Efficacy of dual-specific Bcr-Abl and Src-family kinase inhibitors in cells sensitive and resistant to imatinib mesylate.
ABSTRACT Monotherapy of chronic myeloid leukemia (CML) with imatinib mesylate has been cast into shadow by the evolution of clinical resistance during therapy. Resistance to imatinib can arise by multiple mechanisms including amplification or mutation of Bcr-Abl, and continuity of imatinib therapy is probably a poor option for either of these patient groups. Recently, however, a structurally distinct new class of drugs, the pyrido[2,3-d]pyrimidines, has been described, and these compounds are predicted to make different molecular contacts in the Abl kinase domain. These drugs potently target both the Bcr-Abl and Src-family kinase activities, both of which are thought to be relevant to survival of the leukemic cell. We asked whether these drugs could selectively induce cell death in murine cell line models of CML cells sensitive and resistant to imatinib by different mechanisms. We show that whereas the pyrido[2,3-d] pyrimidines are indeed highly potent in suppressing proliferation of Bcr-Abl-overexpressing imatinib-resistant cells, they are almost completely ineffective against cells expressing the T315I mutant. This implies that despite structural differences from imatinib, these drugs are unlikely to be useful in patients expressing this mutant Bcr-Abl protein, but may be effective in cases where selection of cells overexpressing the oncoprotein leads to refractoriness to imatinib.
- SourceAvailable from: Gary E Gallick[Show abstract] [Hide abstract]
ABSTRACT: Despite its discovery nearly a century ago, the functions of the Src family of protein tyrosine kinases (SFKs) remain incompletely understood. While much has been learned regarding the functions of Src family kinases in the last few years, new roles for Src, particularly in promoting the progression of cancer towards the metastatic phenotype, continue to emerge. SFKs, through their functions as kinases and adapter proteins in signaling complexes, regulate such diverse cellular events as proliferation, migration, cell cycle control, and apoptosis. In tumor cells, the kinase activity of Src is frequently activated, with greater increases during progressive stages. Likewise, resistance to chemotherapy also corresponds with Src kinase activity. Thus, Src activation is predictive of poor prognosis in several tumors. Recently, selective SFK inhibitors are showing promise in clinical trials in imatinib mesylate (Gleevec, Novartis) resistant chronic myelogenous leukemia. However, in vitro studies have suggested that Src inhibitors may hold promise in the treatment of solid tumors such as colon and pancreatic cancer in which new therapeutic inhibitors are desperately needed. This review will summarize briefly the structure and function of Src and the evidence for Src in promoting tumor progression and metastasis. As recent work in this laboratory and others has demonstrated that Src is a regulator of expression of diverse pro-angiogenic factors produced by tumor cells, and a regulator of the endothelial cells that respond to these factors, this review will focus on the role of Src in angiogenesis and potential roles of Src inhibitors as antiangiogenic agents.Current Cancer Therapy Reviews 12/2004; 1(1):45-50.
- [Show abstract] [Hide abstract]
ABSTRACT: Although conventional high-throughput screens performed in vitro with purified protein kinases are powerful tools to discover new kinase inhibitors, they are far from ideal for determining efficacy in vivo. As a complementary approach, cell-based, target-driven secondary screens may help predict in vivo compound potency and specificity as well as evaluate bioavailability and toxicity. Here the authors report a simple protocol for treating K562 Bcr-Abl-expressing cells with small-molecule kinase inhibitors in 96-well filter-bottom plates followed by in-plate cell lysis. The lysates were assayed via a solid-phase kinase assay, allowing determination of apparent IC(50) for known Bcr-Abl inhibitors as well as facilitating the screening of a small kinase inhibitor library. This approach may have further applications in generating lysates for analyzing kinase activity and inhibition in other nonadherent suspension cell lines.Journal of Biomolecular Screening 03/2010; 15(4):434-40. · 2.01 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Chronic myelogenous leukemia is characterized by the presence of the chimeric BCR-ABL gene, which is expressed as the constitutively active Bcr-Abl kinase. Although kinase activity is directly responsible for the clinical phenotype, current diagnostic and prognostic methods focus on a genetic classification system in which molecularly distinct subcategories are used to predict patient responses to small-molecule inhibitors of the Bcr-Abl kinase. Point mutations in the kinase domain are a central factor regulating inhibitor resistance; however, compensatory signaling caused by the activation of unrelated kinases can influence inhibitor efficacy. Kinase activity profiling can be used as a complementary approach to genetic screening and allows direct screening of small-molecule inhibitors. We developed a quantitative assay to monitor tyrosine kinase activities and inhibitor sensitivities in a model of chronic myelogenous leukemia using peptide reporters covalently immobilized on Luminex beads. Kinase activity is quantified by nonlinear regression from well-specific internal standard curves. Using optimized synthetic substrates and peptides derived from native substrates as probes, we measured kinase inhibition in cell lysates by the signal transduction inhibitors imatinib and dasatinib. Taking advantage of a convenient 96-well plate format, this assay also allows a straightforward and quantitative analysis of the differential effects of ATP and inhibitors on kinase activity. This method for analyzing a focused signaling network benefits from rigorous statistical analysis and short processing times, thereby offering a powerful tool for drug discovery and clinical testing.Molecular Cancer Therapeutics 05/2010; 9(5):1469-81. · 5.60 Impact Factor
MOLECULAR TARGETS FOR THERAPY (MTT)
Efficacy of dual-specific Bcr-Abl and Src-family kinase inhibitors in cells sensitive
and resistant to imatinib mesylate
AJ Tipping1, S Baluch1, DJ Barnes1, DR Veach2, BM Clarkson2, WG Bornmann2, FX Mahon3, JM Goldman1and JV Melo1
1Department of Haematology, Imperial College London, Hammersmith Hospital, London, UK;2Memorial Sloan-Kettering Cancer
Center, New York, USA; and3Laboratoire Greffe de Moelle, Universite ´ Victor Segalen, Bordeaux, France
Monotherapy of chronic myeloid leukemia (CML) with imatinib
mesylate has been cast into shadow by the evolution of clinical
resistance during therapy. Resistance to imatinib can arise by
multiple mechanisms including amplification or mutation of
Bcr-Abl, and continuity of imatinib therapy is probably a poor
option for either of these patient groups. Recently, however, a
structurally distinct new class of drugs, the pyrido[2,3-d]pyr-
imidines, has been described, and these compounds are
predicted to make different molecular contacts in the Abl
kinase domain. These drugs potently target both the Bcr-Abl
and Src-family kinase activities, both of which are thought to be
relevant to survival of the leukemic cell. We asked whether
these drugs could selectively induce cell death in murine cell
line models of CML cells sensitive and resistant to imatinib by
different mechanisms. We show that whereas the pyrido[2,3-d]
pyrimidines are indeed highly potent in suppressing prolifera-
tion of Bcr-Abl-overexpressing imatinib-resistant cells, they are
almost completely ineffective against cells expressing the T315I
mutant. This implies that despite structural differences from
imatinib, these drugs are unlikely to be useful in patients
expressing this mutant Bcr-Abl protein, but may be effective in
cases where selection of cells overexpressing the oncoprotein
leads to refractoriness to imatinib.
Leukemia (2004) 18, 1352–1356. doi:10.1038/sj.leu.2403416
Published online 17 June 2004
Keywords: chronic myeloid leukemia; imatinib mesylate; pyrido[2,3-d]
pyrimidines; drug resistance
Chronic myeloid leukemia (CML) is a hematopoietic stem cell
disorder caused by the oncogenic activity of the Bcr-Abl protein,
a deregulated tyrosine kinase. Inhibition of this activity with the
2-phenylaminopyrimidine imatinib mesylate (STI571, Glivecs,
Gleevect) selectively induces leukemic cell death in vitro and
in vivo. However, clinical resistance can develop by at least two
main mechanisms: overexpression of Bcr-Abl and mutation of
the Bcr-Abl kinase domain. The highly defined molecular
contacts between imatinib and the ATP-binding pocket of the
kinase domain appear to enforce exquisite selective pressure for
pre-existing mutations that abrogate drug binding.1–3Selection
of these mutant cells by exposure to imatinib can allow
outgrowth of the mutant subclone and clinical relapse. More-
over, rare cells carrying multiple amplified copies of the Bcr-Abl
fusion gene can similarly be selected from the bulk population
by the action of the drug.4–6
Dose escalation of imatinib7may be a good clinical option in
only a restricted group of patients who carry cells with mutant
Bcr-Abl molecules still sensitive to a higher concentration of the
inhibitor.8An apparent paradox is the observation that there
may be a potentially useful effect of withdrawing imatinib in
cases where, in the absence of the inhibitor, the Bcr-Abl kinase
activity in the resistant clone is much greater than that of the
original leukemic cells: as we have described for a Bcr-Abl-
overexpressing cell line model, uninhibited Bcr-Abl signaling
can be toxic to imatinib-resistant cells.9Key anecdotal cases
suggest that a similar phenomenon of regression of the mutant
clone may operate clinically.10,11
resistance such as independence from Bcr-Abl signaling4,12,13
are, by definition, also likely to be subject to selective pressure
in the presence of imatinib; thus, any therapy targeted to Bcr-Abl
activity will be ineffective in these leukemic cells.
Given the likelihood that withdrawal of imatinib will not be
able to ‘deselect’ most mutant subclones, other options are
clearly required for management of clinical imatinib resistance.
The use of other drugs, singly or in combination with imatinib,
has been reported in several in vitro studies of cell lines resistant
to imatinib (reviewed in Tipping and Melo14); however, most of
these studies have utilized drugs with relatively nonspecific
modes of action. Recently, new pyrido[2,3-d]pyrimidine com-
pounds have been described which are equally inhibitory to
Abl- and Src-family kinases in vitro. The latter are proposed to
play roles in the transduction of oncogenic signals from Bcr-
Abl.15–20This dual inhibition of multiple targets in the same
oncogenic pathway is an attractive option, as it may effectively
allow ‘combination’ therapy using a single drug. Moreover, the
smaller size of the pyrido[2,3-d]pyrimidines compared to
imatinib may circumvent the unfavourable steric blockade of
drug binding exhibited by some Bcr-Abl mutants.21The dual
specificity of these compounds may contribute to their increased
potency relative to imatinib in cellular inhibition assays in
primary and cell line models of CML. However, the main reason
for their increased potency is probably due to their ability to
inhibit Bcr-Abl irrespective of its activation status, in contrast to
imatinib, which can only bind Bcr-Abl with its activation loop in
the active conformation.22,23
Working forward from lead compounds, variants have been
prepared at the Sloan-Kettering Institute for Cancer Research
and tested for their activity and specificity. We describe here our
experiments with four representative drugs from this library in
murine cell line models of CML expressing wild-type Bcr-Abl
Other mechanisms of
Received 16 February 2004; accepted 29 April 2004; Published
online 17 June 2004
Correspondence: Professor JV Melo, Department of Haematology,
Imperial College, Hammersmith Hospital, Du Cane Road, London
W12 0NN, UK; Fax: þ 44 (0)20 8742 9335
Leukemia (2004) 18, 1352–1356
& 2004 Nature Publishing Group All rights reserved 0887-6924/04 $30.00
(both imatinib sensitive and imatinib resistant via overexpres-
sion of Bcr-Abl) and the T315I mutant Bcr-Abl. We show that
while these drugs are unable to effectively inhibit the prolifera-
tion of cells expressing the T315I mutant, they are highly potent
against cells expressing wild-type Bcr-Abl, including those in
which Bcr-Abl is overexpressed.
Materials and methods
Baf/BCR-ABL-s was generated by electroporation of Ba/F3 cells
and from this culture Baf/BCR-ABL-r was derived after a period
of selection in 1mM imatinib as described previously.4The Baf/
BCR-ABL-T315I cell line was created by transfection of a
construct engineered to express the Bcr-Abl T315I tyrosine
kinase mutant variant.24AR230-s and AR230-r are imatinib-
sensitive and -resistant clones of the CML cell line AR230,
established as previously described.4All cell lines were cultured
in RPMI1640 (Gibco, Paisley, UK) supplemented with strepto-
mycin, penicillin, L-glutamine and 10% fetal calf serum, herein
termed RF10. The Baf/BCR-ABL-r cell line was cultured in 1mM
imatinib to maintain selection of Bcr-Abl overexpression. The
Ba/F3 cell line culture was supplemented with 10% conditioned
medium from the WEHI-3B cell line, as a source of murine
IL-3.25All cell lines were kept at 371C and 5% CO2in a fully
Imatinib (kind gift of Dr Elizabeth Buchdunger, Novartis, Basel,
Switzerland) was made up to a stock solution of 1mM in water
and stored at 41C. From this, a 2mM solution was made up (2?
final concentration) in RF10. PD166326, SKI-DV-MO17, SKI-
DV1-10 and SKI-DV2-47 (collectively referred to here as SKI
drugs) were synthesised at the Sloan-Kettering Institute, NY,
USA, and were stored in powder form at ?201C in the dark.
Each was made up to a 10mM stock solution in dimethyl
sulfoxide (DMSO) and aliquoted for storage at ?201C in the
dark. Working dilutions were prepared in RF10 and 0.2mm
filter-sterilized before serial dilutions were prepared in RF10 to
2? final concentrations.
MTS cell proliferation assay
Relative cell proliferation was determined by the MTS (3-(4,5-
phenyl)-2H-tetrazolium, inner salt) assay using the Cell Titer 96
AQueous One Solution Cell Proliferation Assay (Promega,
Southampton, UK) according to the manufacturer’s instructions.
Cells were washed three times in RF10, and the corresponding
dilutions were made up in appropriate media. Cells were only
utilized if their viability was 490% by trypan blue exclusion.
For Ba/F3, 50ml of cells in RF10 þ20% WEHI were seeded at
96-well microtiter plates. For AR230, Baf/BCR-ABL-s, Baf/
BCR-ABL-r and Baf/BCR-ABL-T315I, 50ml of cells in RF10 were
seeded at 5?104cell/ml (final concentration) as above; these
concentrations were determined in pilot experiments to main-
tain a linear response between MTS absorbance and cell
number after 3 days even in the exponentially growing control
wells. Then, 50ml of 2? drug dilutions were added to the wells
cells/ml (final concentration) into flat-bottomed
to a total volume of 100ml. Blank wells containing 100ml of
RF10, and a set of 100% control wells containing 50ml of cells
and 50ml of RF10 were included. All cell lines were plated in
quadruplicate per drug for each independent experiment and
each experiment was performed twice. Owing to the strong
color of the SKI compounds, control plates comprising 50ml of
each drug dilution in 50ml of RF10 were set up and stained with
MTS after 3 days, and the drug dilutions were found not to
enhance the MTS absorbance values.
The plates were placed in an incubator for 3 days at 371C, 5%
CO2in a fully humidified atmosphere, after which 20ml of MTS
reagent were added to each well and the plates returned to the
incubator for a further 3h. The plates were then gently shaken to
spread the contents of each well evenly. Absorbance of each
well at 492nm was measured on a MKII Titertek Multiskans
Plus ELISA reader. Absorbance data were generated from blank-
corrected mean values and related to the absorbance of the
untreated control wells.
Protein lysates were prepared according to the method of
Kabarowski et al.26Protein concentrations were determined by
the Lowry method (Dc Protein Assay; BIO RAD, Hercules, CA,
USA). Approximately, 100mg protein were resolved on 10%
SDS-PAGE gels, blotted onto polyvinylidene difluoride mem-
branes (Immobilon-P; Millipore, Bedford, MA, USA) by semidry
electrophoretic transfer, probed with individual antibodies, and
visualized by the ECL system (Amersham, Little Chalfont, UK).
The following antibodies were used: PY-99 anti-phosphotyr-
osine (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and
A-2066 anti-actin (Sigma Chemical, St Louis, MO, USA).
Secondary antibodies were horseradish peroxidase-conjugated
rabbit anti-mouse IgG and swine anti-rabbit IgG (DAKO,
Glostrup, Denmark), respectively.
As previously reported, 1mM imatinib was nontoxic to Ba/F3
BCR-ABL-negative cells, and to Baf/BCR-ABL-r and Baf/BCR-
in the SKI compounds as assessed by 3-day MTS assays. Each result is
presented as the mean percentage of proliferation of unexposed
control cultures, and represents two independent experiments. Error
bars indicate one standard deviation from the mean.
Dose-dependent proliferation of parental Ba/F3 culture
Src/Bcr-Abl kinase inhibitors in imatinib-resistant cells
AJ Tipping et al
ABL-T315I imatinib-resistant cells.4,27,28These responses served
as an internal control for the behavior of the cells under study.
Exposure of Ba/F3 cells to the four SKI drugs under study
revealed that significant inhibition of proliferation was only
reached at doses greater than 2.5mM, with little difference in
toxicity below this concentration (Figure 1). This result implies
that these drugs may be as nontoxic to normal hematopoiesis as
imatinib. Above 2.5mM, a differential antiproliferative effect in
Ba/F3 was noted between the SKI drugs, with PD166326 being
significantly more cytotoxic than the other three compounds.
The trend for increased potency of PD166326 generally held
true for the Bcr-Abl-positive cell lines under test. However, its
effect in inhibiting proliferation of Baf/BCR-ABL-s and Baf/BCR-
ABL-r is markedly increased, with doses as low as 500nM being
able to induce almost complete cell death after 3 days even in
the imatinib-resistant Bcr-Abl-overexpressing clone (Figure 2).
The effect was even more striking in a human CML cell line
(AR230), where the IC50for inhibition of proliferation were five-
and four-fold lower in the imatinib-sensitive and -resistant sub-
lines, respectively, as compared to those of the corresponding
Baf/BCR-ABL cell lines (Figure 3; Table 1).
The other three SKI drugs are slightly less potent than
PD166326 but similarly act selectively in the submicromolar
range, inducing significant inhibition of proliferation of Baf/
BCR-ABL-s and Baf/BCR-ABL-r (Figure 2), but not in the
untransfected Ba/F3 cells (Figure 1). In contrast, in cells
expressing the Bcr-Abl T315I mutant, none of the SKI drugs
were effective even at concentrations of up to 10mM, a dose that
is also inhibitory to the BCR-ABL-negative Ba/F3 cells (Figure 2;
Table 1). Investigation of the effect of PD166326 treatment on
cellular tyrosine phosphorylation showed clear inhibition of the
phosphotyrosine content of, and driven by, wild-type Bcr-Abl
even at levels conferring resistance to 1mM imatinib (Baf/BCR-
ABL-r, Figure 4). Phosphotyrosine analysis also revealed that this
effect was absent in T315I-expressing cells, presumably because
of poor drug binding to this variant of Bcr-Abl.
Although the current study utilizes only one model cell line
system for imatinib-responsive and imatinib-resistant CML, this
system affords clear insight into the effect of mutation or
overexpression of Bcr-Abl on drug responses. Given that the Baf/
BCR-ABL-T315I cells show similar or even lower sensitivity to
mean percentage of proliferation of unexposed control cultures, and represents two independent experiments. Error bars indicate one standard
deviation from the mean.
Proliferation of the BCR-ABL-positive cell lines in the four drugs as assessed by 3-day MTS assays. Each result is presented as the
as assessed by 3-day MTS assays. Each result is presented as the mean
percentage of proliferation of unexposed control cultures, and
represents two independent experiments. Error bars indicate one
standard deviation from the mean.
Proliferation of the AR230 CML cell lines in PD166326
Src/Bcr-Abl kinase inhibitors in imatinib-resistant cells
AJ Tipping et al
these SKI drugs than the parental Ba/F3 cells, it is unlikely that
these drugs will prove clinically efficacious for patients in whom
imatinib exposure has led to selection of a dominant-resistant
clone expressing this mutation. Our observations are at odds
with a previously reported sensitivity to PD166326 of Baf/BCR-
ABL cells carrying the T315I mutation.29The reasons for this
apparent discrepancy are unknown at present, and can only be
attributed to the different sources of the Baf/BCR-ABL T315I cell
line. In agreement with our data, other pyridopyrimidines have
been shown to be ineffective against the T315I mutant,21and a
very recent publication also finds that these drugs are ineffective
against the T315I variant, although other mutant variants are
inhibited.28Under these circumstances, other drugs from further
derivation may prove to be more effective against the now-
familiar panoply of Bcr-Abl mutant variants with steric
hindrance. However, specificity of drug binding might be
affected if the structure of these drugs is modified to bind even
Bcr-Abl mutant variants; T315 is proposed to confer specificity
of imatinib for Abl kinase domains rather than, for instance, the
insulin receptor kinase (IRK) domain where this residue is not
conserved.23As a result, drugs that bind irrespective of the
identity of residue 315 may be somewhat nonspecific.
Part of the attraction of these SKI drugs is their capacity to
inhibit two key proteins in the pathogenesis of CML with one
therapy. Given the lack of efficacy of these drugs against the
T315I mutant, it is clear that any inhibitory effect against Src-
related proteins is of little significance unless Bcr-Abl is also
effectively inhibited. The importance of the latter is demon-
strated by the marked antiproliferative effect of all four
compounds in the Baf/BCR-ABL-r cells which, in spite of an
increased level of Bcr-Abl expression, experience a clear
reduction in Bcr-Abl autophosphorylation. Our results suggest
that these pyridopyrimidines may prove useful in the treatment
of patients who develop resistance to imatinib due to
amplification and overexpression of the Bcr-Abl gene, in
addition to those with kinase mutations other than the T315I,
as proposed by von Bubnoff et al.28It is also possible that these
dual inhibitors may be effective in overcoming primary
resistance to imatinib, a phenomenon with largely unknown
causes, but unlikely to be due to Abl kinase mutations.30,31
The role of Bcr-Abl in driving CML signaling is uncontro-
versial in early-phase disease, but the exact significance of Src-
family protein activity in disease progression requires further
study. We, along with others, note that the increase in potency
over imatinib and the prior demonstration of Src-family protein
inhibition19,28,32,33may be linked issues, the former conferred
by the latter. Recent history suggests that one could expect a
repeat of the imatinib experience with future signal transduction
inhibitors, and the emergence of a (perhaps novel) set of mutant
protein variants selected for by each drug. However, dual-
inhibition of Src family kinases and Bcr-Abl does imply a much-
reduced rate of mutant clone selection, given the mathematical
rarity, probably as a factorial reduction, of a cell spontaneously
and randomly mutated at specific residues in two proteins. To
this end, the derivation of an arsenal of small-molecule Bcr-Abl
and Src-family protein inhibitors with characterized stereospe-
cificity and a suggested order of clinical utility should be
encouraged in order to supply hematologists with ‘ammunition
for a moving target’.
We are grateful to Dr Elisabeth Buchdunger (Novartis Pharma,
Basel, Switzerland) for the gift of imatinib mesylate, and to Dr
Charles Chuah (Department of Haematology, Imperial College)
for technical help. This work was supported by grants from the
Leukaemia Research Fund, UK (AJT, SB, DJB, JMG, JVM); NCI
Grants CA64593 and CA08748; The Albert C Bostwick Founda-
tion, Mr William H Goodwin and Mrs Alice Goodwin and the
Commonwealth Cancer Foundation for Research, The Experi-
mental Therapeutics Center of Memorial Sloan-Kettering Cancer
Center, New York, The Enid A Haupt Charitable Trust, The
Leukemia & Lymphoma Society, The United Leukemia Fund, The
Westvaco Corporation and MeadWestvaco (DRV, WGB, BMC);
and the Association pour la recherche sur le cancer and Fondation
de la recherche medicale, France (FXM).
1 Gambacorti-Passerini CB, Gunby RH, Piazza R, Galietta A,
Rostagno R, Scapozza L. Molecular mechanisms of resistance to
imatinib in Philadelphia-chromosome-positive leukaemias. Lancet
Oncol 2003; 4: 75–85.
Table 1 IC50values (in mM) for inhibition of proliferation
Cell linePD166326 SKI
IC50values for inhibition of proliferation were interpolated from mean
dose–response curves generated from the MTS data with each drug.
N/A: 50% inhibition of cell proliferation was not achieved with doses of
up to 10mM SKI DVMO17. NT¼not tested.
of the Bcr-Abl-expressing cells after 24-h incubation in 0, 50 or 250nM
PD166326. Bcr-Abl constitutes the highest molecular weight band on
each lane (arrowed), and is progressively dephosphorylated by the
drug in Baf/BCR-ABL-s. Overexpression of Bcr-Abl in Baf/BCR-ABL-r
does not significantly prevent protein inhibition by PD166326;
however, expression of the T315I variant in Baf/BCR-ABL-T315I does.
Relative protein loading is assessed by reprobing the filter for b-actin
and is shown beneath the phosphotyrosine result.
Western blot showing relative phosphotyrosine content
Src/Bcr-Abl kinase inhibitors in imatinib-resistant cells
AJ Tipping et al
2 von Bubnoff N, Peschel C, Duyster J. Resistance of Philadelphia-
chromosome positive leukemia towards the kinase inhibitor
imatinib (STI571, Glivec): a targeted oncoprotein strikes back.
Leukemia 2003; 17: 829–838.
3 Luzzatto L, Frassoni F, Melo JV. Imatinib: can one outwit chronic
myeloid leukemia? Haematologica 2002; 87: 898–901.
4 Mahon FX, Deininger MW, Schultheis B, Chabrol J, Reiffers J,
Goldman JM et al. Selection and characterization of BCR-ABL
positive cell lines with differential sensitivity to the tyrosine kinase
inhibitor STI571: diverse mechanisms of resistance. Blood 2000;
5 le Coutre P, Tassi E, Varella-Garcia M, Barni R, Mologni L, Cabrita
G et al. Induction of resistance to the Abelson inhibitor STI571 in
human leukemic cells through gene amplification. Blood 2000; 95:
6 Weisberg E, Griffin JD. Mechanism of resistance to the ABL
tyrosine kinase inhibitor STI571 in BCR/ABL-transformed hemato-
poietic cell lines. Blood 2000; 95: 3498–3505.
7 Kantarjian HM, Talpaz M, O’Brien S, Giles F, Garcia-Manero G,
Faderl S et al. Dose escalation of imatinib mesylate can overcome
resistance to standard-dose therapy in patients with chronic
myelogenous leukemia. Blood 2003; 101: 473–475.
8 Corbin AS, Rosee PL, Stoffregen EP, Druker BJ, Deininger MW.
Several Bcr-Abl kinase domain mutants associated with imatinib
mesylate resistance remain sensitive to imatinib. Blood 2003; 101:
9 Tipping AJ, Mahon FX, Lagarde V, Goldman JM, Melo JV.
Restoration of sensitivity to STI571 in STI571-resistant chronic
myeloid leukemia cells. Blood 2001; 98: 3864–3867.
10 Gorre ME, Mohammed M, Ellwood K, Hsu N, Paquette R, Rao PN
et al. Clinical resistance to STI-571 cancer therapy caused by
BCR-ABL gene mutation or amplification. Science 2001; 293:
11 Hochhaus A, Kreil S, Corbin AS, La Rosee P, Muller MC, Lahaye T
et al. Molecular and chromosomal mechanisms of resistance to
imatinib (STI571) therapy. Leukemia 2002; 16: 2190–2196.
12 Donato NJ, Wu JY, Stapley J, Gallick G, Lin H, Arlinghaus R et al.
BCR-ABL independence and LYN kinase overexpression in
chronic myelogenous leukemia cells selected for resistance to
STI571. Blood 2003; 101: 690–698.
13 Tipping AJ, Deininger MW, Goldman JM, Melo JV. Comparative
gene expression profile of chronic myeloid leukemia cells innately
resistant to imatinib mesylate. Exp Hematol 2003; 31: 1073–1080.
14 Tipping AJ, Melo JV. Imatinib mesylate in combination with other
chemotherapeutic drugs: in vitro studies. Semin Hematol 2003; 40
(2 Suppl 3): 83–91.
15 Danhauser-Riedl S, Warmuth M, Druker BJ, Emmerich B, Hallek
M. Activation of Src kinases p53/56lyn and p59hck by p210bcr/abl
in myeloid cells. Cancer Res 1996; 56: 3589–3596.
16 Plattner R, Kadlec L, DeMali KA, Kazlauskas A, Pendergast AM. c-
Abl is activated by growth factors and Src family kinases and has a
role in the cellular response to PDGF. Genes Dev 1999; 13:
17 Warmuth M, Forster K, Stanglmaier M, Schuster C, Hallek M. PP1,
a tyrosine kinase inhibitor specific for Src-family kinases,
selectively inhibits survival of BCR-ABL expressing myeloid cells.
Blood 1999; 94 (Suppl.1): 387a.
18 Lionberger JM, Wilson MB, Smithgall TE. Transformation of
myeloid leukemia cells to cytokine independence by Bcr-Abl is
suppressed by kinase-defective Hck. J Biol Chem 2000; 275:
19 Clarkson B, Strife A, Wisniewski D, Lambek CL, Liu C. Chronic
myelogenous leukemia as a paradigm of early cancer and possible
curative strategies. Leukemia 2003; 17: 1211–1262.
20 Golas JM, Arndt K, Etienne C, Lucas J, Nardin D, Gibbons J et al.
SKI-606, a 4-anilino-3-quinolinecarbonitrile dual inhibitor of Src
and Abl kinases, is a potent antiproliferative agent against chronic
myelogenous leukemia cells in culture and causes regression of
K562 xenografts in nude mice. Cancer Res 2003; 63: 375–381.
21 La Rosee P, Corbin AS, Stoffregen EP, Deininger MW, Druker BJ.
Activity of the Bcr-Abl kinase inhibitor PD180970 against
clinically relevant Bcr-Abl isoforms that cause resistance to
imatinib mesylate (Gleevec, STI571). Cancer Res 2002; 62:
22 Nagar B, Bornmann WG, Pellicena P, Schindler T, Veach DR,
Miller WT et al. Crystal structures of the kinase domain of c-Abl in
complex with the small molecule inhibitors PD173955 and
imatinib (STI-571). Cancer Res 2002; 62: 4236–4243.
23 Schindler T, Bornmann W, Pellicena P, Miller WT, Clarkson B,
Kuriyan J. Structural mechanism for STI-571 inhibition of Abelson
tyrosine kinase. Science 2000; 289: 1938–1942.
24 Azam M, Latek RR, Daley GQ. Mechanisms of autoinhibition and
STI-571/imatinib resistance revealed by mutagenesis of BCR-ABL.
Cell 2003; 112: 831–843.
25 Ihle JN, Keller J, Henderson L, Klein F, Palaszynski E. Procedures
for the purification of interleukin 3 to homogeneity. J Immunol
1982; 129: 2431–2436.
26 Kabarowski JH, Allen PB, Wiedemann LM. A temperature sensitive
p210 BCR-ABL mutant defines the primary consequences of BCR-
ABL tyrosine kinase expression in growth factor dependent cells.
EMBO J 1994; 13: 5887–5895.
27 Hoover RR, Mahon FX, Melo JV, Daley GQ. Overcoming STI571
resistance with the farnesyl transferase inhibitor SCH66336. Blood
2002; 100: 1068–1071.
28 von Bubnoff N, Veach DR, Miller WT, Li W, Sanger J, Peschel C
et al. Inhibition of wild-type and mutant Bcr-Abl by pyrido-
pyrimidine-type small molecule kinase inhibitors. Cancer Res
2003; 63: 6395–6404.
29 Huron DR, Gorre ME, Kraker AJ, Sawyers CL, Rosen N, Moasser
MM. A Novel pyridopyrimidine inhibitor of Abl kinase is a
picomolar inhibitor of Bcr-abl-driven K562 cells and is effective
against STI571-resistant Bcr-abl mutants. Clin Cancer Res 2003; 9:
30 Melo JV, Hughes TP, Apperley JF. Chronic myeloid leukemia.
Hematology (Am Soc Hematol Educ Program) 2003, 132–152.
31 Hochhaus A. Cytogenetic and molecular mechanisms of resistance
to imatinib. Semin Hematol 2003; 40 (2 Suppl 3): 69–79.
32 Dorsey JF, Jove R, Kraker AJ, Wu J. The pyrido[2,3-d]pyrimidine
derivative PD180970 inhibits p210Bcr-Abl tyrosine kinase and
induces apoptosis of K562 leukemic cells. Cancer Res 2000; 60:
33 Wisniewski D, Lambek CL, Liu C, Strife A, Veach DR, Nagar B
et al. Characterization of potent inhibitors of the Bcr-Abl and the
c-kit receptor tyrosine kinases. Cancer Res 2002; 62: 4244–4255.
Src/Bcr-Abl kinase inhibitors in imatinib-resistant cells
AJ Tipping et al