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Experimental Therapeutics |
Division of Hematology/Oncology [C. Y., G. K., M. R., S. G.], and the Department of Radiation Oncology [P. D., R. M.], Medical College of Virginia, Richmond, Virginia 23298, and the Department of Medicine, Tufts University, Boston, Massachusetts 02135 [L. V.]
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ABSTRACT |
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200 nM) resulted in early suppression (i.e., at 6 h) of p42/44 MAPK phosphorylation followed at later intervals (i.e., 
24 h) by a marked increase in p42/44 MAPK phosphorylation/activation. Coadministration of a nontoxic concentration of the MEK1/2 inhibitor PD184352 (5 µM) prevented STI571-mediated activation of p42/44 MAPK. Cells exposed to STI571 in combination with PD184352 for 48 h demonstrated a very dramatic increase in mitochondrial dysfunction (e.g., loss of 
m and cytosolic cytochrome c release) associated with procaspase-3 activation, poly(ADP-ribose) polymerase cleavage, and the appearance of the characteristic morphological features of apoptosis. Similar results were obtained using other pharmacological MEK1/2 inhibitors (e.g., PD 98059 and U0126) as well as another leukemic cell line that expresses Bcr-Abl (e.g., LAMA 84). However, synergistic induction of apoptosis by STI571 and PD184352 was not observed in human myeloid leukemia cells that do not express the Bcr-Abl kinase (e.g., HL-60 and U937) nor in normal human peripheral blood mononuclear cells. Synergistic potentiation of STI571-mediated lethality by PD184352 was associated with multiple perturbations in signaling and apoptotic regulatory pathways, including caspase-dependent down-regulation of Bcr-Abl and Bcl-2; caspase-independent down-regulation of Bcl-xL and Mcl-1; activation of JNK, p38 MAPK, and p34cdc2; and diminished phosphorylation of Stat5 and CREB. Significantly, coexposure to PD184352 strikingly increased the lethality of a pharmacologically achievable concentration of STI571 (i.e., 1–2 µM) in resistant K562 cells expressing marked increases in Bcr-Abl protein levels. Together, these findings raise the possibility that treatment of Bcr-Abl-expressing cells with STI571 elicits a cytoprotective MAPK activation response and that interruption of the latter pathway (e.g., by pharmacological MEK1/2 inhibitors) is associated with a highly synergistic induction of mitochondrial damage and apoptosis. They also indicate that in the case of Bcr-Abl-positive cells, simultaneous interruption of two signal transduction pathways may represent an effective antileukemic strategy. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B (9)
.
Given the well-defined role of the Bcr/Abl kinase in CML and related disorders, it represents a very attractive molecular target for pharmacological intervention. Recently, considerable attention has focused on CGP57148B, currently referred to as STI571, an inhibitor of the Bcr/Abl, Kit, and platelet-derived growth factor receptor kinases (10)
. In in vitro studies, STI571 has been shown to inhibit the growth of Bcr/Abl-positive leukemic cells at micromolar concentrations (11)
. Interestingly, exposure of such cells to STI571 promotes leukemic cell apoptosis (12)
, suggesting that Bcr/Abl not only confers on these cells a growth advantage but is at the same time required for their survival. Importantly, clinical trials have now demonstrated that STI571, when administered at doses of 300 mg/day, achieves clinical remissions in the large majority of patients with CML (e.g., 96%; Ref. 13
). In addition, preclinical studies have demonstrated that the combination of STI571 with established chemotherapeutic drugs (e.g., ara-C) results in enhanced toxicity in Bcr/Abl-positive leukemias (14
, 15)
. These findings raise the possibility that combining STI571 with such agents might lead to enhanced activity in CML and/or circumvention of drug resistance. In this context, Vigneri and Wang (16)
reported recently that coadministration of STI571 with leptomycin, an inhibitor of the nuclear export sequence receptor, resulted in increased killing of cells expressing Bcr/Abl. However, in this study optimal killing occurred in cells exposed to 10 µM STI571, which is above concentrations obtained in the plasma of patients receiving this agent (13)
.
In addition to tyrosine kinases such as Bcr/Abl, apoptosis and survival are also regulated by the activity of multiple other signal transduction pathways, particularly the MAPK cascade. The MAPK cascade consists of a superfamily of three parallel signal transduction modules converging on the serine/threonine kinases JNK, p42/44 MAPK (ERK), and p38 MAPK (17) . These kinases are activated by a variety of stimuli and are intimately involved in diverse cellular processes including responses to DNA damage or osmotic shock, mitogenic stimuli, cell differentiation and survival, among others (18) . Although exceptions occur, activation of JNK and p38 MAPK are generally associated with induction of apoptosis, whereas p42/44 MAPK exerts cytoprotective effects (19) . Efforts to delineate the role of p42/44 MAPK in various cellular functions has been facilitated by the development of several pharmacological inhibitors of the enzymes that activate p42/44 (i.e., the MAPK kinases MEK1/2), including PD98059 (20) , U0126 (21) , and PD184352. The latter agent has attracted attention in view of its ability to inhibit p42/44 MAPK activity and to block human colon carcinoma growth in an in vivo model (22) . The observation that MEK1/2 inhibitors potentiate the antitumor activity of various cytotoxic agents, including ara-C (23) , cisplatin (24) , and paclitaxel (25 , 26) , suggests a possible role for MEK1/2 inhibitors in the treatment of human malignancies.
The contribution of the ERK/MAPK cascade in the antiapoptotic actions of Bcr/Abl remains to be fully elucidated. For example, it has been shown that in fibroblasts and hematopoietic cells, JNK represents a primary target in Bcr/Abl-mediated transformation, although the Ras/MEK/MAPK pathway may be involved in this process (27) . Other studies have demonstrated that MAPK is phosphorylated in hematopoietic cells constitutively expressing Bcr/Abl (28) . Moreover, interference with MEK/MAPK activation has been implicated in apoptosis induction in Bcr/Abl-positive cells, including that occurring in response to STI571 (12 , 29) . Collectively, such findings raise the possibility that disruption of MEK/MAPK signaling might enhance the lethality of STI571 toward CML progenitors. To address this issue, we have examined interactions between pharmacological MEK1/2 inhibitors and STI571 in Bcr/Abl-positive human leukemia cells. Our results indicate that contrary to expectations, treatment of these cells with STI571 results in a delayed increase in p42/44 MAPK activation. Furthermore, coadministration of a marginally toxic concentration of STI571 with several MEK1/2 inhibitors is associated with a very marked increase in mitochondrial dysfunction and caspase activation, as well as a highly synergistic potentiation of apoptosis. Significantly, similar interactions occur in STI571-resistant cells that express high levels of the Bcr/Abl protein. Together, these findings suggest that the MEK/MAPK cascade exerts a cytoprotective effect in Bcr/Abl-positive leukemia cells exposed to STI571, and raise the possibility that combining this agent with pharmacological MEK1/2 inhibitors may represent a novel therapeutic strategy in CML and related disorders.
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MATERIALS AND METHODS |
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4 x 105 cells/ml). Multidrug-resistant K562R cells were derived from the parental line by subculturing in progressively higher concentrations of doxorubicin as described previously (30) . They were cultured in the absence of doxorubicin before all of the experimental procedures. In addition, STI571-resistant K562 cells, designated K562-R-STI, were generated by subculturing K562 cells in progressively higher concentrations of STI571. These cells are maintained under selection pressure in medium containing 1 µM of STI571. For studies involving the K562-R-STI line, cells are washed free of drug and resuspended in drug-free medium 48 h before experimentation.
Reagents.
STI571 was kindly provided by Dr. Elizabeth Buchdunger, Novartis Pharmaceuticals, Basel, Switzerland, and prepared as a 10 mM stock solution in sterile DMSO (Sigma Chemical Co., St. Louis, MO). Verapamil was purchased from Sigma Chemical Co., stored in light-protected containers at -20°C, and dissolved in sterile 100% ethanol as a 0.1 M stock solution before use. PD98059, U0126, SB203580, and SB202190 were purchased from Calbiochem, La Jolla, CA. Each of these was formulated in DMSO as above. Stock solutions were then diluted in RPMI medium to achieve the desired final concentration. In all of the cases, final concentrations of DMSO were < 0.1% and did not modify responses of cells to STI571. PD184352 was kindly provided by Dr. Judy Sebolt-Leopold, Pfizer/Parke-Davis Pharmaceuticals, Ann Arbor, MI, and was formulated as above. IETD-fmk and ZVAD-fmk were purchased from Enzyme Products, Ltd., Livermore, CA, and formulated in sterile DMSO before use.
Experimental Format.
Logarithmically growing cells were placed in sterile plastic T-flasks (Corning, Corning, NY) to which were added the designated drugs and the flasks replaced in the incubator for intervals ranging from 6 to 72 h. At the end of the incubation period, cells were transferred to sterile centrifuge tubes, pelleted by centrifugation at 400 x g for 10 min at room temperature, and prepared for analysis as described below.
Assessment of Apoptosis.
After drug exposures, cytocentrifuge preparations were stained with Wright-Giemsa and viewed by light microscopy to evaluate the extent of apoptosis (i.e., cell shrinkage, nuclear condensation, formation of apoptotic bodies, and so forth) as described previously (23)
. For these studies, the percentage of apoptotic cells was determined by evaluating 500 cells/condition in triplicate. To confirm the results of morphological analysis, TUNEL staining was used. For TUNEL staining, cytocentrifuge preparations were obtained and fixed with 4% formaldehyde. The slides were treated with acetic acid/ethanol (1:2), stained with terminal transferase reaction mixture containing 1 x terminal transferase reaction buffer (0.25 units/µl terminal transferase, 2.5 mM CoCl2, and 2 pmol fluorescein-12-dUTP; Boehringer Mannheim, Indianapolis, IN), and visualized using fluorescence microscopy. For cell viability studies, CellTiter 96 Aqueous One Solution (Promega, Madison, WI) was used as per the manufacturer’s instructions and the absorbance at 490 nm recorded using a 96-well plate reader (Molecular Devices, Sunnyvale, CA).
Determination of MMP(m).
MMP was monitored using DiOC6 (31)
. For each condition, 4 x 105 cells were incubated for 15 min at 37°C in 1 ml of 40 nM DiOC6 (Calbiochem) and subsequently analyzed using a Becton Dickinson FACScan cytofluorometer with excitation and emission settings of 488 and 525 nm, respectively. Control experiments documenting the loss of m were performed by exposing cells to 5 µM of carbamoyl cyanide m-chlorophenylhydrazone (Sigma Chemical Co.; 15 min, 37°C), an uncoupling agent that abolishes the MMP.
Preparation of S-100 Fractions and Assessment of Cytochrome C Release.
K562 cells were harvested after drug treatment as described previously (31)
by centrifugation at 600 x g for 10 min at 4°C and washed in PBS. Cells (4 x 106) were lysed by incubating for 3 min in 100 µl of lysis buffer containing 75 mM NaCl, 8 mM Na2HPO4, 1 mM NaH2PO4, 1 mM EDTA, and 350 µg/ml digitonin. The lysates were centrifuged at 12,000 x g for 5 min, and the supernatant was collected and added to an equal volume of 2 x LAEMMLI buffer. The protein samples were quantified and separated by 15% SDS-PAGE.
Western Analysis.
A minor modification of a method described previously was used (32)
. After treatment, whole cell pellets (1 x 107 cells/condition) were washed twice in PBS, resuspended in 50 µl of PBS, lysed by the addition of 50 µl 2 x Laemmli buffer [1X = 30 mM Tris-base (pH 6.8), 2% SDS, 2.88 mM ß-mercaptoethanol, and 10% glycerol], and briefly sonicated. Homogenates were quantified using Coomassie protein assay reagent (Pierce, Rockford, IL). Equal amounts of protein (20 µg) were boiled for 10 min, separated by SDS-PAGE (5% stacker and 10% resolving), and electroblotted to nitrocellulose. The blots were stained in 0.1% amido black and destained in 5% acetic acid to ensure transfer and equal loading. After blocking in PBS-T (0.05%) and 5% milk for 1 h at 22°C, the blots were incubated in fresh blocking solution with an appropriate dilution of primary antibody for 4 h at 22°C. The source and dilution of antibodies were as follows: Bcl-2 1:200, mouse monoclonal, Dako, Carpinteria, CA; Bcl-xL 1:1000, rabbit polyclonal, Santa Cruz Biotechnology; XIAP 1:1000, rabbit polyclonal, R & D Systems, Minneapolis, MN; Mcl-1 1:1000, mouse monoclonal, PharMingen, San Diego, CA; ERK 1/2 1:1000, rabbit polyclonal, Cell Signaling Technology, Beverly, MA; phospho-ERK 1/2 (thr202/tyr204) 1:1000, rabbit polyclonal, Cell Signaling Technology; JNK 1:1000, rabbit polyclonal, Santa Cruz Biotechnology; phospho-JNK 1:1000, mouse monoclonal, Santa Cruz Biotechnology; phospho-p38 MAPK 1:1000, rabbit polyclonal, Cell Signaling Technology; phospho-cdc2 1:1000, rabbit polyclonal, Cell Signaling Technology; phospho-CREB 1:1000, rabbit polyclonal, Upstate Biotechnology, Lake Placid, NY; phospho-tyrosine (PY20) 1:300, mouse monoclonal, Transduction Laboratories, Lexington, KY; c-Abl (24–11) 1:1000, mouse monoclonal, Santa Cruz Biotechnology; PARP (C-2–10) 1:3000, mouse monoclonal, BioMol Research Laboratories, Plymouth, MA; procaspase-3 1:1000, mouse monoclonal, Transduction Laboratories; cleaved caspase 3 and phospho-Stat5, 1:1000, rabbit polyclonal, Cell Signaling Technology; cytochrome c 1:500, mouse monoclonal; caspase 8 1:1000, rabbit polyclonal, PharMingen; and 
-tubulin 1:2000, Calbiochem. Blots were washed 3 x 5 min in PBS-T and then incubated with a 1:2000 dilution of horseradish peroxidase-conjugated secondary antibody (Bio-Rad Laboratories, Hercules, CA) for 1 h at 22°C. Blots were again washed 3 x 5 min in PBS-T and then developed by enhanced chemiluminescence (Pierce, Rockford, IL).
Cell Cycle Analysis.
After treatment, cells were pelleted at 500 x g and resuspended in 70% ethanol. The cell pellets were incubated on ice for 1 h and resuspended in 1 ml of cell cycle buffer (0.38 mM sodium citrate, 0.5 mg/ml RNase A, and 0.01 mg/ml propidium iodide (all Sigma Chemical Co.) at a concentration of 106 cells/ml as described previously (31)
. Samples were stored in the dark before analysis at 4°C (generally within 24 h) and analyzed on a Becton Dickinson FACScan flow cytometer (Cambridge, MA) using a commercially available software program (ModFit LT 2.0; Verity Software, Topsham, ME; Ref. 31
).
Akt Activity.
Akt activity in treated samples was determined using a method published previously (33)
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EMSA.
Nuclear extracts were prepared as described previously (34)
. Double-stranded oligonucleotides corresponding to STAT5 binding site of ß-casein promoter (5'- AGATTTCTAGGAATTCAATCC-3') were obtained from Santa Cruz Biotechnology and labeled with [
32P]ATP (5,000 Ci/mmol, Amersham Pharmacia Biotech) using T4 polynucleotide kinase (Promega) and purified using a G-25 column (Amersham Pharmacia Biotech). Nuclear extracts (5 µg) were incubated at 4°C for 20 min with 50,000 cpm of labeled oligonucleotide probe dissolved in 15 µl of binding buffer [20 mM HEPES (pH 7.9), 5 mM MgCl2, 4 mM DTT, 20% glycerol, 0.1 mM phenylmethylsulfonyl fluoride, 5 mM benzamidine, 2 mM levamisol, 0.1 µg/ml aprotinin, 0.1 µg/ml bestatin, 2 µg poly(dI·dC)]. The reaction mixtures were then loaded onto 6% native polyacrylamide gels in 0.09 M Tris borate, 2 mM EDTA (pH 8.0) buffer and electrophoresed for 1.5 h at 150 V. The gels were dried at 80°C and exposed to X-ray film for autoradiography.
Normal Peripheral Blood Mononuclear Cells.
Peripheral blood was obtained with informed consent from normal volunteers, in syringes containing preservative-free heparin, diluted 1:3 with RPMI 1640 medium, and layered over a cushion of 10 ml of Ficoll-Hypaque (specific gravity, 1.077; Sigma Chemical Co.) in sterile 50-ml plastic centrifuge tubes. These studies have been approved by the Human Investigations Committee of Virginia Commonwealth University, Richmond, VA. After centrifugation for 40 min at 400 x g at room temperature, the interface layer, consisting of mononuclear cells, was extracted with a sterile Pasteur pipette and diluted in fresh RPMI 1640 containing 10% FCS in 25-cm2 tissue culture flasks at a cell density of 106 cells/ml.
Statistical Analysis.
The significance of differences between experimental conditions was determined using the two-tailed Student t test.
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RESULTS |
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10 µM (percentage apoptosis < 3%), whereas 200 nM STI571 induced apoptosis in 6% of cells (Fig. 1A)
. However, coadministration of PD184352 (1 µM) with STI571 resulted in a very substantial potentiation of apoptosis (e.g., to 20%), with values exceeding 75% for PD184352 concentrations 5 µM. Roughly parallel results were obtained when loss of MMP (
m) was monitored (Fig. 1B)
, although the loss of m in PD184352-treated cells was somewhat greater than the extent of apoptosis.
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. STI571 concentrations of 150–250 nM were minimally toxic to these cells but resulted in marked lethality when combined with PD184352 (Fig. 1C)
. The extent of apoptosis increased to 20% in cells treated with 300 nM STI571 but approached 75% when PD184352 was coadministered. Finally, whereas 500 nM STI571 triggered apoptosis in 50% of cells, values increased to > 90% with PD184352. Similar results were noted when changes in MMP were monitored (Fig. 1D)
. Together, these findings indicate that PD184352 strikingly increases STI571-induced mitochondrial damage and apoptosis in K562 cells in a dose-dependent manner.
A time course analysis of cells exposed to 200 nM STI571 + 5 µM PD184352 revealed a modest increase in apoptosis after 24 h of cotreatment but a very substantial increase by 36 h (e.g., 50%), with nearly 100% apoptotic cells at 72 h (Fig. 2A)
. Thus, essentially subtoxic concentrations of PD184352 dramatically increased STI571-induced mitochondrial damage and apoptosis in K562 cells, most notably at exposure intervals 36 h. Median dose effect analysis was used to characterize interactions between STI571 and PD184352 in regard to both induction of apoptosis and loss of m (Fig. 2B)
. Combination index values significantly < 1.0, corresponding to synergistic interactions, were obtained in each case. In addition, the MTS assay, which reflects both cell proliferation and viability, was used to assess interactions between very low concentrations of PD184352 (1.5 µM) and STI571 (50 nM). Whereas the drugs were nontoxic when given alone for 72 h, the combination reduced viability by > 33% (Fig. 2C)
. Finally, coadministration of two other pharmacological MEK1/2 inhibitors (PD98059 or U0126), which by themselves were nontoxic, with STI571 (200 nM; 48 h) also resulted in a striking increase in apoptosis in K562 cells (Fig. 2D)
consistent with a role for inhibition of MEK1/2 in enhanced lethality.
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. Treatment of K562 cells for 48 h with 200 nM of STI571 or 5 µM of PD184352 individually resulted in only a minimal degree of TUNEL positivity, reflecting cells containing DNA with free 3'-OH ends. In contrast, cells exposed to both agents exhibited a striking increase in the number of TUNEL-positive cells.
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. The Bcr/Abl+ cell line LAMA 84 is somewhat more sensitive to STI571 than K562 cells (35)
. However, coadministration of 5 µM of PD184352 with 200 nM of STI571, both of which were only modestly toxic to LAMA 84 cells when given alone, resulted in a marked increase in the percentage of apoptotic cells after 36 h (e.g., 70%). Parallel results were obtained when loss of m was monitored (data not shown). In contrast, 200 nM of STI571 was nontoxic to Bcr/Abl- U937 cells, and cotreatment with PD184352 did not result in an increase in lethality. PD184352 alone exerted some toxicity toward HL-60 cells, as we have reported previously (23)
, but STI571 by itself did not induce apoptosis in these cells, nor did it enhance the lethal effects of PD184352. Finally, normal peripheral blood mononuclear cells were immune to the effects of these agents administered individually or together. Collectively, these findings suggest the synergism between STI571 and MEK1/2 inhibition is restricted to Bcr/Abl+ cells.
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. At the 24 or 48 h exposure intervals, STI571 or PD184352 administered alone exerted minimal effects on PARP degradation, procaspase 3 and 8 cleavage, or cytochrome c release. However, in cells exposed to the combination of STI571 and PD184352 for 24 h, PARP degradation, procaspase 3 and 8 activation, and cytosolic cytochrome c release could be faintly discerned, whereas at 48 h, each of these events was very pronounced. (Fig. 5B)
. A more detailed time course study revealed that a small amount of cytochrome c release was first noted at 24 h in STI571/PD184352-treated cells but that extensive cytochrome c redistribution occurred at 36 h (Fig. 5C)
, consistent with the time course study of apoptotic morphology (Fig. 2A)
. To gain insights into the hierarchy of events accompanying cell death, cells were exposed to STI571 + PD184352 for 48 h in the presence or absence of the general caspase inhibitor ZVAD-fmk or the caspase 8 inhibitor IETD-fmk (Fig. 6)
. It can be seen that the broad caspase inhibitor ZVAD-fmk was highly effective in blocking STI571/PD184352-induced apoptosis, whereas IETD-fmk was significantly less so (Fig. 6A)
. Parallel results were obtained when PARP degradation and pro-caspase 3 activation were monitored (Fig. 6B)
. This indicates that activation of the extrinsic, caspase 8-related pathway in all likelihood represents a secondary process in cells exposed to STI571 in combination with MEK1/2 inhibitors. However, neither caspase inhibitor blocked cytochrome c release, suggesting that redistribution of cytochrome c represents an upstream event in STI571/PD184352-mediated apoptosis.
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. Administration of STI571(200 nM) or 5 µM PD184352 individually for 24 h resulted in small decreases in expression of Bcl-2 and Bcl-xL but little change in XIAP or Mcl-1 protein levels (Fig. 7A)
. However, coadministration of these agents resulted in very marked declines in Bcl-2, Bcl-xL, and Mcl-1 protein levels, but no effect on XIAP. Moreover, whereas down-regulation of Bcl-2 expression in STI571/PD184352-treated cells was substantially diminished by ZVAD-fmk, suggesting that this process occurs secondary to caspase activation, down-regulation of Bcl-xL and Mcl-1 was not reduced, indicating that the latter events occur upstream of activation of the caspase cascade (Fig. 7B)
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, a time point shortly preceding the extensive induction of apoptosis accompanying combined drug treatment. PD184352 by itself exerted minimal effects on cell cycle distribution. STI571 (200 nM) modestly increased the G0G1 and G2M fractions, and decreased the S phase fraction. In contrast, combined exposure to STI571 and PD184352 resulted in a substantial increase in the G0G1 population (e.g., to 65%), which was significantly greater than that observed in cells exposed to STI571 alone (P < 0.005). On the other hand, the decline in the S phase fraction was not significantly greater than that noted in STI571-treated cells (P > 0.05). However, STI571/PD184352-treated cells exhibited a reduction rather than an increase in the percentage of cells in G2M (P < 0.002 versus STI571 alone). These findings indicate that MEK1/2 inhibition potentiates the ability of STI571 to induce G0G1 arrest in K562 cells.
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. Unexpectedly, levels of phospho-ERK were increased in STI571-treated cells at this time, whereas total ERK expression remained unchanged (Fig. 9A)
. As anticipated, activation (phosphorylation) of ERK was abrogated by coadministration of PD184352. Interestingly, exposure of cells to STI571 alone failed to induce phosphorylation (activation) of p38 MAPK and JNK, whereas both stress-related kinases were robustly activated in cells exposed to the combination of STI571 and PD184352. PD184352 alone modestly decreased phospho-CREB levels, an effect that was markedly potentiated by coadministration of STI571. STI alone induced a modest reduction in expression of phospho-Stat5, a downstream Bcr/ABL target, but combination with PD184352 resulted in an even more pronounced decline in Stat5 phosphorylation. The combination of STI571 and PD184352 also resulted in a striking increase in p34cdc2 activation (dephosphorylation), whereas STI571 alone exerted only a minimal effect. Interestingly, STI571 alone failed to reduce levels of the Mr 210,000 Bcr-Abl protein, whereas expression was barely detectable in cells exposed to both PD184352 and STI571. In separate studies, phospho(tyrosine)-Bcr-Abl levels were diminished in cells exposed to STI571 and, as anticipated given the extensive reduction in Bcr/Abl protein levels, were essentially undetectable in cells treated with the combination of STI571 and PD184352 (data not shown). However, whereas treatment with STI571 alone (200 nM) significantly reduced Akt activity in K562 cells (e.g., by 42 ± 7%), this decline was not additionally enhanced by coadministration of PD184352 (e.g., 29 ± 0.9.%; P > 0.05; data not shown).
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. Coadministration of 10 µM of U0126 alone had little effect on DNA binding to a Stat5 consensus oligonucleotide in the gel shift assay, whereas 200 nM of STI571 modestly reduced binding. However, combined treatment of cells with both STI571 and U0126 or PD184352 resulted in a marked reduction in the association of Stat5 and the labeled probe. Essentially identical results were obtained when 5 µM of PD184352 was used instead of U0126 (data not shown)
In view of evidence that apoptotic caspases can induce cleavage of various signaling proteins (36)
, parallel studies were performed in STI571/PD184352-treated cells exposed to the caspase inhibitor ZVAD-fmk (Fig. 9C)
. Down-regulation of the Bcr/Abl protein in drug-treated cells was essentially abrogated by ZVAD, suggesting that this effect largely reflects caspase-mediated protein degradation. In contrast, down-regulation of phospho-ERK, phospho-CREB, and phospho-p34cdc2, or up-regulation of phospho-JNK and p38 MAPK was unperturbed by ZVAD-fmk, indicating that these events proceed through a caspase-independent pathway.
The effects of higher concentrations of STI571 on Bcr/Abl degradation were then examined (Fig. 9D)
. Exposure of K562 cells to an STI571 concentration of 1 µM alone for 48 h resulted in a marked reduction in Bcr/Abl levels, an effect that was blocked by the caspase-3 inhibitor DEVD-fmk and the pan-caspase inhibitor Boc-fmk (25 µM each). Taken together with the preceding findings, these observations suggest that MEK1/2 inhibition enhances the ability of STI571 to promote the caspase-3-mediated cleavage of the Bcr/Abl protein.
Treatment of K562 Cells with STI571 Initially Inhibits and Subsequently Promotes ERK1/2 Phosphorylation, an Effect That Is Abrogated by Coadministration of PD184352.
To additionally characterize the effects of STI571 and PD184352 on MAPK activation, a more detailed time course analysis was carried out (Fig. 10)
. In these studies, ratios of expression of activated (phosphorylated) p42/44 MAPK versus total MAPK were determined by densitometric analysis of Western blots over a 48-h exposure interval (Fig. 10A)
. Western blots depicting phosphorylation of MAPK at 6 and 24 h are also shown in Fig. 10B
. Interestingly, at the 6-h interval, 200 nM of STI571 significantly reduced MAPK activation, consistent with results of previous reports (12)
. However, at later intervals (e.g., 24 and 36 h), STI571-treated cells displayed a significant increase in MAPK activation (Fig. 10A)
. By 48 h, MAPK activation had declined to control levels. In all of the cases, coadministration of PD184352 essentially abrogated phospho-MAPK expression. To rule out the possibility that activation of MAPK occurs only in response to a low concentration of STI571, phospho-ERK1/2 expression was monitored in K562 cells exposed to 2 µM of STI571 for 24 h. As in the case of lower drug concentrations, treatment with 2 µM of STI571 resulted in clear evidence of enhanced ERK1/2 phosphorylation/activation (Fig. 10C)
, an effect that was prevented by PD184352 (data not shown). Together, these findings suggest that at early intervals, STI571, by inhibiting the Bcr/Abl kinase, opposes MAPK phosphorylation/activation, whereas subsequently, MAPK activation is enhanced, presumably through a Bcr/Abl-independent pathway. They also raise the possibility that interruption of the latter event (e.g., by pharmacological MEK1/2 inhibitors) contributes to or is responsible for the observed marked potentiation of apoptosis.
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, coadministration of either p38 MAPK inhibitor (10 µM each) partially but significantly reduced the extent of apoptosis in STI571/PD184352-treated cells (P 0.01) versus STI571 + PD184352 alone). These findings suggest that the reciprocal activation of p38 MAPK contributes to potentiation of STI571-related lethality by MEK1/2 inhibition.
K562R Cells Display Increased Basal Bcr-Abl and Phospho-ERK1/2 Expression and Are Resistant to STI571-induced Apoptosis.
An attempt was then made to characterize interactions between MEK inhibitors and STI571 in Bcr/Abl-positive cells resistant to the latter agent. To this end, a multidrug-resistant K562 cell line (K562R) characterized previously, isolated by culturing cells in progressively higher concentrations of doxorubicin, was used (30)
. Unexpectedly, K562R cells expressed an 3-fold increase in Bcr/Abl protein levels relative to the parental line (K562S) and a slightly greater relative increase in the expression of phospho-MAPK (Fig. 11A)
. In addition, basal Akt activity was increased by a factor of 1.9 ± 0.2 in K562R cells compared with controls (data not shown). A dose-response study revealed that K562R cells were substantially more resistant to STI571-induced apoptosis than the wild-type line, with an 10-fold higher IC50 value (Fig. 11B)
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, a pharmacologically achievable STI571 concentration (e.g., 1.5 µM) was largely ineffective in inducing apoptosis in K562R cells. However, when 10 µM of PD184352, which was nontoxic to these cells, was coadministered, the extent of apoptosis in the resistant cells increased dramatically (e.g., to 60%). Interestingly, verapamil (2 µM), which has been shown previously to increase the susceptibility of multidrug-resistant LAMA 84 cells to STI571 (35)
, was ineffective in the K562R line. In separate studies, coadministration of this concentration of verapamil increased by 5-fold the extent of apoptosis induced by 10 µM of doxorubicin (data not shown). These findings suggest that factors other than or in addition to alterations in drug uptake are involved in potentiation of STI571 lethality by PD184352. The shift in the STI571 dose-response curve after treatment of resistant cells with a nontoxic concentration of PD184352 is shown in Fig. 12B
. Finally, whereas 1.5 µM STI571 alone minimally induced PARP degradation, procaspase-3 cleavage, and cytochrome c release in K562R cells, addition of PD184352, which was inactive by itself, resulted in a marked potentiation of mitochondrial damage and caspase activation (Fig. 12C)
. Together, these findings indicate that MEK1/2 inhibition is able to reverse, at least in part, STI571 resistance in leukemic cells overexpressing the Bcr/Abl protein.
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, these cells, designated K562-R-STI, exhibited a greater relative increase in Bcr/Abl protein expression compared with parental cells (e.g., 6-fold) than the K562R cell line. A time course study (Fig. 13A)
revealed that K562-R-STI cells were for all practical purposes immune to apoptosis induced by 2 µM of STI571 over a 72-h interval, whereas essentially 100% of parental cells were apoptotic after 48 h of drug exposure. Whereas 10 µM of PD184352 by itself exerted modest toxicity toward K562-R-STI cells (i.e., 9% apoptotic), combination with a nontoxic concentration of STI571 (2 µM) for 48 h resulted in a very marked increase in cell death (40% apoptotic; Fig. 13B
). This finding indicates that MEK1/2 inhibitors can enhance the lethal effects of STI571 in Bcr/Abl+ cells developed specifically for resistance to this agent.
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DISCUSSION |
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Interactions between MEK1/2 inhibitors and STI571 were noteworthy in several respects, including the extent of synergism, particularly at low drug concentrations, as well as the observed activity against otherwise STI571-resistant cells. Whereas activation of the MEK/MAPK module has generally been associated with antiapoptotic actions (19)
, the relationship between MEK/MAPK induction and Bcr/Abl expression is unclear. For example, MAPK has been reported to be a downstream target of the Bcr/Abl kinase (26)
, although Morgan et al. (43)
could find no clear relationship between Bcr/Abl expression and MAPK activation in a panel of human leukemic cell lines. On the other hand, Kang et al. (29)
reported that interruption of MEK1/2 (e.g., by PD98050) was a potent inducer of apoptosis in the K562 line, suggesting an important role for the MAPK cascade in the survival of Bcr/Abl-positive cells. Similarly, Woessmann and Miveschi (44)
observed that disruption of ERK signaling, either by transfection with a dominant-negative ERK1 mutant or treatment with the MEK1/2 inhibitor UO126, induced apoptosis in K562 cells. Consistent with the results of Dan et al. (12)
, we also found that exposure of Bcr/Abl+ cells to STI571 resulted in down-regulation of activated MAPK, at least at early time points (e.g., 6 h). However, at later intervals, a significant increase in MAPK activation was observed in STI571-treated cells, an effect that was abrogated by addition of MEK1/2 inhibitors. Consequently, it is tempting to speculate that disruption of the Bcr/Abl pathway (e.g., by STI571) represents a stress for such cells, and in so doing elicits a compensatory cytoprotective MEK/MAPK response, which may operate, at least in part, through a Bcr/Abl-independent mechanism. Moreover, interference with the latter process may lead, through an as yet to be defined mechanism, to mitochondrial damage (e.g., cytochrome c release, loss of m) and subsequent activation of the apoptotic caspase cascade. However, the present results do differ from those of Oetzel et al. (45)
who reported that the MEK1/2 inhibitor PD98059 failed to enhance STI571 lethality in murine pro-B lymphocytic Baf-1 cells transfected with a p185 Bcr/Abl construct. This discrepancy may stem from intrinsic differences in the responses of Bcr/Abl+ murine and human hematopoietic cells to interruption of the MEK/MAPK cascade. Alternatively, such differences might reflect the fact that lethality was examined at relatively early time intervals in the latter study (e.g., 24 h). In this regard, cell death induced by the combination of STI571 and PD184352 was also quite limited in K562 cells after a 24-h drug treatment, consistent with the results of Oetzel et al. (45)
but increased dramatically at exposure intervals 36 h. In any case, it is apparent that the lethal consequences of combined Bcr/Abl kinase and MEK1/2 inhibition in K562 and LAMA 84 cells result from relatively late rather than early events.
Aside from the MEK1/2/MAPK pathway, several Bcr/Abl downstream targets have been postulated to promote survival of Bcr/Abl+ cells, including Bcl-xL, nuclear factor B, and Akt, among others (8
, 9
, 46)
. It is possible that STI571 acts, at least in part, by down-regulating expression of these putative prosurvival proteins. This phenomenon may occur either through direct inhibition of the Bcr/Abl kinase or indirectly through cleavage of target proteins by apoptotic caspases. In this regard, it is noteworthy that the combination of STI571 + PD184352 (or STI571 alone) inhibited phosphorylation/activation of Bcr/Abl as anticipated (9)
but also resulted in reduced levels of Bcr/Abl protein through a caspase-dependent process. Whereas exposure of Bcr/Abl+ cells to STI571 has not in general been associated with down-regulation of the Bcr/Abl protein, several other agents have been shown to act in this way, including arsenic trioxide (42
, 47)
, geldanamycin (48)
, proteasome inhibitors (49)
, and the kinase inhibitor AG957 (50)
. Although reduction in Bcr/Abl protein expression by STI571/PD184352 was sensitive to the general caspase inhibitor ZVAD as well as the caspase-3 inhibitor DEVD, the possibility that proteasomal degradation may also be involved in this process cannot be excluded. Whatever the mechanism, the present findings raise the possibility that coadministration of MEK1/2 inhibitors with a low concentration of STI571 triggers an amplification loop in which Bcr/Abl protein expression is diminished through a caspase-dependent process. On the other hand, our inability to detect an additional reduction in Akt activity in cells exposed to the combination of STI571 and PD184352 argues against a contribution of this pathway to enhanced lethality.
Analogously, the combination of PD184352 and STI571 resulted in caspase-dependent down-regulation of the antiapoptotic protein Bcl-2. Cleavage of Bcl-2 into a proapoptotic fragment during the course of apoptosis has been reported previously (51) and may serve to ensure that the apoptotic process, once initiated, proceeds to completion. Coadministration of PD184352 with STI571 also resulted in a marked reduction in protein expression of Bcl-xL and Mcl-1 compared with the effects of STI571 alone, although the insensitivity of these events to ZVAD-fmk, in contrast to Bcl-2 down-regulation, suggests a primary mechanism of action. The finding that STI571/PD184352 treatment was associated with reduced levels of Mcl-1 is also consistent with previous reports demonstrating a requirement for MEK/MAPK activation in sustained expression of this antiapoptotic protein (52) . In addition, it is important to note that Bcr/Abl lies upstream of the Stat family of transcription factors, which have been implicated in the regulation of antiapoptotic proteins such as Bcl-xL and Mcl-1 in hematopoietic cells (53, 54, 55) . The observation that MEK1/2 inhibition enhanced STI571-mediated attenuation of Stat5 phosphorylation and DNA binding is consistent with such a model. Collectively, these findings suggest that combined inhibition of the MEK/MAPK and Bcr/Abl pathways results in diminished expression of multiple antiapoptotic proteins through both caspase-dependent and -independent mechanisms, which together may serve to amplify the cell death process.
Combined exposure of K562 cells to STI571 and PD184352 resulted in perturbations in other signaling cascades, although the functional contribution of these events to lethality remains to be determined. For example, cells exposed to both agents but not each agent individually exhibited a marked activation of both JNK and p38 MAPK. However, whereas activation of such stress-related pathways have been linked to cell death in some systems (19) , in others (23) , including K562 cells (29) , their role in cell death may be less critical than that of inhibition of MEK/MAPK. Nevertheless, the finding that the p38 MAPK inhibitors SB203580 and SB202190 partially attenuated STI571/PD184352-mediated apoptosis suggests a functional role for activation of this stress pathway in the lethal actions of this drug combination. Treatment of K562 cells with the combination of STI571 and PD184352 also resulted in diminished phosphorylation of CREB and p34cdc2, both of which have been associated with promotion of apoptosis (56 , 57) . Elucidation of the functional role of these events, as well as that of JNK activation, in STI571/MEK1/2 inhibitor-related apoptosis awaits additional analysis, and studies designed to address this issue are currently in progress.
Resistance of Bcr/Abl+ cells to STI571 may occur via multiple mechanisms, including amplification of the Bcr/Abl gene, mutations in the Bcr/Abl kinase ATP binding site, or reduction in intracellular drug uptake, among others (35 , 58, 59, 60) . Although gene amplification is not universally encountered in resistant cells, increases in Bcr/Abl protein expression commonly occurs, at least in cultured cell lines (58 , 60) . The K562R line was originally developed by subculturing cells in progressively higher concentration of doxorubicin and was initially characterized as an overexpressor of the Pgp protein (30) . However, in view of the ability of the Bcr/Abl protein to confer resistance to multiple cytotoxic drugs (7) , it is not surprising that such cells would in addition exhibit increased Bcr/Abl protein expression. K562R cells also displayed a significant increase in basal MAPK activity, consistent with the notion that MAPK is a downstream target of the Bcr/Abl kinase (28 , 29) . The degree of resistance of K562R cells to STI571-mediated lethality is similar to if not greater than that exhibited by lines described previously in the literature (35 , 58 , 59) . Significantly, coadministration of a nontoxic concentration of PD184352 with a pharmacologically achievable concentration of STI571 (e.g., 1.5 µM) resulted in a substantial increase in mitochondrial damage (e.g., cytochrome c release), caspase activation, and apoptosis in otherwise resistant K562 cells. In fact, the response of resistant cells to the combination was comparable with that of sensitive cells exposed to STI571 alone. In contrast to the results of Mahon et al. (35) who found that STI571-resistant LAMA 84 cells exhibited overexpression of Bcr/Abl and Pgp, and that verapamil increased STI571 sensitivity, coadministration of verapamil had a negligible effect on STI571 lethality in K562R cells. Nevertheless, the possibility that PD184352 acts by increasing the intracellular accumulation STI571 cannot presently be excluded. Taken together, the present findings indicate that inhibition of the MEK/MAPK pathway can restore STI571 sensitivity in at least some Bcr/Abl+ cell types that are otherwise resistant to this agent. In this regard, the finding that PD184352 promoted STI571-mediated lethality in K562 cells selected specifically for resistance to STI571, and which exhibit a high level of Bcr/Abl overexpression, may have particular relevance for patients who have become drug resistant after in vivo exposure to the latter agent.
The observation that MEK inhibitors potentiate the lethal effects of very low concentrations of STI571 against Bcr/Abl+ leukemic cells could have additional clinical implications. For example, it has been proposed that 1-acidic glycoprotein, which binds avidly to the staurosporine derivative UCN-01 (57)
, may exert the same effect in regard to STI571 (61)
. Consequently, binding to 1-acidic glycoprotein and other proteins, by diminishing free plasma STI571 concentrations, could have the net effect of reducing the antileukemic efficacy of this agent. In this regard, Gambacorti-Passerini et al. (62)
reported recently that the antibiotic erythromycin induced displacement of STI571 from 1-acidic glycoprotein, and in so doing increased its activity against Bcr/Abl+ KU812 cells in a nude mouse model system. Whether such a strategy would be effective in humans remains to be determined. However, an alternative approach would be to administer STI571 in conjunction with a MEK1/2 inhibitor such as PD184352, which is capable of inhibiting MAPK activation in tumor cells in mice (22)
and which is currently entering clinical trials in humans. Thus, if 90% of STI571 were bound to 1-acidic glycoprotein or other plasma proteins and presumably inactive, free concentrations of 100–200 nM might, under conditions of MEK/MAPK inhibition, be sufficient to kill Bcr/Abl+ leukemic cells, as suggested by the present findings. Pending results of ongoing Phase I trials of MEK/MAPK inhibitors in humans, it would clearly be desirable to test this strategy in an animal model system. Accordingly, plans to examine the in vivo potential of this approach are under development.
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FOOTNOTES |
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1 Supported by Grants CA63753 and CA 83705 from the NIH, by a Merit Award from the Veteran’s Administration, and Grant 6630-01 from the Leukemia and Lymphoma Society of America. 
2 To whom requests for reprints should be addressed, at Division of Hematology/Oncology, Medical College of Virginia, MCV Station Box 230, Richmond VA, 23298. Phone: (804) 828-5211; Fax: (804) 828-8079; E-mail: stgrant{at}hsc.vcu.edu
3 The abbreviations used are: BCR, breakpoint cluster region; MEK, mitogen-activated protein/extracellular signal-regulated kinase kinase; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; MAPK, mitogen-activated protein kinase; PARP, poly(ADP-ribose) polymerase; Abl, Ableson murine leukemia; CML, chronic myelogenous leukemia; JNK, c-Jun-NH2-terminal kinase; TUNEL, terminal deoxynucleotidyl transferase (Tdt)-mediated nick end labeling; DiOC6, 3,3-dihexyloxacarbocyanine iodide; PBS-T, PBS-Tween; EMSA, electrophoretic mobility shift assay; MMP, mitochondrial membrane potential.
Received 6/25/01. Accepted 11/ 1/01.
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C. Yu, M. Rahmani, J. Almenara, M. Subler, G. Krystal, D. Conrad, L. Varticovski, P. Dent, and S. Grant Histone Deacetylase Inhibitors Promote STI571-mediated Apoptosis in STI571-sensitive and -resistant Bcr/Abl+ Human Myeloid Leukemia Cells Cancer Res., May 1, 2003; 63(9): 2118 - 2126. [Abstract] [Full Text] [PDF]
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J. M. Golas, K. Arndt, C. Etienne, J. Lucas, D. Nardin, J. Gibbons, P. Frost, F. Ye, D. H. Boschelli, and F. Boschelli 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., January 15, 2003; 63(2): 375 - 381. [Abstract] [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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P. La Rosee, A. S. Corbin, E. P. Stoffregen, M. W. Deininger, and B. J. Druker Activity of the Bcr-Abl Kinase Inhibitor PD180970 against Clinically Relevant Bcr-Abl Isoforms That Cause Resistance to Imatinib Mesylate (Gleevec, STI571) Cancer Res., December 15, 2002; 62(24): 7149 - 7153. [Abstract] [Full Text] [PDF]
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C. Yu, G. Krystal, P. Dent, and S. Grant Flavopiridol Potentiates STI571-induced Mitochondrial Damage and Apoptosis in BCR-ABL-positive Human Leukemia Cells Clin. Cancer Res., September 1, 2002; 8(9): 2976 - 2984. [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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) or absence (
) of 200 nM of STI571, after which cytospin preparations were stained with Wright-Giemsa and the percentage of apoptotic cells determined by morphological assessment of 
