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The Farnesyltransferase Inhibitor L744832 Potentiates UCN-01–Induced Apoptosis in Human Multiple Myeloma Cells

Authors' Affiliations: Departments of ¹ Medicine, ² Biochemistry, ³ Pharmacology, and ⁴ Radiation Oncology, Virginia Commonwealth University/Medical College of Virginia, Richmond, Virginia

Requests for reprints: Steven Grant, Division of Hematology/Oncology, Virginia Commonwealth University/Medical College of Virginia, Medical College of Virginia Station Box 230, Richmond, VA 23298. Phone: 804-828-5211; Fax: 804-828-8079; E-mail: stgrant{at}hsc.vcu.edu.

	Abstract

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Purpose: The purpose of this study was to characterize interactions between the farnesyltransferase inhibitor L744832 and the checkpoint abrogator UCN-01 in drug-sensitive and drug-resistant human myeloma cell lines and primary CD138⁺ multiple myeloma cells.

Experimental Design: Wild-type and drug-resistant myeloma cell lines were exposed to UCN-01 ± L744832 for 24 hours, after which mitochondrial injury, caspase activation, apoptosis, and various perturbations in signaling and survival pathways were monitored.

Results: Simultaneous exposure of myeloma cells to marginally toxic concentrations of L744832 and UCN-01 resulted in a synergistic induction of mitochondrial damage, caspase activation, and apoptosis, associated with activation of p34^cdc2 and c-Jun-NH₂-kinase and inactivation of extracellular signal-regulated kinase, Akt, GSK-3, p70^S6K, and signal transducers and activators of transcription 3 (STAT3). Enhanced lethality for the combination was also observed in primary CD138⁺ myeloma cells, but not in their CD138^– counterparts. L744832/UCN-01–mediated lethality was not attenuated by conventional resistance mechanisms to cytotoxic drugs (e.g., melphalan or dexamethasone), addition of exogenous interleukin-6 or insulin-like growth factor-I, or the presence of stromal cells. In contrast, enforced activation of STAT3 significantly protected myeloma cells from L744832/UCN-01–induced apoptosis.

Conclusions: Coadministration of the farnesyltransferase inhibitor L744832 promotes UCN-01–induced apoptosis in human multiple myeloma cells through a process that may involve perturbations in various survival signaling pathways, including extracellular signal-regulated kinase, Akt, and STAT3, and through a process capable of circumventing conventional modes of myeloma cell resistance, including growth factor– and stromal cell–related mechanisms. They also raise the possibility that combined treatment with farnesyltransferase inhibitors and UCN-01 could represent a novel therapeutic strategy in multiple myeloma.

Key Words: Apoptosis • multiple myeloma • UCN-01 • farnesyltransferase inhibitor • drug resistance

UCN-01 (7-hydroxystaurosporine) is a derivative of the kinase inhibitor staurosporine that was originally developed as protein kinase C inhibitor (13). It has subsequently been shown to inhibit the activity of other kinases, including cyclin-dependent kinases (14), Chk1 (15), and most recently, PDK1 (16). In malignant hematopoietic cells, UCN-01 induces apoptosis at nanomolar concentrations in association with dephosphorylation of CDK1 and CDK2 (17). It also potentiates the lethal effects of DNA-damaging agents by abrogating both G₂-M and G₁ checkpoints (18, 19). Several clinical trials involving UCN-01 have been carried out (20), and suggestions of activity when combined with conventional cytotoxic chemotherapy in a patient with non-Hodgkin's lymphoma have been described (21).

Previously, our group reported that exposure of human leukemia and myeloma cells to subtoxic concentrations of UCN-01 induces activation/phosphorylation of ERK, and that interference with this process by coadministration of pharmacologic MEK1/2 inhibitors results in a striking increase in mitochondrial injury and apoptosis (22, 23). Antileukemic synergism between UCN-01 and agents that interrupt the Akt pathway, including the Hsp90 antagonist 17-AAG, has been observed (24). As inhibition of Ras can theoretically disrupt both the MEK1/2/ERK1/2 and the Akt pathways (25), the possibility arose that farnesyltransferase inhibitors might be particularly effective in potentiating UCN-01–mediated lethality. In fact, we have very recently observed that farnesyltransferase inhibitors enhance the lethal effects of UCN-01 in human myeloid leukemia cells (e.g., U937; ref. 26). Because signaling pathways involved in the survival of myeloid leukemia and multiple myeloma cells differ markedly, and in view of current interest in the use of farnesyltransferase inhibitors in the treatment of multiple myeloma (27), it would be important to determine whether this strategy could be extended to plasma cell dyscrasias. The purpose of the present study was to establish whether and to what extent pharmacologic farnesyltransferase inhibitors interact synergistically with UCN-01 in human multiple myeloma cells, and to characterize perturbations in survival signaling pathways that might accompany such interactions. A second goal was to determine whether such a regimen would be effective in myeloma cells exhibiting both conventional and more novel forms of drug resistance, including that associated with growth factors implicated in myeloma cell survival [e.g., interleukin-6 (IL-6) and insulin-like growth factor-I (IGF-I)] as well as stromal cell adhesion–mediated drug resistance (28). Our results indicate that farnesyltransferase inhibitors interact in a highly synergistic manner with UCN-01 to induce apoptosis in human multiple myeloma cells in association with perturbations in multiple signaling pathways (e.g., inactivation of ERK, Akt, and particularly STAT3), and that this process occurs in myeloma cells exhibiting both conventional and growth factor/stromal cell–related mechanisms of drug resistance.

	Materials and Methods

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Cells and reagents. The dexamethasone-sensitive (MM.1S) and dexamethasone-resistant (MM.1R) human multiple myeloma cell lines were kindly provided by Dr Steven T. Rosen (Northwestern University, Chicago, IL; ref. 29). The human multiple myeloma cell lines U266, NCI-H929, and RPMI8226 were purchased from American Type Culture Collection (Rockville, MD). Cells were maintained in RPMI 1640 containing 10% fetal bovine serum as reported previously (23). Melphalan-resistant sublines (LR5) of 8226 cells were kindly provided by Dr William S. Dalton (University of South Florida, Tampa, FL; ref. 30) and maintained in RPMI 1640, containing 5 µmol/L melphalan, as described above. U266/caspase 9 (dominant negative) and U266/STAT3 (constitutive active) cells were obtained by stable transfection of cells with dominant-negative caspase 9 cDNA (mutation of the active site Cys²⁸⁶ to an alanine; ref. 31) or Flag-tagged constitutive active STAT3 (active mutation of the site Ala⁶⁶¹ and Asp⁶⁶³ to cysteines; refs. 32, 33) cDNA, after which clones were selected with G418. Dominant-negative caspase 9 and constitutive active STAT3 cDNAs were kindly provided by Drs. Kapil Bhalla and Richard Jove (University of South Florida, Tampa, FL), respectively. For coculture experiments described below, U266 cells were stably transfected with a vector (pEGFP-C2, BD Clontech, Palo Alto, CA) encoding green fluorescence protein (GFP), and clones were selected with G418 and confirmed by flow cytometry/fluorescence microscopy. Logarithmically growing cells (4 x 10⁵-6 x 10⁵ cells/mL) were utilized in all experiments.

UCN-01 (7-hydroxystauronsporine) was provided by Dr. Edward Sausville (Developmental Therapeutics Program, National Cancer Institute, Bethesda, MD). L744832, a potent and selective thiol-containing peptidomimetic farnesyltransferase inhibitor (34), was purchased from Calbiochem (San Diego, CA). They were dissolved in DMSO as a stock solution, stored at –80°C, and diluted with serum-free RPMI medium before use. Dexamethasone (Sigma, St. Louis, MO) was dissolved in DMSO, aliquoted, and stored at –20°C. Recombinant human IL-6 (Sigma) and IGF-I (R&D Systems, Minneapolis, MN) were rehydrated in PBS containing 10 mmol/L acetic acid and 0.1% bovine serum albumin, respectively, aliquoted, and stored at –80°C. In all experiments, the final concentration of DMSO did not exceed 0.1%.

Isolation of CD138⁺ myeloma cells. Bone marrow samples were obtained with informed consent from four patients with multiple myeloma undergoing routine diagnostic aspirations. Approval was obtained from the institutional review board of Virginia Commonwealth University. Informed consent was provided according to the Declaration of Helsinki. CD138⁺ and CD138^– cells were separated using an MS⁺/LS⁺ column and a magnetic separator according to the manufacturer's instructions (Miltenyi Biotech, Auburn, CA; ref. 23). The purity of CD138⁺ cells (>90%) was monitored by CD138-phycoerythrin staining and flow cytometry. Viability of the cells was regularly >95% by trypan blue exclusion. CD138⁺ and CD138^– cells were cultured in RPMI 1640 containing 10% FCS in 96-well plates under the same condition described above. Following drug treatment, the percentage of apoptotic cells was evaluated by examining Wright-Giemsa–stained cytospin preparations under light microscopy.

Coculture of myeloma cells with stromal cells. HS-5 human stromal cell line, developed from human bone marrow, was obtained from American Type Culture Collection. HS-5 cells were maintained in RPMI 1640 containing 10% fetal bovine serum as described above, and subcultured twice a week by trypsinization as subcultivation ratio of 1:5. For coculture experiments, HS-5 cells were cultured for 48 hours before seeding myeloma cells (GFP/U266). After 24-hour coculturing, equal volume of fresh medium containing the indicated drugs was added. Alternatively, conditioned medium from HS-5 cells was prepared as previously reported (35). Briefly, after HS-5 cells were grown to 80% to 90% confluence, medium was harvested and centrifuged to remove cells and debris, and the supernatant was collected as HS-5–conditioned medium. For drug treatment in the presence of conditioned medium, myeloma cells were cultured in suspension for 48 hours and then equal volumes of the conditioned medium containing drugs was added.

Assessment of apoptosis. The extent of apoptosis was evaluated by assessing Wright-Giemsa–stained cytospin slides under light microscopy and scoring the number of cells exhibiting classic morphologic features of apoptosis. For each condition, 5 to 10 randomly selected fields per slide were evaluated, encompassing at least 800 cells. To confirm the results of morphologic analysis, apoptosis was also determined by Annexin V-FITC staining and flow cytometry as described previously (36). Annexin V⁺/propidium iodide^– and Annexin V⁺/propidium iodide⁺ reflect early and late apoptosis, respectively. In all cases, results of morphologic analysis correlated highly with results of Annexin V/propidium iodide staining (R > 0.90). In some cases, cell death was analyzed by 7AAD staining and flow cytometry. Briefly, cells were incubated with 0.5 µg/mL 7AAD at 37°C for 30 minutes and then subjected to flow cytometric analysis.

Clonogenic assays. Colony-forming ability following drug treatment was evaluated using a soft agar cloning assay as described preciously (37). Briefly, cells were washed thrice with serum-free RPMI medium. Subsequently, 5,000 cells/well were mixed with RPMI medium containing 20% fetal bovine serum and 0.3% agar, and plated on 12-well plates (three wells per condition). The plates were then maintained in a 37°C/5% CO₂ fully humidified incubator. After 15-day incubation, colonies consisting of >50 cells were scored using an Olympus Model CK inverted microscope, and colony formation for each condition calculated in relation to values obtained for untreated control cells.

Analysis of mitochondrial membrane potential (_m). Cells (2 x 10⁵) were incubated with 40 nmol/L 3,3-dihexyloxacarbocyanine (DiOC₆; Molecular Probes, Inc., Eugene, OR) in PBS at 37°C for 20 minutes, and then analyzed by flow cytometry. The percentage of cells exhibiting decreased level of DiOC₆ uptake, which reflects loss of _m, was determined using Becton Dickinson FACScan (Becton Dickinson, San Jose, CA).

Western blot analysis. Western blot samples were prepared from whole-cell pellets as described previously (37). Total protein was quantified using Coomassie Protein Assay Reagent (Pierce, Rockford, IL). Equal amount of protein (30 µg) were separated by SDS-PAGE and electrotransferred onto nitrocellulose membrane. For analysis of protein phosphorylation, 1 mmol/L each of Na vanadanate and Na PPi were added to 1x sample buffer; no SDS was included in the transfer buffer and TBS was used instead of PBS throughout. The blots were probed with primary antibodies as follows. Where indicated, the blots were reprobed with actin antibody (BD PharMingen, San Diego, CA) or tubulin antibody (BD Transduction Lab., San Diego, CA) to ensure equal loading and transfer of proteins. The following primary antibodies were used: caspase-8 (Alexis, San Diego, CA), caspase-3 (BD Transduction Lab.), cleaved caspase-3 (17 kDa; Cell Signaling, Beverly, MA), caspase-7 (BD PharMingen), caspase 9 (BD PharMingen), PARP (Biomol, Plymouth Meeting, PA), H-Ras (Santa Cruz Biotech, Santa Cruz, CA), phospho-p44/42 MAP kinase (ERK, Thr²⁰²/Tyr²⁰⁴; Santa Cruz Biotech), p44/42 MAP kinase (Santa Cruz Biotech), phospho–c-Jun-NH₂-kinase (JNK) (Thr¹⁸³/Tyr¹⁸⁵; Santa Cruz Biotech), JNK (Cell Signaling), phospho-cdc2 (Tyr¹⁵; Cell Signaling), cdc2 (Cell Signaling), phospho-Akt (Ser⁴⁷³; Santa Cruz Biotech), Akt (Santa Cruz Biotech), phospho-GSK-3/ß (Ser^21/9; Cell Signaling), phospho-p70 S6 kinase (Thr³⁸⁹; Cell Signaling), p70 S6 kinase (Santa Cruz Biotech), phospho-Bad (Ser¹³⁶; Santa Cruz Biotech), phospho-STAT3 (Upstate Biotechnology, Lake Placid, NY), STAT3 (Santa Cruz Biotech), and anti-Flag (Sigma).

Analysis of cytosolic cytochrome c and Smac/DIABLO. Cells (4 x 10⁶) were lysed by incubating in digitonin lysis buffer (75 mmol/L NaCl, 8 mmol/L Na₂HPO₄, 1 mmol/L NaH₂PO₄, 1 mmol/L EDTA, and 350 µg/mL digitonin). The lysates were centrifuged at 12,000 x g for 1 minute, and the supernatant, consisting of the cytosolic S-100 fraction, was collected in an equal volume of 2x sample buffer. The proteins were quantified, separated by 15% SDS-PAGE, and subjected to Western blotting as described above. Cytochrome c antibody (BD PharMingen) and Smac/DIABLO antibody (BD PharMingen) were used as primary antibodies.

Akt kinase assay. An Akt kinase assay kit (Cell Signaling) was used to monitor the activity of Akt kinase as per the manufacturer's instructions. Briefly, 2 x 10⁷ cells were lysed in 1x cell lysis buffer plus 1 mmol/L phenylmethylsulfonyl fluoride by sonication. Protein, 200 µg/200 µL per condition, was incubated with 20 µL of immobilized Akt (1G1) monoclonal antibody beads with gentle rocking overnight at 4°C. The beads were washed twice each with 1x cell lysis buffer and 1x kinase buffer, and then incubated with 200 µmol/L ATP and 1 µg GSK-3/paramyocin fusion protein in kinase buffer at 30°C for 30 minutes. The reaction was terminated by adding 3x SDS sample buffer. GSK-3/paramyocin was separated by 15% SDS-PAGE and visualized by Western blot by using phospho-GSK-3/ß (Ser^21/9) antibody (Cell Signaling) as primary antibody. As positive control, before lysis, MM.1S and H929 cells were serum starved for 2 hours in RPMI 1640 without fetal bovine serum and then exposed to IGF-I (100 ng/mL) for 10 minutes at 37°C as reported previously (38).

Signal transducers and activators of transcription 3 activity assay. STAT3 TransLucent Reporter Vector (STAT3-Luc), which is designed to monitor transcription factor binding activity of STAT3 through the use of the standard luciferase assay, was purchased from Panomics (Redwood City, CA). Cells (4 x 10⁶) were transfected with 5 µg of STAT3-Luc using the Amaxa Nucleofector Device (program T-01) with Cell Line Specific Nucleofector Kit R (Amaxa GmbH, Cologne, Germany). After drug treatment, cells were harvested and subjected to luciferase assay using Luciferase Reporter Assay kit (BD Clontech) as per manufacturer's instructions. Relative light units reflect STAT3 activity.

Statistical analysis. For morphologic assessment of apoptosis, _m, Annexin V-FITC, 7AAD, STAT3/Luc reporter, and clonogenic assays, values represent the means ± SD for at least three separate experiments done in triplicate experiments. The significance of differences between experimental variables was determined using the Student's t test. Analysis of synergism was done according to median dose effect analysis (39) using a commercially available software program (Calcusyn; Biosoft, Ferguson, MO).

	Results

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Because glucocorticoids play an important role in the treatment of plasma cell dyscrasias, an attempt was made to determine whether cross-resistance to the L744832/UCN-01 regimen might occur. To this end, multiple myeloma cells sensitive (MM.1S) and resistant (MM.1R) to steroids were exposed to L744832 (10 µmol/L) and UCN-01 (150 nmol/L) alone and in combination for 18 hours after which apoptosis was assessed by Annexin V/propidium iodide analysis (Fig. 1A). Whereas L744832 and UCN-01 administered individually exerted only modest toxicity, combined treatment resulted in cell death in 80% of cells in both cell lines. Dose-response studies revealed that a minimally toxic concentration of L744832 (10 µmol/L) dramatically increased apoptosis (as show in Annexin V/propidium iodide) and loss of mitochondrial membrane potential (_m) in MM.1S cells exposed to UCN-01 concentrations 75 nmol/L (Fig. 1B); similarly, a marginally toxic concentration of UCN-01 (150 nmol/L) markedly increased the lethality of L744832 concentrations 5 µmol/L. Time course studies revealed that combined treatment was associated with a modest increase in apoptosis after 6 hours, which became very pronounced after 12 to 18 hours of exposure (Fig. 1D). Lastly, whereas individual exposure of MM.1S cells to UCN-01 (150 nmol/L) or L744832 (10 µmol/L) modestly reduced colony formation, combined treatment essentially abrogated myeloma cell self-renewal capacity (Fig. 1E).

View larger version (39K): [in this window] [in a new window]   Fig. 1. Combined treatment with UCN-01 and L744832 potently induces mitochondrial dysfunction, apoptosis, and loss of clonogenicity in human multiple myeloma cells. A, MM.1S cells and its dexamethasone-resistant counterparts (MM.1R) were exposed to 10 µmol/L L744832 (L) ± 150 nmol/L UCN-01 (UCN) for 18 hours, after which the percentage of apoptotic cells was monitored by Annexin V-FITC staining and flow cytometry as described in Materials and Methods. B, MM.1S cells were incubated for 18 hours with the indicated concentration of UCN-01 in either presence or absence of 10 µmol/L L744832, after which the percentage of apoptotic cells exhibiting Annexin V+ and loss of mitochondrial membrane potential (m), respectively, was determined by flow cytometry as described in Materials and Methods. C, MM.1S cells were treated with the indicated concentration of L744832 in either the presence or absence of 150 nmol/L UCN-01 for 18 hours, after which the percentage of apoptotic cells was determined as described in (B). D, MM.1S cells were exposed to 10 µmol/L L744832 ± 150 nmol/L UCN-01. At the indicated intervals, cells were harvested and the percentage of cells exhibiting apoptotic morphology was determined by evaluating Wright-Giemsa–stained cytospin preparations as described in Materials and Methods. E, MM.1S cells were treated with 10 µmol/L L744832 (L) ± 150 nmol/L UCN-01 (UCN) for 18 hours, after which cells were washed free of drug and plated in soft agar as described in the Materials and Methods. After 15 days of incubation, colonies (consisting of groups of >50 cells) were scored, and colony formation for each condition expressed relative to untreated control cells. For (A) to (E), values represent the means ± SD for three separate experiments done in triplicate.

View larger version (24K): [in this window] [in a new window]   Fig. 2. The L744832/UCN-01 regimen highly synergistically induces apoptosis in multiple myeloma cells, including drug-resistant cell lines and primary CD138+ multiple myeloma cells. A, MM.1S, MM.1R, 8226, and H929 cells were exposed to a range of L744832 (5-20 µmol/L) and UCN-01 (50-250 nmol/L) concentrations alone and in combination at fixed ratio (66.7:1 for all cell lines) for 18 hours (MM.1S and MM.1R) or 36 hours (8226 and H929). At the end of this period, the percentage of Annexin V+ cells was determined for each condition; fractional effect values were determined by comparing results to those of untreated controls, and median dose effect analysis was used to characterize the nature of the interaction between L744832 and UCN-01. Combination index (C.I.) values <1.0 denote a synergistic interaction. Two additional studies yielded equivalent results. B, MM.1S and its dexamethasone-resistant counterparts (MM.1R), 8226 and its melphalan-resistant subline (8226/LR), H929, and U266 cells, were exposed to L744832 (10 µmol/L for all lines except 12.5 µmol/L for U266) ± UCN-01 (MM.1S and MM.1R, 150 nmol/L; 8226, 8226/LR, and H929, 200 nmol/L; U266, 100 nmol/L) for 18 hours (MM.1S and MM.1R), 36 hours (8226, 8226/LR, H929), or 48 hours (U266) after which the percentage of cells exhibiting apoptotic morphology was determined by evaluation of Wright Giemsa–stained cytospin preparations. Columns, means for three separate experiments done in triplicate; bars, SD. C, CD138+ and CD138- cells were isolated from bone marrow samples of four patients with multiple myeloma as described in Materials and Methods. Cells were exposed to 10 µmol/L L744832 (L) ± 150 nmol/L UCN-01 (UCN) for 24 hours, after which the percentage of cells exhibiting apoptotic morphology was determined by evaluation of Wright-Giemsa–stained cytospin preparations. Columns, means for the experiments done in triplicate; bars, SD.

View larger version (40K): [in this window] [in a new window]   Fig. 3. The combination of L744832 and UCN-01 triggers activation of the mitochondrial dysfunction and caspase cascade in multiple myeloma cells. MM.1S, MM.1R, 8226, 8226/LR, and H929 cells were treated with L744832 ± UCN-01 as described in Fig. 2B. At the end of this period, the cells were lysed and subjected to Western blot analysis using the indicated primary antibodies. CF, cleavage fragment. Alternatively, cells were treated as described above, after which cytosolic (S-100) fractions were prepared as described in Materials and Methods, and expression of cytochrome c and Smac/DIABLO in the cytosol was monitored by Western blot. Each lane was loaded with 30 µg of protein; the blots were stripped and reprobed with antiactin or antitubulin antibody to ensure equal loading and transfer. The results of a representative experiment are shown; two additional studies yielded equivalent results.

View larger version (46K): [in this window] [in a new window]   Fig. 4. Coadministration of L744832 and UCN-01 results in inactivation of MEK/ERK and Akt pathways while promoting activation of SEK/JNK and p34cdc2. A and B, MM.1S (left) and H929 cells (right) were treated with L744832 (10 µmol/L) ± UCN-01 (MM.1S, 150 nmol/L; H929, 200 nmol/L) for 18 hours (MM.1S) or 36 hours (H929), after which Western blot analysis was done to evaluate processing of H-Ras (UP, unprocessed; P, processed), expression of total and/or phosphorylated ERK, JNK, cdc2/Cdk1, Akt, and its downstream targets (e.g., GSK-3/ß, p70 S6 kinase, and Bad). C, Alternatively, MM.1S and H929 cells were treated as described in (A) and (B), after which an Akt kinase assay was done as described in Materials and Methods. D, MM.1S cells were incubated with 10 µmol/L L744832 ± 150 nmol/L UCN-01 for the indicated intervals, after which cells were lysed and subjected to Western blot analysis to monitor PARP cleavage and phosphorylation of ERK, JNK, cdc2, GSK-3/ß, and p70 S6 kinase. For Western blot analysis, each lane was loaded with 30 µg of protein; blots were subsequently stripped and reprobed for expression of tubulin to ensure equivalent loading and transfer of protein. Two additional studies yielded equivalent results.

Time course studies were carried out to characterize the kinetics of changes in signaling pathways in MM.1S cells. Combined treatment resulted in PARP degradation, down-regulation of phospho-ERK, activation of JNK, and diminished phosphorylation of p34^cdc2, GSK-3, and p70^S6K as early as 3 hours following drug exposure, when apoptosis was minimal (Fig. 1D), and these effects were considerably more pronounced after 6 hours (Fig. 4D). These findings suggest that the effects of L744832/UCN-01 on signaling pathways represent relatively early events in the cell death process.

Attempts were then made to determine whether IL-6 and IGF-I, growth factors that promote myeloma cell survival (41, 42), could attenuate the lethality of the UCN-01/L744832 regimen. As shown in Fig. 5A, coadministration of IL-6 (100 ng/mL) or IGF-I (400 ng/mL) failed to diminish UCN-01/L744832–mediated apoptosis in either MM.1S or U266 cells (P > 0.05 in each case). In marked contrast, IL-6 and IGF-I significantly reduced apoptosis induced by dexamethasone in both cell lines (P < 0.02 and P < 0.05, respectively). Consistent with these results, IL-6 and IGF-I failed to block UCN-01/L744832–mediated PARP degradation, ERK inactivation, p34^cdc2 dephosphorylation, and down-regulation of phospho-GSK-3 and phospho-p70^S6K in MM.1S cells (Fig. 5B).

View larger version (33K): [in this window] [in a new window]   Fig. 5. The L744832/UCN-01 regimen is not susceptible to drug-resistance conferred by IL-6, IGF-I, or stromal cells. A, MM.1S and U266 cells were incubated with 10 µmol/L L744832 + 150 nmol/L UCN-01 for 24 hours (MM.1S) or 48 hours (U266) in either the presence or absence of 100 ng/mL IL-6 or 400 ng/mL IGF-I. At the end of this period, the percentage of cells exhibiting apoptotic morphology was determined by evaluating Wright-Giemsa–stained cytospin preparations. For comparison, multiple myeloma cells were exposed to 50 µmol/L (MM.1S) or 100 µmol/L dexamethasone (U266) for 24 hours in the presence or absence of IL-6 or IGF-I. Columns, means for three separate experiments done in triplicate; bars, SD. *, significantly less than values for dexamethasone-treated cells in the absence of IL-6 or IGF-I (**, P < 0.01 and *, P < 0.05). B, MM.1S cells were incubated with 10 µmol/L L744832 + 150 nmol/L UCN-01 for 24 hours in either the presence or absence of 100 ng/mL IL-6 (left) or 400 ng/mL IGF-I (right), after which Western blot analysis was used to monitor PARP cleavage and phosphorylation statues of ERK, JNK, cdc2, GSK-3/ß, and p70 S6 kinase. Each lane was loaded with 30 µg of protein; the blots were stripped and reprobed with antitubulin antibody to ensure equal loading and transfer. Results are representative of three separate experiments. C, HS-5 cells were precultured in 12-well plates for 48 hours, after which stable GFP-transfected U266 cells (GFP/U266) were seeded to HS-5 culture as described in Materials and Methods. After a 24-hour incubation, equal volumes of fresh medium containing the indicated drugs (final concentration, L744832 = 10 µmol/L and UCN-01 = 200 nmol/L) was added to cocultures of GFP/U266 and HS-5 cells, followed by incubation in the presence of drugs for an additional 36 hours. In parallel experiments, equal volume of fresh medium (M) and conditioned medium (CM) from HS-5 cell culture, both containing the indicated drugs (final concentration, L744832 = 10 µmol/L and UCN-01 = 200 nmol/L), were added to GFP/U266 culture, which were then incubated for an additional 36 hours. At this time, cells were harvested and the percentages of GFP+/U266 cells exhibiting apoptotic morphology (left) and 7AAD positivity (right) were determined by evaluating cytospin preparations under fluorescent microscope and by flow cytometry, respectively. Columns (A and C), means for three separate experiments done in triplicate; bars, SD.

The STAT3 transcription factor has been implicated in multiple myeloma cell survival as well as in stromal cell–related mechanisms of drug resistance (45, 46). Attempts were then made to determine whether STAT3 might be implicated in UCN-01/L744832–mediated lethality. The Western blot shown in Fig. 6A shows that exposure of U266 and MM.1S cells to UCN-01 diminished expression of phospho-STAT3, whereas expression was essentially abrogated following exposure to the combination of UCN-01 and L744832 (Fig. 6A, top). Furthermore, UCN-01/L744832 blocked IL-6–mediated up-regulation of phospho-STAT3 in both U266 and MM.1S cells (Fig. 6A, bottom). In separate studies, treatment of U266 cells with L744832/UCN-01 did not modify the expression of Bcl-x_L, a downstream target of Stat3, in untreated cells or in cells exposed to IL-6 (data not shown). To assess the functional significance of this phenomenon, U266 ectopically expressing constitutively active STAT3 were used (STAT3/U266; Fig. 6B, inset). These cells displayed a very marked (e.g., 7-fold) increase in basal STAT3 activity compared with their empty vector counterparts (PRC-CMV/U266) as determined by luciferase reporter assay (Fig. 6B). Furthermore, following treatment with UCN-01/L744832, STAT3 activity remained 6-fold higher in mutant cells than in their control cells (Fig. 6B). Notably, STAT3/U266 cells were also significantly more resistant to UCN-01/L744832–mediated lethality compared with controls (P < 0.02; Fig. 6C). Western analysis revealed that ectopic expression of STAT3 failed to prevent UCN-01/L744832–mediated ERK inactivation and dephosphorylation of p34^cdc2 and GSK-3, although JNK activation was clearly attenuated in mutant cells (Fig. 6D). Another clone of STAT3/U266 cells yielded equivalent results (data not shown). Together, these findings suggest that inactivation of STAT3 by UCN-01/L744832 regimen may be involved in the lethality of this drug combination and possibly circumvention of IL-6–mediated resistance.

View larger version (33K): [in this window] [in a new window]   Fig. 6. Stable transfection with constitutively active STAT3 or dominant-negative caspase-9 attenuates the lethality of the L744832/UCN-01 regimen but fails to block inactivation of ERK and Akt or activation of p34cdc2. A, top, MM.1S and U266 cells were incubated with L744832 (MM.1S, 10 µmol/L; U266, 12.5 µmol/L) ± UCN-01 (MM.1S, 150 nmol/L; U266, 100 nmol/L) for 18 hours (MM.1S, top) or 24 hours (U266, bottom), after which Western blot analysis was done to evaluate expression of total and phosphorylated STAT3. A, bottom, MM.1S and U266 cells were treated with L744832 + UCN-01 in either presence or absence of 100 ng/mL IL-6 for 18 or 24 hours, after which Western blot analysis was done as described above. B, U266 cells were stably transfected with Flag-tagged constitutive active STAT3 cDNA and empty-vector (PRC-CMV). Western blot analysis was done to monitor expression of Flag-STAT3 (inset). STAT3/U266 and its empty vector counterparts were transfected with STAT3/Luc. After 6 hours of recovery, cells were treated with 12.5 µmol/L L744832 + 100 nmol/L UCN-01 for 48 hours, after which cells were lysed and subjected to luciferase assay for monitoring STAT3 activity (RLU, relative light units). C, STAT3/U266 cells and their empty vector counterparts were treated with 12.5 µmol/L L744832 + 100 nmol/L UCN-01 for 48 hours, after which the percentage of cells exhibiting 7AAD staining were determined by flow cytometry. D, empty vector U266 cells and U266/STAT3 cells were exposed to 12.5 µmol/L L744832 + 100 nmol/L UCN-01 for 48 hours, after which cells were lysed and Western blot analysis used to monitor expression of PARP, phospho-ERK, phospho-JNK, phospho-cdc2, and GSK-3. E, caspase-9 (dominant negative) cells and empty-vector (pcDNA3.1)-transfected U266 cells were treated with L744832 and UCN-01 as above, after which the extent of apoptosis was determined by 7AAD staining and flow cytometry. F, alternatively, caspase-9 (DN, dominant negative) cells and empty vector (pcDNA3.1)–transfected U266 cells were treated as described in (E), after which cells were lysed and subjected to Western blot analysis to monitor release of mitochondrial cytochrome c and Smac/DIABLO, cleavage of caspase-9 and PARP, and expression of phosphorylated ERK, cdc2, GSK-3, and STAT3. Columns (B, C, and E), means for three separate experiments done in triplicate; bars, SD. **, significantly lower than values for parental cells (C) or cells transfected with the empty vector (E; P < 0.02). For Western blot analysis in (A), (D), and (F), each lane was loaded with 30 µg of protein; the blots were stripped and reprobed with antiactin or antitubulin antibodies to ensure equal loading and transfer. Results are representative of three separate experiments.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The results of this study indicate that the farnesyltransferase inhibitor L744832 synergistically increases the lethality of the Chk1 inhibitor UCN-01 in human multiple myeloma cell lines as well as in primary myeloma cells (CD138⁺); moreover, this regimen does not seem to be susceptible to mechanisms, including those related to stromal cell factors, that confer resistance to conventional agents active in multiple myeloma. Furthermore, these events are associated with multiple perturbations in signaling pathways, including inactivation of ERK, STAT3, Akt, and its downstream targets, activation of JNK, and dephosphorylation (activation) of p34^cdc2, all of which have been associated with promotion of apoptosis (47–49). Whereas farnesyltransferase inhibitors have been shown to be potent inducers of apoptosis in certain malignant cell types (50) and to lower the threshold for cell death induced by more conventional cytotoxic agents (51), recent studies indicate that they may also promote cell death by more novel agents targeting cell survival pathways (e.g., imatinib mesylate; ref. 52). The results presented herein suggest that combining farnesyltransferase inhibitors with agents such as UCN-01, which disrupt both cell cycle and survival pathways, warrants further examination in multiple myeloma and potentially other hematologic malignancies.

Farnesyltransferase inhibitors were initially thought to exert their lethal effects by preventing farnesylation of Ras, which is necessary for its translocation to the plasma membrane and activation of downstream targets, including the Raf/MEK/ERK and Akt pathways (53). However, several lines of evidence suggest that the lethal effects of farnesyltransferase inhibitors involve events other than or in addition to interference with Ras prenylation. For example, in contrast to H-Ras, which is dependent upon farnesylation for activation, K-Ras and N-Ras can be prenylated by alternative means (i.e., geranylgeranylation; ref. 54). The finding that responses to farnesyltransferase inhibitors can occur independently of Ras mutational status (55) suggest that alternative targets are likely to be involved in responsiveness to these agents. Such candidate targets include members of the Rho family and centromeric proteins (e.g., CENP-E and CENP-F), which are also subject to farnesylation (56). Analogously, in multiple myeloma, whereas the status of Ras may influence the response of cells to farnesyltransferase inhibitors, an absolute correlation between these events has not been observed (11), suggesting that other factors are in all likelihood involved in lethality. It should be noted that regardless of direct effects on Ras farnesylation, coadministration of L744832 with UCN-01 was associated with a clear diminution in activation of ERK as well as Akt and several of its downstream targets, including GSK-3 and p70S6K. In view of evidence that UCN-01 inhibits the PDK1/Akt pathway (16) and that ERK activation attenuates UCN-01 lethality (22), it is tempting to speculate that disruption of these cytoprotective pathways are involved in the lethality of the UCN-01/L744832 regimen. It is also important to note that coadministration of UCN-01 did not clearly enhance L744832-mediated interference with Ras prenylation. Nevertheless, whereas this finding argues against the view that enhanced disruption of Ras farnesylation is primarily responsible for synergistic interactions between these agents, the possibility that interference with this process contributes to or is necessary for synergistic interactions cannot be excluded.

The results of this study are in many respects reminiscent of previous findings indicating that pharmacologic inhibitors of the MEK1/2/ERK1/2 pathway (e.g., U0126, PD184352) dramatically increase UCN-01–induced apoptosis in both human leukemia and myeloma cells (22, 23). In each case, exposure of cells to UCN-01 resulted in an increase ERK activation, which was blocked or attenuated by either MEK1/2 inhibitors or L744832. By inhibiting Chk1, UCN-01 spares the cdc25C phosphatase from proteasomal degradation, resulting in activation of p34^cdc2 through removal of inhibitory phosphorylation on Thr¹⁴ and Tyr¹⁵ residues (57). Because unscheduled or inappropriate activation of p34^cdc2 is a potent inducer of apoptosis (49), it is tempting to postulate that activation of the MEK/ERK cascade in response to this event protects the cell from apoptosis and that pharmacologic interruption of this pathway lowers the threshold for cell death. Consistent with this model, coexposure of myeloma cells to UCN-01 and L744832 was associated with dephosphorylation of p34^cdc2. It is also noteworthy that combined exposure to UCN-01 and 744832 resulted in a pronounced increase in JNK phosphorylation in myeloma cells. Activation of JNK, possibly through facilitation of mitochondrial injury (cytochrome c release), opposes the antiapoptotic actions of the MEK/ERK pathway, and it has been proposed that the net output of the cytoprotective MEK/ERK and stress-related JNK pathways determines cell fate (58). Consequently, the possibility that UCN-01/L744832–mediated JNK activation cooperates with MEK/ERK inactivation to promote apoptosis in myeloma cells must be considered.

It is noteworthy that the combined exposure to L744832 and UCN-01 effectively killed myeloma cells refractory to standard chemotherapeutic agents (e.g., melphalan) as well as dexamethasone, suggesting that this regimen is able to bypass conventional resistance mechanisms in activating the apoptotic cascade. Significantly, the L744832 regimen exerted selective toxicity toward CD138⁺ bone marrow myeloma cells while sparing their CD138^– counterparts. The basis for this in vitro selectivity, which is similar to that previously observed in the case of regimens combining UCN-01 and MEK1/2 inhibitors (23), is not clear, but might be related to disruption of checkpoint control characteristic of neoplastic cells (59). According to this model, neoplastic cells may be more susceptible to further dysregulation of cell cycle checkpoints (e.g., by agents such as UCN-01), or, alternatively, more dependent upon survival signaling pathways (e.g., MEK/ERK and Akt) to escape the lethal consequences of checkpoint abrogation or inappropriate p34^cdc2 activation.

Interestingly, L744832/UCN-01–mediated lethality toward myeloma cells was minimally affected by stromal cell–related forms of drug resistance, which have been implicated in cell adhesion–mediated drug resistance (27). Consistent with these findings, the growth factors IL-6 and IGF-I, which have been implicated in resistance of multiple myeloma cells to various cytotoxic agents (60), were relatively ineffective in protecting cells from this regimen. Analogously, human stromal cells, which promote the survival of myeloma cells and protect them from various cytotoxic agents (43), were unable to confer resistance to L744832/UCN-01. Taken together, these findings indicate that perturbations induced by the L744832/UCN-01 regimen operate downstream of IL-6/IGF-I and other stromal cell cytoprotective pathways. In this context, the findings that coadministration of inhibitors of MEK1/2 (23) or, more recently, nuclear factor-B (36), similarly circumvent stromal cell or growth factor resistance in multiple myeloma raise the possibility that these interventions share a common mechanism in promoting UCN-01–mediated lethality. Of potential relevance to this issue are the observations that (a) UCN-01/L744832 down-regulated the expression of phospho-STAT3, an important survival factor for myeloma cells (61, 45), and (b) that ectopic expression of activated STAT3 significantly protected myeloma cells from the lethal effects of this drug combination. Notably, STAT3 has been implicated in IL-6 and stromal cell survival signaling in myeloma cells, a process that may involve interactions between the PI3K/Akt and Ras pathways (62). Furthermore, UCN-01, which is known to inhibit Akt (16), has been reported to inactivate STAT3 in colon cancer cells (63). Together, these findings raise the possibility that inactivation of STAT3 by UCN-01/L744832 may represent a common pathway by which this drug regimen overcomes stromal cell–mediated forms of drug resistance. Additional studies will be required to confirm or refute this hypothesis.

The findings that farnesyltransferase inhibitors, such as L744832, dramatically increase the lethality of UCN-01 toward human myeloid leukemia cells (26), and circumvent several conventional forms of drug resistance in myeloma cells, have potentially significant translational implications. Whereas preclinical studies show highly synergistic interactions between UCN-01 and both MEK1/2 and nuclear factor–B inhibitors (23, 36), clinical development of agents, such as PD184352 (CI-1040), are at an early stage of development, and IKK inhibitors, such as Bay 11-7082, have not yet entered clinical trials. In contrast, there is considerable experience with farnesyltransferase inhibitors, such as R115777, in hematologic malignancies, and encouraging initial results in patients with acute leukemia (64) and multiple myeloma (7). Given the present findings, as well the possible activity of UCN-01 in certain hematologic malignancies (14, 17), the concept of combining clinically relevant farnesyltransferase inhibitors with UCN-01 in the treatment of multiple myeloma and leukemia suggests itself. However, the rationale for pursuing this approach clinically would be strengthened by evidence of in vivo activity for this drug combination. Accordingly, animal studies to test the in vivo relevance of these findings are currently under development.

	Footnotes

 
Grant support: NIH grants CA 63753, CA 100866, and CA 93738; award 6045-03 from the Leukemia and Lymphoma Society of America; a Translational Research award from the V Foundation; and award DAMD-17-03-1-0209 from the Department of Defense.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 11/17/04; revised 3/ 2/05; accepted 3/18/05.

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