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NEOPLASIA
From the Division of Hematology, Department of Internal
Medicine, the Division of Oncology Research, Department of Oncology,
Mayo Clinic, Rochester, MN; the Department of Adult Oncology,
Dana-Farber Cancer Institute, Boston, MA; and the Developmental
Therapeutics Program, Division of Cancer Treatment and Diagnosis,
National Cancer Institute, Bethesda, MD.
The adenosine triphosphate binding-site-directed agent STI571 and
the tyrphostin adaphostin are undergoing evaluation as bcr/abl kinase inhibitors. The current study compared the effects of these agents on the survival of K562 cells, bcr/abl-transduced FDC-P1 cells,
and myeloid progenitors from patients with chronic myelogenous leukemia
(CML) compared with healthy donors. Treatment of K562 cells with 10 µM adaphostin resulted in decreased p210bcr/abl
polypeptide levels in the first 6 hours, followed by caspase activation
and accumulation of apoptotic cells in less than 12 hours. By 24 hours,
90% of the cells were apoptotic and unable to form colonies. In
contrast, 20 µM STI571 caused rapid inhibition of bcr/abl
autophosphorylation without p210bcr/abl degradation.
Although this was followed by the inhibition of Stat5 phosphorylation
and the down-regulation of Bcl-xL and Mcl-1, only
7% ± 3% and 25% ± 9% of cells were apoptotic at 16 and 24 hours, respectively. Instead, the cytotoxic effects of STI571 became
more pronounced with prolonged exposure, with IC90
values greater than 20 µM and 1.0 ± 0.6 µM after 24 and 48 hours, respectively. Consistent with these results, 24-hour adaphostin
exposure inhibited CML granulocyte colony-forming units (CFU-G) (median
IC50, 12 µM) but not normal CFU-G (median
IC50, greater than 20 µM), whereas 24-hour STI571
treatment had no effect on CML or normal CFU-G. Additional
experiments revealed that STI571-resistant K562 cells remained sensitive to adaphostin. Moreover, the combination of STI571 + adaphostin induced more cytotoxicity in K562 cells and in
CML CFU-G than either agent alone did. Collectively, these results
identify adaphostin as a mechanistically distinct CML-selective agent
that retains activity in STI571-resistant cell lines.
(Blood. 2002;99:664-671) Approximately 4500 new cases of chronic myelogenous
leukemia (CML) occur in the United States each year. In most patients, a characteristic t(9;22) translocation juxtaposes the 5' end of the
bcr gene with the 3' end of the abl gene,
resulting in a unique 210-kd fusion protein,
p210bcr/abl.1,2 This constitutively active
cytoplasmic kinase is capable of not only transforming murine
fibroblasts and hematopoietic cell lines, but also causing a chronic
myeloproliferative disorder resembling CML on transduction into mouse
marrow.3,4 This p210bcr/abl-induced
transformation appears to involve the activation of signaling through
the Ras-Raf and phosphatidylinositol-3 kinase/Akt pathways as well as
transcription mediated by signal transducer and activator of
transcription 5 (Stat5) and nuclear factor Until recently, treatment options for CML, which included the use of
hydroxyurea, The most widely studied p210bcr/abl inhibitor is STI571
(formerly known as CGP 57148),17 a reversible inhibitor
that occupies the adenosine triphosphate-binding pocket of
p210bcr/abl and stabilizes the kinase in an inactive
conformation.18 Preclinical studies demonstrated that
STI571 also inhibits the kinase activities of c-abl,
platelet-derived growth factor receptor, and the c-kit receptor.15,19,20 Phase 1 studies suggest that STI571 has impressive activity against chronic-phase CML21 but more
limited activity against p190bcr/abl-expressing acute
lymphocytic leukemia and the blast crisis phase of CML.22
Recent studies have demonstrated that mutation and amplification of
p210bcr/abl are observed in samples from patients who have
relapsed after STI571 therapy.23 Additional preclinical
and clinical studies of STI571, alone and in combination with
conventional cytotoxic agents, are ongoing.7,24,25
An alternative approach to inhibiting protein kinases involves the use
of small molecules that alter the binding of peptide substrates rather
than adenosine triphosphate. A chemically diverse group of agents,
generically termed tyrphostins, has been synthesized and evaluated as
potential tyrosine kinase inhibitors.26 The tyrphostin
AG957 has previously been reported to inhibit p210bcr/abl
activity in immune complex kinase assays14 and to cause
decreased p210bcr/abl autophosphorylation followed by
bcr/abl degradation in intact cells.27,28 Interestingly,
AG957 also inhibits T-cell receptor-mediated phosphorylation of the
adaptor protein c-Cbl,29 suggesting that kinases
other than bcr/abl might also be affected. Despite the lack of absolute
specificity for bcr/abl-transformed cell lines, AG957 selectively
inhibits the proliferation of CML progenitors compared with normal
myeloid progenitors.28,30 Subsequent animal testing has
revealed that AG957 has a short serum half-life (S. Stinson, V.L.N.,
E.A.S., unpublished observations, December 2000). Examination
of a series of analogues has demonstrated that adaphostin, the
adamantyl ester of AG957, has greater potency in vitro28 and a longer serum half-life in vivo (S. Stinson, V.L.N., E.A.S., unpublished observations, December 2000).
In the current study, we compared the actions of STI571 and
adaphostin in a number of preclinical models. These studies focused on
determining whether similar events occur downstream of bcr/abl kinase
inhibition after treatment with the 2 compounds. We also examined the
possibility that both of these p210bcr/abl-directed agents
might exhibit synergistic or non-cross-resistant effects.
Materials
Antibodies were purchased from the following suppliers: phosphotyrosine
and phospho-Stat5 from Upstate Biotechnology (Lake Placid, NY); c-abl
from Oncogene Research (Cambridge, MA); Stat5, procaspase-3, XIAP, and
Mcl-1 from Transduction Labs (Lexington, KY); and Bcl-xL
and FADD from PharMingen (San Diego, CA). Rabbit antiserum that
recognizes cleaved caspase-3 was from Tamie Chilcote (Elan
Pharmaceutics, San Francisco, CA). All other materials were obtained as
previously indicated.28
Cell lines
To derive FDC-P1bcr/abl cells, log-phase FCD-P1 murine interleukin-3 (IL-3)-dependent myeloid cells (kindly provided by Larry Karnitz, Mayo Clinic, Rochester, MN) growing in medium B, which consisted of RPMI 1640, 10% (vol/vol) heat-inactivated fetal calf serum, 10% (vol/vol) WEHI-conditioned medium, 100 U/mL penicillin G, 100 µg/mL streptomycin, and 2 mM glutamine, were incubated for 20 hours with the retrovirus pBabe/Puro containing p210bcr/abl cDNA behind the SV40 early promoter (kindly provided by Ruibao Ren, Brandeis University, Waltham, MA), washed twice, incubated for 48 hours in medium B, and diluted with medium B containing 5 µg/mL puromycin. Puromycin-resistant clones were subsequently isolated by limiting dilution and subjected to immunoblotting to confirm the expression of p210bcr/abl. Thereafter, cells were grown in medium B in the absence (parental FDC-P1) or presence (FDC-P1bcr/abl) of 5 µg/mL puromycin. Clonogenic assays Aliquots containing 0.5 × 106 K562 cells in 1 mL medium A were incubated with diluent, STI571, or adaphostin for the indicated lengths of time, sedimented at 100g for 5 minutes, diluted, and plated in gridded 35-mm plates in the medium of Pike and Robinson32 containing 0.3% (wt/vol) Bacto agar. After incubation for 10 to 14 days at 37°C, colonies containing at least 50 cells were counted on an inverted phase-contrast microscope.Clinical samples were studied under the aegis of a protocol approved by the Institutional Review Board of the Mayo Clinic in accordance with the policies of the US Department of Health and Human Services. To evaluate the effects of adaphostin and STI571 on normal versus CML CFU-G, 10 mL peripheral blood was drawn from healthy volunteers and consenting patients with previously untreated chronic-phase CML using EDTA as an anticoagulant. Mononuclear cells (density = 1.077 g/cm3) harvested from Ficoll-Hypaque step gradients were cultured for 24 hours at a density of 1 × 106/mL in Iscoves modified Dulbecco medium containing 20% (vol/vol) fetal bovine serum (medium C) supplemented with increasing concentrations of adaphostin or STI571. At the completion of the incubation, samples were sedimented at 200g for 10 minutes, resuspended at a concentration of 1 × 106 cells/mL in medium C containing 50 ng/mL G-CSF, plated in 0.3% agar, and incubated for 7 to 8 days. Colonies containing at least 32 cells were counted on an inverted phase-contrast microscope. Immunoblotting K562 cells were incubated with the indicated concentration of STI571 or adaphostin in medium A for 1 to 48 hours as indicated, sedimented at 200g for 10 minutes, washed in serum-free RPMI 1640 containing 10 mM HEPES (pH 7.4), and lysed in 6 M guanidine hydrochloride under reducing conditions.33 Aliquots containing 50 µg total cellular protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) containing 5% to 15% polyacrylamide gradients, transferred to nitrocellulose, and probed with primary antibodies followed by horseradish peroxidase-coupled secondary antibodies using standard procedures.34Immunoprecipitation Ten million cells were incubated in the absence or presence of 10 µM adaphostin or 20 µM STI571 for 3 hours, sedimented at 200g for 10 minutes, resuspended in 1 mL lysis buffer containing 0.5% Nonidet P-40, 50 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 150 mM NaCl, 1 mM sodium orthovanadate and 1 mM dithiothreitol supplemented with one Complete Mini protease inhibitor tablet (Boehringer Mannheim, Indianapolis, IN) per 10 mL of lysis buffer immediately before use, incubated on a rotating shaker at 4°C for 1 hour, and centrifuged at 16 000g for 15 minutes. Additional incubations were performed at 4°C with gentle rotation. After pellets were discarded, supernatants were supplemented with 20 µL protein A and 20 µL protein G beads, incubated for 1 hour, and sedimented for 5 minutes at 700g to preclear the lysates. Supernatants were transferred to fresh tubes, incubated overnight with 0.2 µg anti-Stat5 antibody, supplemented with 20 µL protein A and protein G beads, and incubated for 1 hour. After sedimentation for 5 minutes at 700g, bound immunoprecipitates were washed 3 times in 1 mL lysis buffer, solubilized in 50 µL SDS sample buffer, heated to 100°C for 5 minutes, and loaded onto 7.5% polyacrylamide gels. After transfer to nitrocellulose, immunoprecipitates were probed with antiphospho-Stat5 or anti-Stat5 antibodies essentially as described above.Fluorescence microscopy For morphologic analysis, cells were fixed in 3:1 (vol/vol) methanol:acetic acid, stained with 1 µg/mL Hoechst 33258 in 50% (vol/vol) glycerol, and examined under epi-illumination using a Zeiss Axioplan microscope (Carl Zeiss, Thornwood, NY). Two hundred to 300 cells per sample were scored for apoptotic changes (peripheral chromatin condensation or nuclear fragmentation) as described.28Transient transfections Plasmids encoding enhanced green fluorescence protein (pEGFP-N1), dominant negative caspase-9, or crmA were obtained from Clontech (Palo Alto, CA), Emad Alnemri (Thomas Jefferson University, Philadelphia, PA), and Charles Young (Mayo Clinic, Rochester, MN), respectively. Log-phase K562 or Jurkat cells were transfected in the buffer described by van den Hoff et al35 using a T840 square-wave electroporator (BTX, San Diego, CA) delivering a 240-V pulse for 10 msec. After 24-hour incubation, 30% to 40% of the cells displayed green fluorescence. The brightest 10% to 12% of the total cell population was isolated by fluorescence-activated cell sorting (FACS), exposed to drug or diluent as indicated in the legend to Figure 4A, fixed, and examined for apoptotic morphologic changes.Statistical analysis Data are expressed as the mean ± standard deviation of the indicated number of replicate experiments. Differences between samples were analyzed using 2-sided t tests. Changes in paired samples were analyzed using 2-sided paired t tests.36 The distributions of IC50 values for CFU-G from control subjects and patients with CML were compared using a 2-sided Mann-Whitney-Wilcoxon U test.36
Delayed induction of apoptosis by STI571 To compare the cellular effects of adaphostin and STI571, we initially incubated K562 cells, a cell line that expresses p210bcr/abl,37 with varying concentrations of adaphostin or STI571 for 24 hours. At the completion of the treatment, cells were sedimented and examined for apoptotic morphologic changes. Results of this analysis indicated that adaphostin induced apoptosis in a dose- and time-dependent manner. Representative experiments are shown in Figure 1A and 1B. After the addition of 10 µM adaphostin to the culture, 67% ± 7% of the cells (mean ± SD, n = 3) exhibited apoptotic morphologic changes at 16 hours and 85 ± 10% (n = 10) appeared apoptotic at 24 hours. Moreover, after treatment with lower doses of adaphostin, the percentage of apoptotic cells continued to increase even after drug withdrawal (data not shown). In striking contrast, 20 µM STI571 induced apoptosis more slowly, with only 7% ± 3% (n = 3) and 25% ± 9% (n = 9) of the cells appearing apoptotic at 16 hours and 24 hours, respectively (Figure 1A-B). If STI571 was removed after 24 hours, the percentage of apoptotic cells did not increase over time (Figure 1C). Prolonging the STI571 exposure to 48 hours or more, however, dramatically enhanced the induction of apoptosis (Figure 1C). These results suggest that the induction of apoptosis by the 2 agents is fundamentally different. In particular, adaphostin relatively rapidly initiates a process that continues to proceed even after drug withdrawal. In contrast, the induction of apoptosis by STI571 appears to be slower and to depend on the continuous presence of drug.
These differences in the kinetics of induction of apoptosis were
also reflected in the results of colony-forming assays. Treatment with
adaphostin for 24 hours reduced the ability of K562 cells to form
colonies in soft agar by several logs (Figure
2A), with an IC90 of
10 ± 2 µM (n = 17). In contrast, treatment with varying concentrations of STI571 for 24 hours had a much less dramatic effect
(Figure 2A-B), with 70% ± 10% (n = 7) of clonogenic cells retaining the ability to form colonies after 24-hour exposure to 10 µM STI571. Nonetheless, K562 cells were relatively sensitive to more
prolonged STI571 exposures, with an IC90 of 1.0 ± 0.6 µM after 48 hours (n = 5) and less than 1 µM after 72 hours
(Figure 2B).
Effect of adaphostin and STI571 on p210bcr/abl To investigate the biochemical basis for the differences seen in Figures 1 and 2, we examined the effects of adaphostin and STI571 on p210bcr/abl phosphorylation, p210bcr/abl polypeptide content, and selected downstream signals. K562 cells were incubated with the drugs for increasing lengths of time, harvested, and subjected to immunoblotting with reagents that recognize phosphotyrosine or the p210bcr/abl polypeptide (top and middle panels, respectively, Figure 3A-B). Cells treated with 10 µM adaphostin showed gradual and progressive decreases in phosphorylation of bcr/abl and other tyrosine phosphorylated polypeptides, with readily detectable phosphorylation persisting for at least 6 hours (Figure 3A, top panel). In contrast, 20 µM STI571 abolished p210bcr/abl phosphorylation within the first hour and caused marked decreases in tyrosine phosphorylation of many other polypeptides over the same time frame (Figure 3B). Despite its lower toxicity (Figures 1, 2), STI571 appeared to produce more robust inhibition of bcr/abl kinase activity in situ. Consistent with this conclusion, STI571 inhibited phosphorylation of the p210bcr/abl substrate Stat538,39 by more than 85% within 3 hours, whereas adaphostin had significantly less effect (Figure 3C).
These 2 agents also differed in their effects on p210bcr/abl polypeptide levels. Treatment with 10 µM adaphostin resulted in a gradual decrease of p210bcr/abl in the first 8 hours (Figure 3A). In contrast, STI571 had no effect on p210bcr/abl levels in the first 24 hours, though p210bcr/abl diminished markedly at 48 hours when 99% of the cells were apoptotic (Figure 3B). Apoptotic biochemical changes induced by adaphostin and STI571 Additional experiments were performed to identify changes occurring between the drug-induced alteration of p210bcr/abl and the appearance of apoptotic morphologic changes. When cells were transiently transfected with a plasmid encoding dominant-negative caspase-9,40 the induction of apoptosis by either adaphostin or STI571 was prevented (Figure 4A). In contrast, transfection with the caspase-8 inhibitor crmA41 had no effect on the induction of apoptosis by either of these agents (Figure 4A), even though the same crmA construct prevented Fas-mediated apoptosis in Jurkat cells (Figure 4A, bottom panel). These results suggest that adaphostin and STI571 both induce apoptosis through the mitochondrial pathway of caspase activation.42,43
Recent results have suggested that decreased synthesis of
Bcl-xL, a Stat5-induced antiapoptotic regulator of the
mitochondrial pathway, contributes to STI571-induced
apoptosis.44 Additional observations have suggested that
survivin and XIAP, 2 antiapoptotic products of
p210bcr/abl-induced45 NF As previously reported,7 treatment with STI571 resulted in
decreased levels of the antiapoptotic protein XIAP. This was most
prominent, however, at 48 hours (Figure 4B, lane 17), a time when 99%
of the cells in this experiment were apoptotic. In contrast, the Stat
transcriptional target Bcl-x Despite the down-regulation of Bcl-xL and Mcl-1, most STI571-treated cells remained viable for up to 24 hours, as demonstrated by the limited loss of proliferative potential (Figures 1A, 2). Consistent with this interpretation, we observed that cleavage of procaspases-9 and -3 to active fragments (Figure 4F-H) and digestion of the caspase substrates poly(ADP-ribose) polymerase and lamin A (Figure 4I-J) was limited in the first 24 hours but became more extensive at 48 hours. After treatment with adaphostin, XIAP levels again did not change until
most of the cells were apoptotic (Figure 4B, lane 8). The behavior of
other polypeptides, however, was considerably different after
adaphostin treatment. In contrast to the down-regulation of
Bcl-xL and Mcl-1 seen with STI571, adaphostin treatment
caused a consistent up-regulation of these antiapoptotic proteins at 2 to 6 hours (Figure 4C-D, lanes 3-5), followed by a down-regulation at
later time points as the cells became apoptotic. Because these changes
occurred at a time when Stat5 phosphorylation was minimally altered
(Figure 3C, lane 2), these changes appeared to be independent of
increased Stat5 activation. Despite the elevation of these antiapoptotic proteins, proteolytic cleavage of procaspases-9 and -3 to
active species was evident by 8 hours (Figure 4F, H, lane 6). This was
accompanied by cleavage of poly(ADP-ribose) polymerase and lamin A by 8 and 12 hours, respectively. In short, adaphostin was able to induce
apoptosis without the down-regulation of NF Comparison of the effects of adaphostin and STI571 in FDC-P1 and p210bcr/abl-transduced FDC-P1 The striking differences in the manner in which adaphostin and STI571 induced apoptosis in K562 cells raised the possibility that these agents might differ in other respects. To assess this possibility, we examined the selectivity of the agents for bcr/abl-expressing cells in vitro using 2 model systems.The first set of experiments was performed using the murine myeloid
line FDC-P1 transduced with a retrovirus encoding
p210bcr/abl. Consistent with changes reported in other cell
types transduced with bcr/abl kinases,6,49,50 the
expression of p210bcr/abl resulted in the up-regulation of
Bcl-xL (inset, Figure 5A) and decreased sensitivity to IL-3 withdrawal (Figure 5A) relative to
parental FDC-P1 cells. Transduction with p210bcr/abl also
sensitized the FDC-P1 cells to the cytotoxic effects of STI571 (Figure
5B). In contrast, adaphostin was at least as toxic to parental cells as
to p210bcr/abl-expressing cells (Figure 5C).
Effects on normal and CML CFU-G The results obtained in the FDC-P1 model appeared to be at odds with previous reports that AG957 and adaphostin exhibit selectivity for CML myeloid progenitors, including granulocyte erythroid macrophage megakaryocyte-CFU (CFU-GEMM), granulocyte macrophage-CFU (CFU-GM) and CFU-G, compared to the normal counterparts.28,30 To further evaluate this issue, the effects of adaphostin and STI571 on circulating CFU-G from patients with chronic-phase CML and from healthy control subjects were compared. In the 14 patients with CML who provided samples for this study, we observed an overwhelming dependence on CML clones for hematopoiesis. This was evidenced by the fact that 99%± 2.5% of the cells were Philadelphia chromosome-positive in the 12 patients who underwent bone marrow cytogenetics and that 97%± 2.8% of the cells were positive for bcr/abl rearrangement in the 7 patients who underwent fluorescence in situ hybridization studies. Results obtained using cells from 2 CML patients and the simultaneously analyzed healthy control subjects are shown in Figure 6A. A 24-hour exposure to adaphostin inhibited subsequent granulocyte colony formation in a dose-dependent manner, with 90% inhibition at 12 and 17 µM, respectively (Figure 6A, open symbols). In contrast, there was no inhibition of granulocyte colony formation in the corresponding normal samples (Figure 6A, closed symbols). As summarized in Figure 6B, the 24-hour adaphostin exposure inhibited subsequent granulocyte colony formation in 13 of 14 CML samples, with IC50 values ranging from 7 to 16 µM. In contrast, the effects on healthy control subjects were significantly different (P < .02 by Mann-Whitney-Wilcoxon U test). In particular, adaphostin did not affect CFU-G in 8 of 10 healthy control subjects examined. Although adaphostin inhibited colony formation by approximately 50% in 2 other control subjects, this was observed at all concentrations ranging from 6 to 20 µM and appeared to reflect technical difficulties with the diluent-treated samples rather than a true dose-dependent inhibition (data not shown).
When the same samples shown in Figure 6A and 6B were incubated with STI571, a different picture emerged. Even though 24-hour adaphostin treatment inhibited granulocyte colony formation by more than 90% (Figure 6A), 24-hour STI571 exposure had limited effect (Figure 6C). Examination of 12 separate CML samples indicated that treatment with 20 µM STI571 for 24 hours inhibited subsequent granulocyte colony formation only modestly, with 68% ± 23% (median, 61%) of CML CFU-G surviving. Effects on normal CFU-G were even more modest, with a median colony count of 118% of diluent-treated control subjects after 24-hour STI571 exposure. Effect of adaphostin on STI571-resistant cells K562 cell lines resistant to STI571 have been described and characterized.31 To determine whether these cells were cross-resistant to adaphostin, parental and STI-resistant cells were treated with 10 µM adaphostin for 24 hours and examined for apoptotic morphologic changes. As indicated in Figure 7A, STI571-resistant cells retained their sensitivity to adaphostin-induced apoptosis. In colony-forming assays, STI571-resistant cells (Figure 7B) also retained their sensitivity to the antiproliferative effects of adaphostin (Figure 7C).
Synergy between adaphostin and STI571 Because adaphostin works, at least in part, by down-regulating levels of bcr/abl (Figure 3A) and because decreased levels of bcr/abl would be expected to increase the sensitivity of cells to STI571,31,51,52 we evaluated the effects of combining these 2 agents. When parental K562 cells were treated with increasing concentrations of adaphostin, the addition of STI571 enhanced the antiproliferative effects at all adaphostin concentrations analyzed (Figure 8A). This enhancement was evident at STI571 concentrations as low as 2.5 µM (inset, Figure 8A). Further examination revealed that the enhanced inhibition of colony formation reflected an induction of apoptosis in a greater percentage of cells treated with the combination than of cells treated with either drug alone (Figure 8B). During treatment with 2.5 µM adaphostin, for example, the addition of 10 µM STI571 increased the percentage of apoptotic cells from 45% ± 6% to 84% ± 10% (n = 3, P < .01 by t test).
Additional experiments revealed that the effects of combining adaphostin and STI571 were not limited to K562 cells. Treatment with STI571 enhanced the antiproliferative effects of adaphostin on circulating CML myeloid progenitors as well (Figure 8C). When 6 separate CML samples were treated with adaphostin in the absence or presence of STI571, the IC50 values observed in the presence of STI571 were significantly lower (P = .01 using paired t test), with 5 of 6 samples showing readily detectable sensitization (Figure 8D).
In the current study, the effects of the bcr/abl-directed agents adaphostin and STI571 were compared in a number of model systems. Results of this analysis demonstrated that adaphostin was capable of inducing apoptosis relatively rapidly in K562 cells, whereas more prolonged exposures to STI571 were required to induce the same effect. This difference in the tempo of the cytotoxic effects reflected mechanistic differences between the 2 agents. In additional experiments, adaphostin failed to demonstrate selectivity for bcr/abl-transformed tissue culture cell lines, whereas STI571 required bcr/abl expression for its toxicity. Despite this apparent lack of selectivity in established tissue culture cell lines, adaphostin demonstrated selectivity for CML CFU-G compared with normal progenitors in vitro. Additional studies showed that adaphostin was active against STI571-resistant cells. Moreover, enhanced cytotoxic effects resulted when the 2 agents were combined. Collectively, these results not only provide evidence of clear-cut differences between the 2 agents, but also suggest potentially important implications for further development of both agents. Although STI571 and adaphostin both inhibit bcr/abl activity in vitro,14,15 the effects of these agents on p210bcr/abl-expressing K562 cells differed substantially. A 24-hour exposure to adaphostin was capable of reducing survival (Figure 1A-B) and colony-forming ability (Figure 2A) by several logs. In contrast, a 24-hour exposure to STI571 had a more limited effect on cell viability as assessed in colony-forming assays (Figure 2) or assays of apoptosis (Figures 1 and 4). On more prolonged exposure, however, STI571 became highly cytotoxic (Figures 1B, 2B, 4). These results parallel unpublished observations recently cited by Druker17 and support the decision to administer STI571 on prolonged schedules in the clinical studies. It is important to stress that these differences between adaphostin and STI571 did not reflect an inability of STI571 to inhibit p210bcr/abl activity in situ. On the contrary, STI571 induced rapid inhibition of bcr/abl activity, as indicated by the decrease in p210bcr/abl autophosphorylation, the decrease in tyrosine phosphorylation of other polypeptides, and the decrease in phosphorylation of Stat5 (Figure 3B-C). These changes were followed several hours later by down-regulation of the antiapoptotic proteins Bcl-xL and Mcl-1 (Figure 4C-D). These observations not only confirm recent reports that Bcl-xL is down-regulated in STI571-treated cells,7,44 but extend the results in 2 important ways. First, we have observed that Mcl-1, another antiapoptotic protein with a Stat-responsive element in its promoter,48 is also down-regulated by STI571 (Figure 4D). Second, our results indicate that the STI571-induced down-regulation of Mcl-1 is transient, with a subsequent up-regulation that remains unexplained but is unable to rescue the cells (Figure 4D). In contrast to STI571, adaphostin induces a much slower decrease in p210bcr/abl signaling (Figure 3A). This does not lead to an appreciable decrease in Stat5 phosphorylation in the first several hours (Figure 3C), nor does it result in an initial decrease in Bcl-xL or Mcl-1 (Figure 4C-D). On the contrary, Bcl-xL and Mcl-1 transiently increase during the first few hours of adaphostin treatment. Our results are consistent with previous reports that Mcl-1 can be induced by various types of cellular stress,53,54 and they raise the possibility that Bcl-xL might be similarly regulated. These elevated levels decline at later time points when caspases are activated (Figure 4C, lanes 6-8). Such differences in the actions of STI571 and adaphostin suggest that adaphostin is killing cells, at least in part, by a mechanism that does not involve the inhibition of p210bcr/abl-mediated signaling. Consistent with this conclusion, Losiewicz et al29 reported that AG957, a closely related analogue of adaphostin, is capable of inhibiting T-cell receptor-mediated c-Cbl phosphorylation in Jurkat cells. Because c-Cbl is a substrate of bcr/abl,55 the observation that c-Cbl phosphorylation is inhibited in AG957-treated lymphoid cells raises the possibility that AG957 and its derivative, adaphostin, are exerting their effects by inhibiting other kinases that phosphorylate c-Cbl and p210bcr/abl. The possibility of an entirely different and unsuspected target for adaphostin, however, cannot be ruled out. Although adaphostin does not require bcr/abl expression to kill established tissue culture cell lines (Figure 5), this agent does exhibit selectivity for CFU-G from CML patients compared with healthy donors (Figure 6). These results parallel previous reports that the parent drug AG957 selectively inhibits proliferation of CFU-GEMM, CFU-GM and CFU-G from CML patients.28,30 The basis for this selectivity is unknown. Resistance to STI571 has been observed in tissue culture cell lines31,51,52 and in patients treated for chronic-phase CML or blast crisis.23,56,57 The apparent differences in mechanisms of action prompted us to determine whether STI571-resistant cells were cross-resistant to adaphostin. Interestingly, STI571-resistant K562 cells retained their sensitivity to adaphostin (Figure 7), raising the possibility that these agents might have different mechanisms of resistance. In additional experiments, the effect of combining these 2 agents was examined. Treatment with the combination killed many more K562 cells than either agent did alone (Figure 8A-B). In addition, the combination inhibited the outgrowth of CML CFU-G more than either agent alone (Figure 8C-D). Combined with the results in Figure 7, these observations suggest that adaphostin should undergo further preclinical testing to evaluate its potential as a therapeutic agent in settings in which STI571 has limited efficacy, including STI571-resistant chronic-phase CML, blast-crisis CML, and bcr/abl-positive acute lymphocytic leukemia. In addition, the ability of adaphostin to sensitize p210bcr/abl-expressing cells to STI571 (Figure 8) suggests that this combination might be worthy of further preclinical and possible clinical testing.
We thank Novartis for providing STI571, Tamie Chilcote and Guy Poirier for gifts of antibodies, Ruibao Ren for viruses, Larry Karnitz for FDC-P1 cells, Judith Karp for encouragement, and Deb Strauss for secretarial assistance.
Submitted May 14, 2001; accepted September 14, 2001.
Supported in part by R01 CA85972, R01 CA69008, and T32 CA09441.
B.M.F.M. and J.C. contributed equally to this work.
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: Scott H. Kaufmann, Div of Oncology Research, Guggenheim 1301, Mayo Clinic, 200 First St SW, Rochester, MN 55901; e-mail: kaufmann.scott{at}mayo.edu.
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K. Ban, Y. Gao, H. M. Amin, A. Howard, C. Miller, Q. Lin, X. Leng, M. Munsell, M. Bar-Eli, R. B. Arlinghaus, et al. BCR-ABL1 mediates up-regulation of Fyn in chronic myelogenous leukemia Blood, March 1, 2008; 111(5): 2904 - 2908. [Abstract] [Full Text] [PDF]
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K. J. Aichberger, M. Mayerhofer, M.-T. Krauth, H. Skvara, S. Florian, K. Sonneck, C. Akgul, S. Derdak, W. F. Pickl, V. Wacheck, et al. Identification of mcl-1 as a BCR/ABL-dependent target in chronic myeloid leukemia (CML): evidence for cooperative antileukemic effects of imatinib and mcl-1 antisense oligonucleotides Blood, April 15, 2005; 105(8): 3303 - 3311. [Abstract] [Full Text] [PDF]
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T. D. Shanafelt, Y. K. Lee, N. D. Bone, A. K. Strege, V. L. Narayanan, E. A. Sausville, S. M. Geyer, S. H. Kaufmann, and N. E. Kay Adaphostin-induced apoptosis in CLL B cells is associated with induction of oxidative stress and exhibits synergy with fludarabine Blood, March 1, 2005; 105(5): 2099 - 2106. [Abstract] [Full Text] [PDF]
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G. Bandyopadhyay, T. Biswas, K. C. Roy, S. Mandal, C. Mandal, B. C. Pal, S. Bhattacharya, S. Rakshit, D. K. Bhattacharya, U. Chaudhuri, et al. Chlorogenic acid inhibits Bcr-Abl tyrosine kinase and triggers p38 mitogen-activated protein kinase-dependent apoptosis in chronic myelogenous leukemic cells Blood, October 15, 2004; 104(8): 2514 - 2522. [Abstract] [Full Text] [PDF]
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M. Okada, S. Adachi, T. Imai, K.-i. Watanabe, S.-y. Toyokuni, M. Ueno, A. S. Zervos, G. Kroemer, and T. Nakahata A novel mechanism for imatinib mesylate-induced cell death of BCR-ABL-positive human leukemic cells: caspase-independent, necrosis-like programmed cell death mediated by serine protease activity Blood, March 15, 2004; 103(6): 2299 - 2307. [Abstract] [Full Text] [PDF]
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C. Yu, M. Rahmani, D. Conrad, M. Subler, P. Dent, and S. Grant The proteasome inhibitor bortezomib interacts synergistically with histone deacetylase inhibitors to induce apoptosis in Bcr/Abl+ cells sensitive and resistant to STI571 Blood, November 15, 2003; 102(10): 3765 - 3774. [Abstract] [Full Text] [PDF]
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E. A. Sausville Is Another Bcr-Abl Inhibitor Needed for Chronic Myelogenous Leukemia? Clin. Cancer Res., April 1, 2003; 9(4): 1233 - 1234. [Full Text] [PDF]
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J. V. Melo, T. P. Hughes, and J. F. Apperley Chronic Myeloid Leukemia Hematology, January 1, 2003; 2003(1): 132 - 152. [Abstract] [Full Text] [PDF]
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B. J. Druker, S. G. O'Brien, J. Cortes, and J. Radich Chronic Myelogenous Leukemia Hematology, January 1, 2002; 2002(1): 111 - 135. [Abstract] [Full Text]
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Top
Abstract
Introduction
B
(NF
-interferon with or without cytarabine, or stem cell
transplantation, were less than satisfactory.
