oncogene in chronic myelogenous leukemia. Oncogene. 1996;13:247-254[Medline] [Order article via Infotrieve].
14.
Reuther JY, Reuther GW, Cortez D, Pendergast AM, Baldwin ASJ.
A requirement for NF-kappaB activation in Bcr-Abl-mediated transformation.
Genes Dev.
1998;12:968-981
15.
Varticovski L, Daley G, Jackson P, Baltimore D, Cantley L.
Activation of PI 3-kinase in cells expressing abl oncogene variants.
Mol Cell Biol.
1991;11:1107-1113
16.
Skorski T, Kanakaraj P, Nieborowska-Skorska M, et al.
Phosphatidylinositol 3-kinase activity is regulated by BCR/ABL and is required for the growth of Philadelphia chromosome-positive cells.
Blood.
1995;86:726-736
17.
Jain SK, Susa M, Keeler ML, et al.
PI 3-kinase activation in BCR/abl-transformed hematopoietic cells does not require interaction of p85 SH2 domains with p210 BCR/abl.
Blood.
1996;88:1542-1550 18. Harrison-Findik D, Susa M, Varticovski L. Association of phosphatidylinositol 3-kinase with SHC in chronic myelogenous leukemia cells. Oncogene. 1995;10:1385-1391[Medline] [Order article via Infotrieve]. 19. Jain SK, Langdon WY, Varticovski L. Tyrosine phosphorylation of p120cbl in BCR/abl transformed hematopoietic cells mediates enhanced association with phosphatidylinositol 3-kinase. Oncogene. 1997;14:2217-2228[CrossRef][Medline] [Order article via Infotrieve]. 20. Sattler M, Salgia R, Okuda K, et al. The proto-oncogene product p120CBL and the adaptor proteins CRKL and c-CRK link c-ABL, p190BCR/ABL and p210BCR/ABL to the phosphatidylinositol 3'-kinase pathway. Oncogene. 1996;12:839-846[Medline] [Order article via Infotrieve].
21.
Carpenter CL, Auger KL, Chanudhuri M, et al.
Phosphatidylinositol 3-kinase is activated by phosphopeptides that bind to the SH2 domains of the p85 kD subunit.
J Biol Chem.
1993;268:9478-9483 22. Rodriguez-Viciana P, Warne PH, Dhand R, et al. Phosphotidylinositol-3-OH kinase as a direct target of Ras. Nature. 1994;370:527-532[CrossRef][Medline] [Order article via Infotrieve].
23.
Mayer BJ, Jackson PK, Van Etten RA, Baltimore D.
Point mutations in the abl SH2 domain coordinately impair phosphotyrosine binding in vitro and transforming activity in vivo.
Mol Cell Biol.
1992;12:609-618
24.
Afar DEH, Goga A, McLaughlin J, Witte ON, Sawyers CL.
Differential complementation of Bcr-Abl point mutants with c-myc.
Science.
1994;264:424-426 25. Oda T, Tamura S, Matsuguchi M, Griffin JD, Druker BJ. The SH2 domain of ABL is not required for factor-independent growth induced by BCR-ABL in a murine myeloid cell line. Leukemia. 1995;9:295-301[Medline] [Order article via Infotrieve].
26.
Anderson SM, Mladenovic J.
The BCR/ABL oncogene requires both kinase activity and src-homology 2 domain to induce cytokine secretion.
Blood.
1996;87:238-244 27. Goga A, McLaughlin J, Afar DE, Saffran DC, Witte ON. Alternative signals to RAS for hematopoietic transformation by the BCR-ABL oncogene. Cell. 1995;82:981-988[CrossRef][Medline] [Order article via Infotrieve]. 28. Skorski T, Bellacosa A, Nieborowska-Skorska M, et al. Transformation of hematopoietic cells by BCR/ABL requires activation of a PI-3K/Akt-dependent pathway. EMBO J. 1997;16:6151-6161[CrossRef][Medline] [Order article via Infotrieve].
29.
Susa M, Keeler ML, Varticovski L.
Platelet-derived growth factor activates membrane-associated phosphatidylinositol 3-kinase and mediates its translocation from the cytosol.
J Biol Chem.
1992;267:22951-22956 30. Pendergast AM, Muller AJ, Havlik MH, Maru Y, Witte ON. BCR sequences essential for transformation by the BCR-ABL oncogene bind to the ABL SH2 regulatory domain in a non-phosphotyrosine-dependent manner. Cell. 1991;66:161-171[CrossRef][Medline] [Order article via Infotrieve].
31.
Million RP, Van Etten RA.
The Grb2 binding site is required for induction of chronic myeloid leukemia-like disease in mice by the Bcr/Abl tyrosine kinase.
Blood.
2000;96:664-670 32. Cantley LC, Auger KR, Carpenter C, et al. Oncogenes and signal transduction. Cell. 1991;64:281-302[CrossRef][Medline] [Order article via Infotrieve]. 33. Gu H, Pratt JC, Burakoff SJ, Neel BG. Cloning of p97/Gab2, the major SHP2-binding protein in hematopoietic cells, reveals a novel pathway for cytokine-induced gene activation. Mol Cell. 1998;2:729-740[CrossRef][Medline] [Order article via Infotrieve]. 34. Wong KK, Zou X, Merrell KT, et al. v-Abl activates c-myc transcription through the E2F site. Mol Cell Biol. 1995;15:6535-6544[Abstract].
35.
Afar DE, McLaughlin J, Sherr CJ, Witte ON, Roussel MF.
Signaling by ABL oncogenes through cyclin D1.
Proc Natl Acad Sci U S A.
1995;92:9540-9544 36. Mahlmann S, McLaughlin J, Afar DE, et al. Dissection of signaling pathways and cloning of new signal transducers in tyrosine kinase-induced pathways by genetic selection. Leukemia. 1998;12:1858-1865[CrossRef][Medline] [Order article via Infotrieve].
© 2001 by The American Society of Hematology.
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|
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|
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|
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|
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|
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|
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|
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|
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J. Dierov, Q. Xu, R. Dierova, and M. Carroll TEL/platelet-derived growth factor receptor beta activates phosphatidylinositol 3 (PI3) kinase and requires PI3 kinase to regulate the cell cycle Blood, March 1, 2002; 99(5): 1758 - 1765. [Abstract] [Full Text] [PDF]
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S. Ghaffari, C. Kitidis, M. D. Fleming, H. Neubauer, K. Pfeffer, and H. F. Lodish Erythropoiesis in the absence of janus-kinase 2: BCR-ABL induces red cell formation in JAK2-/- hematopoietic progenitors Blood, November 15, 2001; 98(10): 2948 - 2957. [Abstract] [Full Text] [PDF]
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N. C. Wolff and R. L. Ilaria Jr Establishment of a murine model for therapy-treated chronic myelogenous leukemia using the tyrosine kinase inhibitor STI571 Blood, November 1, 2001; 98(9): 2808 - 2816. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
B,
80°C until use. The reaction mixture contained sonicated standard phospholipid with equal parts PS, PI, and PIP2 at a
concentration of 0.2 mg/mL in a 50-µL reaction, incubated at 37°C
for 20 minutes after addition of 150 µmol/L and 0.55 MBq (15 µCi) 
-[32P]-ATP. Reactions were terminated
by addition of methanol:1 N HCl (1:1), phospholipids extracted twice
with chloroform, and fractionated by thin-layer chromatography and
autoradiography. PI 3-kinase activity was expressed as picomoles (pmol)
of phosphate incorporated into PI 3,4,5-P3, and presented
as fold increase relative to activity in control cells.
, macrophages) from an animal with simultaneous CML-like
disease and B-lymphoid leukemia was digested with
BglII and hybridized with a radioactive probe from
the neomycin resistance gene. Control DNA (Con) from a cell line
containing a single BCR/ABL provirus is in the first lane on
the left. DNA size markers (in kb) are at left, whereas the positions
of 4 unique proviral clones in peripheral blood neutrophils and
peritoneal macrophages are indicated by asterisks at the
right.
