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NEOPLASIA
From the Departments of Bioimmunotherapy, Cancer
Biology, and Molecular Pathology, University of Texas, M D Anderson
Cancer Center, Houston, TX.
Clinical studies have shown that the tyrosine kinase inhibitor
STI571 effectively controls BCR-ABL-positive chronic myelogenous leukemia (CML). However, disease progression while on STI571 therapy has been reported, suggesting de novo or intrinsic resistance to
BCR-ABL-targeted therapy. To investigate possible mediators of
acquired STI571 resistance, K562 cells resistant to 5 µM STI571 (K562-R) were cloned and compared to the parental cell population. K562-R cells had reduced BCR-ABL expression and limited activation of
BCR-ABL signaling cascades (Stat 5, CrkL, MAPK). STI571 failed to
activate caspase cascades or to suppress expression of survival genes
(bcl-xL) in resistant cells. Gene sequencing and tyrosine kinase
activity measurements demonstrated that K562-R cells retained wild-type
and active BCR-ABL tyrosine kinase that was inhibitable by in vitro
incubation with STI571, suggesting that BCR-ABL was not coupled to
proliferation or survival of K562-R cells. The src-related kinase LYN
was highly overexpressed and activated in K562-R cells, and its
inhibition reduced proliferation and survival of K562-R cells while
having limited effects of K562 cells. Specimens taken from patients
with advanced CML that progressed on STI571 therapy also were analyzed
for LYN kinase expression, and they were found to be elevated to a
level similar to that of K562-R cells. Comparison of samples from
patients taken prior to and following STI571 failure suggested that
expression and/or activation of LYN/HCK occurs during disease
progression. Together, these results suggest that acquired STI571
resistance may be associated with BCR-ABL independence and mediated in
part through overexpression of other tyrosine kinases.
(Blood. 2003;101:690-698) Cytogenetic abnormalities are common in
adult-onset leukemias, and much attention has been focused on
understanding both the cause and consequence of specific
alterations.1 One of the first and most highly
investigated cytogenetic changes is the 9:22 reciprocal chromosomal
translocation in chronic myelogenous and, to a lesser extent, acute
lymphocytic leukemias.1,2 Translocation places the c-abl
gene under the transcriptional control of the bcr locus, allowing
expression of a hybrid protein encoded by 1 to 3 exons of the bcr gene
and all but the first exon of c-abl.3 This chimeric
BCR-ABL protein (p190 or p210) expresses intrinsic tyrosine kinase
activity with altered compartmentalization and distinctions in
substrate accessible when compared to the predominantly nuclear c-abl
protein.4 Tyrosine kinase activity is essential for the
transforming function of BCR-ABL, and expression of BCR-ABL in stem
cells of immune-deficient mice results in altered hematopoiesis
resembling human leukemialike disorders.5,6 These
observations support a role for BCR-ABL in early leukemogenesis and as
a specific target for therapeutic intervention in chronic myelogenous
leukemia (CML).
BCR-ABL expression alters many signaling pathways that increase cell
survival and cell cycle progression.3,7 Many of these
pathways are used by cytokines that regulate hematopoiesis, and
constitutive enforcement of these cascades by BCR-ABL prolongs survival
and provides a proliferative advantage early in
leukemogenesis.8 Altered survival and cell cycle
regulation may promote additional chromosomal alterations and mutations
that parallel or amplify BCR-ABL transformation. These changes may lead
to acceleration of the disease and play a role in the aggressive nature
of late-stage CML. Although many changes have been described in
late-stage disease, some evidence suggests that additional tyrosine
kinases that function downstream of BCR-ABL or are activated in
leukemic blasts (LYN, HCK) contribute to late-stage
disease.9,10
STI571 (Gleevec, imatinib mesylate; Novartis AG, Basel, Switzerland) is
a tyrosine kinase inhibitor, active against BCR-ABL and other specific
kinase targets.11,12 The drug has effective clinical
activity in CML and other BCR-ABL (+) leukemias and has recently been
approved by the Food and Drug Administration for patients with
BCR-ABL(+) leukemia. Patients recently diagnosed (< 1 year ago) or in
early phases of the disease achieve early and stable hematologic
remission with loss of the Philadelphia (Ph) chromosome in some
patients.13 Patients with late-stage disease (accelerated
phase or blast crisis) can achieve hematologic remission but frequently
progress on therapy.11-13 These results suggest that
although BCR-ABL expression is retained, STI571 responsiveness may be
reduced. Several mechanisms have been proposed that account for loss of
effective STI571 therapy in advanced disease, including pharmacologic
barriers or BCR-ABL gene amplification/mutation, as suggested in recent
studies.14-18 Cell model studies of minimally STI571-resistant leukemic cell clones have shown that BCR-ABL overexpression may account for loss of STI571 sensitivity, but other
cell models suggest mechanisms unrelated to changes in BCR-ABL expression.14-18 More mechanistic studies of STI571
resistance are necessary to understand cellular and clinical
responsiveness to STI571.
To define alternate mechanisms of STI571 resistance, K562 erythroid
leukemic cells were selected for high-level resistance to STI571
(IC50 > 5 µM). Protein, signaling, and inhibitor
studies suggest that these cells had become resistant to STI571 through loss of cellular dependence on BCR-ABL and not through mutations or
loss of sensitivity to STI571-mediated kinase inhibition. Growth and
survival in these cells was controlled by overexpression and/or activation of tyrosine kinases that are not inhibited by STI571, and
analysis of clinical specimens support a role for the src-family of
kinases in STI571 resistance and progressive disease. This cell model
predicts that chronic BCR-ABL inhibition may promote outgrowth of
BCR-ABL-independent CML cells, allowing cells to evade STI571-mediated apoptosis.
Cell lines, kinase inhibitors, drugs, and antibodies
Antibodies used in these studies include poly (ADP-ribose) polymerase
(PARP), phosphoMAPK, mitogen-activated protein kinase (MAPK)
(Cell Signaling, Beverly, MA), phosphotyrosine, phosphoSTAT5, CrkL
(Upstate Biotechnology Institute, Lake Placid, NY),
c-abl8E9, c-src (Oncogene Sciences, San Diego, CA),
bcl-xL, LYN, HCK, phosphoHCK (Santa Cruz Biotechnology,
Santa Cruz, CA), and actin (Sigma, St Louis, MO). Polyclonal anti-STAT5
(a/b) was kindly provided by Dr Robert Kirken (University of Texas,
Health Science Center, Houston, TX).
Isolation of STI571-resistant K562 cells
Apoptosis and cell survival measurement PARP cleavage was used as a measure of apoptosis and was examined in cell lysates by immunoblotting, as previously described.19,24 Methyl-thiazol tetrazolium (MTT) assays were performed as previously described to quantitate changes in cell proliferation/survival.19Analysis of kinase expression and signal transduction Protein levels of BCR-ABL, Stat 5, LYN, HCK, and phosphotyrosine were compared between parental and resistant cells by immunoblotting equal protein cell lysates (determined by bicinchoninic acid protein assay [BCA], Pierce Chemical, Rockford, IL) with specific antibodies. Antibodies against phosphorylated (activated) forms of signaling intermediates also were used to analyze changes in BCR-ABL signaling (Stat 5, MAPK) and other tyrosine kinases (phosphoHCK, HCK, LYN). CrkL was immunoprecipitated from cell lysates (see "Immune complex tyrosine kinase activity assay"), resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotted with anti-p-Tyr to examine its tyrosine phosphorylation. The blot was stripped of primary antibody and reblotted with anti-CrkL to determine its relative expression and recovery by immunoprecipitation. All immunoblots were developed with horseradish-peroxidase-conjugated secondary antibodies (BioRad Laboratories, Hercules, CA) and enhanced chemiluminescence (ECL) reagent (Amersham Pharmacia, Arlington Heights, IL).Immune complex tyrosine kinase activity assay K562 and K562-R cell lysates (400 µg in lysis buffer; as described by Donato and Perez24) were incubated with 2 µg of antibody against c-abl, LYN or HCK (2 hours), and protein A/G-sepharose (40 µL; 50% slurry, 1 hour). Immune complexes were washed (with lysis buffer), and tyrosine kinase activity was measured in immune complexes by resuspension in kinase buffer consisting of 20 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), pH 7.5, 10 mM MnCl2, 0.1 mM Na2VO4, and 10 µg enolase, as previously described.25 Kinase inhibitors (at the concentrations indicated) were preincubated with immune complexes for 30 minutes in some assays. Kinase reactions were initiated by the addition of 10 µCi (0.37 MBq) [32P]-adenosine triphosphate ([32P]-ATP) in a total volume of 50 µL and were incubated for 30 minutes at room temperature. Kinase reactions were quenched by the addition of SDS sample buffer, and after heating to 100°C for 5 minutes reactions were resolved by SDS-PAGE. Phosphoproteins were detected by autoradiography and quantitated by PhosphorImager (Molecular Dynamics, Sunnyvale, CA).RT-PCR amplification of BCR-ABL and sequencing of the ABL kinase domain For bcr-abl reverse transcription-polymerase chain reaction (RT-PCR), mRNA was isolated as described below. RT-PCR reactions were performed in a 50 µL volume using SuperScript One-Step RT-PCR with Platinum Taq from Invitrogen (Carlsbad, CA). Reagents were at the following final concentrations: 1 × reaction mix, 1 µg total RNA, 0.2 µM sense primer, 0.2 µM antisense primer, 4 mM MgSO4, and 2 units RT/Platinum Taq mix. RT-PCR was performed on an MJ Research PTC-200 DNA Engine (Waltham, MA) as follows: for cDNA synthesis, 30 minutes at 55°C followed by 2 minutes at 94°C; for PCR, 40 cycles of 94°C for 15 seconds, 59°C for 30 seconds, and 72°C for 80 seconds. Reactions were run on a 1% agarose gel, and the 1.3-kb bcr-abl bands were excised, purified, and eluted in a 30 µL volume using a gel extraction kit from Qiagen (Valencia, CA). Platinum Taq DNA polymerase was used for nested PCR amplification of the abl kinase domain of the 1.3-kb bcr-abl PCR product. Reaction components were 1 × PCR buffer, 0.2 mM each dNTP, 1.5 mM MgCl2, 0.2 µM sense primer, 0.2 µM antisense), 5 µL of the eluted DNA from above, and 2.5 units of platinum Taq. PCR was performed on an MJ Research PTC-200 DNA Engine as follows: 1 cycle of 94°C for 2 minutes and 30 cycles of 94°C for 15 seconds, 56°C for 30 seconds, and 72°C for 30 seconds. Reaction products were purified as above and sequenced on a Biomeck 3700 automated DNA sequencer (Applied Biosystems, Foster City, CA). Primers were obtained from Sigma-Genosys (The Woodlands, TX), and the sequences used were as follows: forward: 5'-gaagcttctccctggcatcccgt-3' and reverse 5'-gccaggctctcgggtgcagtcc-3'; for amplification of a 1.3-kb bcr-abl product representing the BCR-ABL junction and kinase domain. For nested PCR of the 323-bp kinase domain: forward: 5'-gcgcaacaagcccactgtctatgg-3' and reverse 5'-gtagtccaggaggttcccgt-3'.Lyn and BCR-ABL Northern blot K562 cell RNA was extracted with Trizol reagent as previously described.26 For Northern blot, 15 µg total RNA was separated on a formaldehyde gel and transferred to a Schleicher and Schuell nylon membrane (Keene, NH) using standard protocols. The membrane was probed with 20 ng/µL of a biotinylated 1.3-kb bcr-abl PCR amplification product (as described in "RT-PCR amplification of BCR-ABL and sequencing of the ABL kinase domain") or a biotinylated lyn probe using New England Biolabs' NEBlot Phototope Kit (Beverly, MA) according to the standard hybridization protocol. The template for the lyn probe was a lyn insert separated from the pcDNA3-HA-lyn vector, kindly provided by Dr Seth Corey (University of Pittsburgh, Pittsburgh, PA). The probe was detected using the maximum sensitivity protocol from New England Biolabs' Phototope-Star detection kit.LYN antisense treatment Phosphorothioate antisense oligodeoxynucleotide LYN sequence (Lyn-AS), as previously described,27,28 and a sense oligomer representing the first 7 codons of the LYN gene (Lyn S1; complementary to Lyn-AS) were used in these studies (Sigma-Genosys). To examine cellular effects of oligomers, 20 000 cells growing in 96-well plates in RMPI 1640 media with 10% fetal bovine serum were treated with 20 µM oligomer for 24 hours. Cell media were supplemented with 5 µM oligomers for an additional 48 hours before cell growth and survival were estimated by MTT assays as described above. To monitor effects on LYN expression, cells were treated with 10 µM oligomer for 24 hours and supplemented with the same concentration for an additional 48 hours before lysates were prepared as described above. Equal protein (40 µg) cell lysates were resolved and immunoblotted with anti-LYN or antiactin as a protein loading control.Analysis of LYN expression in specimens from STI571-treated CML patients Blood samples were taken from accelerated-phase and blast-crisis CML patients prior to and during treatment with STI571 and, in some cases, during disease progression but before drug withdrawal. Single specimens from CML patients that had measurable hematologic responses to STI571 but subsequently progressed on therapy were also collected prior to withdrawal from STI571. All patients received 400 to 600 mg STI571 daily. All studies involving human subjects were approved by the Internal Review Board of M D Anderson Cancer Center, and informed consent was obtained from each patient prior to initiation of this procedure.Briefly, fresh peripheral blood (~ 18 mL) was overlaid onto Histopaque-1077 (Sigma) and centrifuged at 400g for 15 minutes. The cells at interphase were removed by aspiration and washed once with phosphate buffered saline. Cell preparations containing significant red blood cell contamination were subjected to treatment with ammonium chloride potassium (ACK) lysis buffer (0.154 M ammonium chloride, 0.01 M KHCO3, 0.13 µM EDTA [ethylenediaminetetraacetic acid]) for 30 minutes. Remaining cells were lysed in solubilization buffer (as described above) for 30 minutes on ice, and lysates were clarified by centrifugation at 12 000g (4°C) for 15 minutes. The supernatant fractions were retained, and protein content was measured by BCA protein dye reagent (Pierce). Next, 20 µg protein were resolved by SDS-PAGE (8% acrylamide), transferred to nitrocellulose membranes, and immunoblotted with anti-phosphoHCK, anti-HCK, or anti-LYN (Santa Cruz Biotechnology). The antigen was detected with secondary antibody and ECL reagent as described above. After primary antigen detection, the membrane was stripped and reprobed with antiactin to determine the relative protein load in each lane.
To examine potential mechanisms of acquired STI571 resistance,
K562 cells were cloned in the presence of 5 µM STI571, and stable
clones were compared to the parental population for STI571 responsiveness. As shown in Figure 1A,
parental K562 cell responsiveness is detected at nM concentrations of
STI571 (IC50 ~ 0.1 µM), while 10 µM STI571
failed to reduce K562-R cell survival or proliferation. These cells
expressed equal sensitivity to doxorubicin (Figure 1B), demonstrating
defects in STI571 responsiveness that were not mediated by expression
of multidrug resistance genes29 or global changes in
responsiveness to an apoptotic stimulus. Because previous studies
suggested that changes in BCR-ABL expression and mutations in the
kinase domain correlate with STI571 responsiveness in resistant
cells,14,15,17,18 BCR-ABL expression, signaling, and gene
mutations were examined in K562 and K562-R cells. As shown in Figure
2, BCR-ABL mRNA, protein expression, and
signaling (Stat5, MAPK, CrkL) were reduced in resistant cells.
Sequencing of the nested PCR product derived from a 1.3-kb BCR-ABL
RT-PCR template (as described in "Materials and methods") failed to
detect mutations in the abl kinase domain (codons 225-328) of K562-R cells. To confirmed wild-type BCR-ABL tyrosine kinase activity in
resistant cells, immune complex kinase assays of BCR-ABL
immunoprecipitates were performed. As shown in Figure
3, BCR-ABL tyrosine kinase activity was
measurable in both parental and resistant cells, and incubation with
STI571 reduced BCR-ABL tyrosine kinase activity and substrate (enolase)
phosphorylation. This analysis suggested that STI571 resistance was not
due to overexpression or mutations affecting STI571 binding in K562-R
cells. Further, down-regulation and inhibition of BCR-ABL were not
associated with an STI571 antiproliferative response in these cells.
Although tyrosine phosphoprotein levels were reduced in resistant
cells, a tyrosine phosphoprotein of ~60 kDa was highly expressed in
K562-R cells (Figure 2). Several techniques were used to identify this
protein and to define its role in K562-R cells.
As previously described, STI571 induces apoptosis through inhibition of
BCR-ABL-mediated tyrosine phosphorylation, changes in survival gene
expression, and activation of caspase cascades.19 As shown
in Figure 4, STI571 reduced tyrosine
phosphorylation and bcl-xL expression, and it induced PARP cleavage in
K562 cells but failed to affect these changes in K562-R
cells.19 However, a tyrosine kinase inhibitor with
reported activity against both src-family and abl kinases (PD180970)
reduced tyrosine phosphorylation in both K562 and K562-R
cells.20,30 Tyrosine phosphorylation of the p60
phosphoprotein was partially reduced by PD180970 in K562-R cells
(Figure 4), suggesting a relationship to src-family kinases or kinase
substrates. PD180970 induced PARP cleavage and growth inhibition in
both STI571-sensitive and -resistant K562 cells (Figure
5A-B), and inhibitors with specificity
for src kinases (PP2) induced greater antiproliferative and apoptotic
effects on K562-R cells (Figure 5C). These results suggested that
expression or activation of a src-family kinase in K562-R plays a role
in STI571 resistance.
Previous studies demonstrated that specific members of the src kinase
family, such as HCK and LYN, are expressed and activated in leukemic
blasts.9 Additional studies demonstrated that these kinases (HCK, LYN) are activated by BCR-ABL kinase,10 and
HCK appears to play an important role in BCR-ABL-mediated cytokine independence.31 Immunodepletion of K562-R cell lysates
demonstrated that anti-LYN but not anti-HCK reduced p60 recovery in
cell supernatants, confirmed by the loss of LYN from depleted lysates
(Figure 6). K562-R cell LYN protein and
mRNA expression were increased 4- to 8-fold, respectively, when
compared to K562 cells (Figure 6B-C). As shown in Figure 6D,
overexpression of LYN in K562-R cells correlated with a 7-fold increase
in LYN tyrosine kinase activity when examined in immune complex kinase
assays with exogenous substrate (enolase). HCK immune complexes from
either K562 or K562-R cells had no detectable tyrosine kinase activity,
demonstrating a specific increase in LYN expression and tyrosine kinase
activity in STI571-resistant K562-R cells. From these results, we
concluded that the p60 tyrosyl-phosphoprotein in STI571-resistant K562
cells is overexpressed LYN kinase.
To determine whether LYN kinase plays a role in K562-R cell growth and
survival, LYN kinase activity or expression was suppressed by
incubation with src-family kinase-specific inhibitor or LYN antisense,
respectively. CGP-76030 (Novartis AG) is representative of a class of
substituted 5,7-diphenyl-pyrrolo [2,3d]pyrimidines previously shown
to inhibit src activity in vitro and to effect osteoclastic activity in
animal models.21,22 The specificity of this compound was
tested in BCR-ABL or LYN immune-complexes from K562 or K562-R cells. As
shown in Figure 7, CGP-76030 inhibited LYN kinase activity from both K562 and K562-R cells with nM
sensitivity. Dose-dependent LYN kinase inhibition was measurable in
K562-R cell-derived immune complexes. However, incubation of BCR-ABL immune complexes with CGP-76030 at LYN inhibitory concentrations had
limited BCR-ABL tyrosine kinase inhibitory affects. Similar results
were obtained with immune complexes derived from K562 cells. These
results demonstrate that CGP-76030 has greater LYN kinase inhibitory
activity when compared to BCR-ABL in vitro. Distinctions in LYN kinase
inhibition by CGP-76030 in immune complexes and intact cells may be due
to the cellular ATP content that can reduce efficacy of kinase
inhibition.
The effects of CGP-76030 on LYN kinase phosphorylation (activation),
growth, and apoptosis were examined in K562-R cells. As shown in Figure
8A, CGP-76030 reduced LYN tyrosine
phosphorylation at a site previously shown to be involved in its
autophosphorylation/activation (Y508; Porter et al32) and
induced PARP proteolysis after extended inhibition of tyrosine kinase
activity (> 12 hours). CGP-76030 treatment resulted in greater
antiproliferative effects on K562-R cells than that measured in K562
cells. As shown in Figure 8B, CGP-76030 at 1.25 µM and lower
concentrations had greater inhibitory effects on K562-R cells
than parental STI571-sensitive K562 cells. Inhibitors of other protein
kinases (50 µM AG-490, 50 µM LY298002) previously shown to play a
role in leukemic cell growth and survival (Janus kinase 2 [Jak 2];
Wilson-Rawls et al33; phosphatidylinositol-3'-kinase, Neshat et al34) failed to induce apoptosis or reduce
proliferation of K562 or K562-R cells by more than 50% (data not
shown). Together with the in vitro effects of CGP-76030, these
data provide evidence of a role for LYN in K562-R cell growth
and survival.
To confirm a role for LYN expression in K562-R cell growth and
survival, the effects of LYN antisense (LYN-AS) oligonucleotide incubation were examined and compared to STI571-sensitive K562 cells.
Incubation with LYN-AS reduced LYN expression by ~ 50% in both
K562 and K562-R cells (Figure 9), while
LYN sense oligomers (LYN-S) had little effect of LYN expression.
Importantly, LYN-AS significantly reduced K562-R cell growth (~ 50% reduction) while having limited effects on K562 cells. In
contrast, LYN-S had only minimal effects on growth of either
population. Together, these results suggest that LYN expression plays a
significant growth regulatory role in K562-R cells.
CML blast-crisis patients frequently progress on STI571 therapy, and
clinical studies suggest that progression may be related to STI571
resistance.13,14,18 Resistance can be mediated by mutations within the ATP binding site in the abl kinase
domain.14 However, other mechanisms may also play a role
in progressive disease.16,18 Based on the K562 cell model,
increased expression of src-family kinases may play a role in acquired
STI571 resistance. To examine this possibility, CML samples from
blast-crisis patients who progressed on STI571 (400-600 mg doses;
progression within 3 months) were subjected to immunoblot and compared
to K562-R cells for LYN and HCK expression. Samples were collected from relapsing patients prior to discontinuing STI571 therapy. As shown in
Figure 10A, LYN kinase alone or LYN and
HCK expression was detected in all samples tested. Expression levels in
all samples were similar to those detected in K562-R cells (lane 1). To
determine whether these changes correlated with disease progression,
clinical samples were taken from blast-crisis patients prior to
initiation of STI571 therapy. These samples were compared to specimens
from the same patient after disease progression on therapy (49 to 186 days of STI571 therapy). Myeloblast contents varied by no more than
25% in these samples. As shown in Figure 10B, a moderate increase in LYN expression (and activation) was detected in patient A, while more
significant changes in LYN (and HCK) were detected in patient B. Increased expression correlated with activation of these kinases when
monitored by immunoblotting with LYN/HCK activation-specific antibody
(p-HCK, p-LYN). These results suggest that src-family kinases are
highly expressed and activated in CML blast-crisis patients and their
increased expression correlates with progressive disease or STI571
resistance in some CML patients. Based on in vitro studies of K562
cells, the results also suggest that chronic STI571 exposure may induce
expression or activation of other tyrosine kinases (that are unaffected
by STI571), which contribute to BCR-ABL-independent growth and STI571
resistance.
STI571 has demonstrated remarkable clinical activity in CML, but chronic use of this inhibitor may result in reduced efficacy, STI571 resistance, and disease progression. Clinical trials with STI571 have shown that blast-crisis patients frequently progress within 3 to 6 months of treatment, suggesting that BCR-ABL inhibition is not sufficient to prevent disease progression or to restrict clonal expansion of resistant cells.35 The underlying mechanisms of STI571 resistance and clonal expansion are not fully understood. Clinical samples from resistant patients suggest that CML cells retain expression of BCR-ABL and dependence on its downstream signaling for sustained growth and survival. BCR-ABL is overexpressed or is unaffected by STI571 due to BCR-ABL point mutations or increased drug efflux, allowing cells to escape STI571-mediated apoptosis and growth inhibition. However, cells expressing BCR-ABL mutations do not appear to predominate in CML patients because high-sensitivity procedures (nested PCR) are required to detect gene mutations.14,36-38 Even with high-sensitivity detection techniques, only a limited percentage of resistant patients express detectable BCR-ABL gene mutations, suggesting other resistance mechanism exist. The results presented in this report suggest that acquired resistance to STI571 is not associated with drug resistance mechanisms or mutations in the BCR-ABL gene but may be a consequence of expansion of BCR-ABL-independent cells. K562-R cells express wild-type BCR-ABL based on sequence analysis (kinase domain) and in vitro kinase measurements. Immune complex kinase assays demonstrated comparable BCR-ABL tyrosine kinase activity in extracts from both K562 and K562-R cells, which was inhibited by in vitro incubation with STI571. Together with other characteristics of K562-R cells (reduced BCR-ABL expression and downstream signaling), these results suggest that BCR-ABL inhibition is not linked to STI571-resistant cell apoptosis. This is aligned with the observed loss of constitutive Stat5 or MAPK activation in K562-R, previously shown to be important contributors to BCR-ABL-mediated transformation.19,39-41 The results presented in Figures 2 and 4 demonstrate that through a reduction in BCR-ABL expression and reduced signaling through chronic STI571 inhibition, K562-R cells are no longer BCR-ABL kinase or BCR-ABL signaling dependent. Expression of the src-related LYN kinase may play a role in acquisition of BCR-ABL independence. This phenotype is rare when compared to other cell models of STI571 resistance, which frequently report increased BCR-ABL expression and signaling activity. Our other cell models (Mo7e, BV-173) did not achieve complete STI571 resistance but rather a 2- to 3-fold shift in STI571 sensitivity and a corresponding increase in BCR-ABL expression (data not shown). Due to its unique characteristics, the K562-R variant was the focus of this report. LYN kinase previously has been shown to be an important component in cytokine signal transduction in a variety of cell types and is reported to play a key role in the growth and apoptotic regulation of hematopoietic cells.42,43 In K562-R cells, overexpression and activation of LYN kinase appear to play a dominant role in their proliferation and survival. This conclusion is based on studies with kinase inhibitors, which were previously reported to target src kinases (PP2) or to inhibit both abl and src kinases (PD180970) in BCR-ABL-expressing cells.20 Growth inhibition and apoptotic studies demonstrate that targeting both kinase families overcomes apoptotic resistance in K562-R cells, while src-selective inhibitors show a consistently greater inhibitory effect on K562-R cells when compared to the parental population. A novel src inhibitory compound (CGP-76030; Missbach et al21,22) was shown to dose-dependently inhibit LYN kinase activity in vitro without significant affects on BCR-ABL (Figure 7). Selective inhibition of LYN in K562-R cells with CGP-76030 may explain their increased sensitivity to this compound when compared to K562 wild-type cells. We have been unable to detect cellular affects of CGP-76030 on LYN phosphorylation in parental K562 cells, perhaps due to its low level of expression and activation in this cell line. However, despite low expression levels, inhibition of LYN kinase activity by CGP-76030 in K562 cells only has limited affects on the growth of these cells when compared to the K562-R cell line (Figure 8). These observations suggest a shift induced through chronic STI571 exposure from BCR-ABL to LYN kinase dependence in STI571-resistant K562-R cells. In support of this conclusion, we also demonstrated that although LYN was expressed at considerably higher levels in K562-R cells, antisense oligonucleotides that suppress LYN expression had greater inhibitory effects on K562-R cells when compared to the K562 parental population (Figure 9). These results further support a role for LYN kinase in the growth and apoptotic protection of STI571-resistant K562-R cells. Of significance to these studies, both LYN and the related HCK kinase
have been shown to be activated by BCR-ABL,9,10 and some
studies suggest that HCK activity is essential for cytokine independence of BCR-ABL-expressing cells.31 However, LYN
and HCK kinase also may be activated through other mechanisms, as shown
in studies of blasts from acute leukemias.9 LYN kinase activity appears to be essential for signal transduction of stress kinase pathways and tyrosine phosphorylation of proteins involved in
DNA repair or damage recognition.44,45 Thus, LYN
overexpression and activation in STI571-resistant cells are likely to
promote growth and apoptotic protection through a signaling cascade
that is distinct from that of BCR-ABL. These pathways are currently being examined, but initial studies have failed to detect a role for
nuclear factor- LYN overexpression also was detected in lysates from STI571-resistant CML patients. However, other STI571-resistant mechanisms, including BCR-ABL mutations, also may exist. Prior to evaluating LYN expression in clinical specimens from these patients (progressed within 3 to 6 months of STI571 therapy), BCR-ABL T315 mutations (using the nested PCR restriction digest analysis as previously reported; Gorre et al14) were analyzed but none were detected. Further sequence analysis of the kinase domain in 4 specimens demonstrated only wild-type BCR-ABL expression (data not shown) and the possible existence of other resistance mechanisms in these patients. In 2 patients where clinical specimens were obtained prior to STI571 therapy, the effects on LYN expression and activation as patients progressed were evaluated. Changes in LYN and HCK expression and activation were detected in both patients with marked increased expression in patient B (Figure 10B). While it is unclear whether expression and/or activation of src kinases are sufficient to confer STI571 resistance in clinical samples, these results support evidence of a role for this kinase family in late-stage disease and BCR-ABL autonomous growth. Targeting both the abl and src kinases in late-stage patients may reduce disease progression and prevent acquired resistance to STI571. As shown in Figure 10, altered signaling pathways, such as LYN, may be engaged in CML cells as a compensatory response to potent or chronic BCR-ABL inhibition. From the current study, it is hypothesized that CML cells may toggle between BCR-ABL and LYN dependence as a means of reducing susceptibility to STI571-induced growth arrest and apoptosis. Each of these kinases appear to signal through independent downstream cascades, providing an exploitable means of evaluating shifts in BCR-ABL dependence in clinical specimens. However, the appropriate signaling pathways engaged by LYN (or related kinases) first must be defined and are currently under investigation. Lineage-specific expression of other tyrosine kinases (such as BTK in acute lymphocytic leukemia patients) may play a parallel role in reducing BCR-ABL dependence and reducing STI571 efficacy in other Ph(+) leukemias.47 Overall, the results presented in this report suggest that STI571 resistance in K562 cells is mediated through BCR-ABL independent activation and overexpression of LYN. Tyrosine kinases with distinct ATP binding pockets that are not accessible to STI571 (such as those of the src family) may underlie development of BCR-ABL independence in some CML cells. Targeted inhibition of LYN kinase may circumvent STI571 resistance and disease progression in CML. Additional studies of BCR-ABL independence and secondary signaling events in STI571-resistant patient-derived cell lines will improve our understanding and therapy for advanced-stage CML.
Submitted November 8, 2001; accepted September 13, 2002.
Supported by a grant from the Leukemia Society of America (6153-02 [N.J.D.]).
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: Nicholas J. Donato, Department of Bioimmunotherapy, University of Texas, M D Anderson Cancer Center, 1515 Holcombe Blvd, Box 422, Houston, TX 77030; e-mail: ndonato{at}mdanderson.org.
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Abstract
Introduction
-actin on the same membrane.
B, Jak kinases, Stat proteins (Stat 1, 3, or 5),
phosphatidylinositol-3'-kinase, or Akt phosphorylation (data not shown)
in LYN signaling in K562-R cells. LYN-mediated phosphorylation of
BCR-ABL itself (as recently reported for the closely related HCK
kinase; Schindler et al