It has been established in preclinical models of multiple myeloma and acute myeloid leukemia (AML) that the bone marrow microenvironment provides protection from chemotherapy- and death receptor–mediated apoptosis. This form of resistance, termed de novo drug resistance, occurs independent of chronic exposure to cancer-related therapies and likely promotes the development of multidrug resistance. Consequently, it is of major interest to identify compounds or drug combinations that can overcome environment-mediated resistance. In this study, we investigated the activity of tipifarnib (Zarnestra, formerly R115777) combined with bortezomib (Velcade, formerly PS-341) in microenvironment models of multiple myeloma and AML. The combination proved to be synergistic in multiple myeloma and AML cell lines treated in suspension culture. Even in tumor cells relatively resistant to tipifarnib, combined activity was maintained. Tipifarnib and bortezomib were also effective when multiple myeloma and AML cells were adhered to fibronectin, providing evidence that the combination overcomes cell adhesion–mediated drug resistance (CAM-DR). Of importance, activation of the endoplasmic reticulum stress response was enhanced and correlated with apoptosis and reversal of CAM-DR. Multiple myeloma and AML cells cocultured with bone marrow stromal cells also remained sensitive, although stromal-adhered tumor cells were partially protected (relative to cells in suspension or fibronectin adhered). Evaluation of the combination using a transwell apparatus revealed that stromal cells produce a protective soluble factor. Investigations are under way to identify the cytokines and/or growth factors involved. In summary, our study provides the preclinical rationale for trials testing the tipifarnib and bortezomib combination in patients with multiple myeloma and AML.

Multiple myeloma and acute myeloid leukemia (AML) are cancers with high mortality rates, where novel strategies are required to improve on current treatment standards. It has been well established in multiple myeloma and other malignancies that the interaction between tumor cells and elements of their microenvironment results in resistance to chemotherapy- and death receptor–mediated apoptosis (1). This form of resistance, termed de novo drug resistance, occurs independent of chronic exposure to chemotherapy and likely promotes the development of multidrug resistance (2).

Two basic components comprise environment-mediated drug resistance: physical contact between tumor cells and microenvironment components (cell adhesion mediated drug resistance, CAM-DR) and the local production of soluble factors. More specifically, in multiple myeloma and AML, it has been found that the adhesion of tumor cells (via integrin receptors) to fibronectin results in a drug-resistant phenotype (1, 3). Of importance, in a small series of AML patients, it was noted that those whose leukemic cells expressed VLA-4 (α4β1 integrin) had a high rate of relapse compared with those with low VLA-4 expression (3). These results imply that the physical interaction between tumor cells and bone marrow constituents provides a refuge for minimal residual disease. Tumor-microenvironment contact also results in the production of soluble factors that can further accentuate the drug resistant phenotype (4). In multiple myeloma and AML, cytokines, such as interleukin-6 (IL-6), IL-1β, and vascular endothelial growth factor, have been implicated as important growth and survival factors, and these molecules may also contribute to environment-mediated resistance. Based on the knowledge that the mechanisms of de novo drug resistance are unique and genetically distinct from those associated with acquired resistance (5), it is of major interest to identify compounds or drug combinations that are specifically active on tumor cells protected by the microenvironment compartment.

The proteasome inhibitor bortezomib (Velcade, formerly PS-341) has been found to have clinical activity in patients with relapsed multiple myeloma (6). Bortezomib is a reversible inhibitor of the 26S proteasome, a complex that plays a major role in protein degradation. Inhibition of this complex ultimately leads to inactivation of the transcription factor nuclear factor-κB, a survival protein that is thought to be one of the drugs main targets. A previous study has reported that bortezomib can reverse the CAM-DR phenotype in a multiple myeloma cell line (7). Interestingly, we made similar observations testing the compound tipifarnib (Zarnestra, formerly R115777) in multiple myeloma and AML cells.3

3

N. Yanamandra et al., AACR Abstract #1722, April 2005.

Tipifarnib is a farnesyl transferase inhibitor (FTI) that inhibits the membrane localization of Ras resulting in a loss of function (8). Tipifarnib has been clinically tested in patients with multiple myeloma and AML and was found to be active in both diseases (9, 10). In this study, we investigate the combination of tipifarnib and bortezomib in microenvironment models of multiple myeloma and AML. Our data provide preclinical evidence of activity for this novel drug combination.

Cell lines. RPMI 8226/S, H929, U266, KG-1, and U937 lines were obtained from the American Type Culture Collection (Manassas, VA). MM1s cells were kindly provided by Steven Rosen (Northwestern University, Chicago, IL). 8226/S, U266, U937, and MM1s cells were maintained in RPMI 1640 supplemented with 100 mmol/L l-glutamine (Mediatech, Inc., Herndon, VA) and 10% fetal bovine serum (FBS; Omega Scientific, Inc., Tarzana, CA). H929 cells were maintained in RPMI 1640 supplemented with 10% FBS and 0.05 mol/L 2-mercaptoethanol (Sigma Chemical, St. Louis, MO). The KG-1 line was maintained in Iscove's modification of DMEM (Mediatech) with 4 mmol/L l-glutamine, 25 mmol/L HEPES, and 20% FBS.

HS-5 bone marrow stromal cells were obtained from the American Type Culture Collection and maintained in RPMI 1640 supplemented with 100 mmol/L l-glutamine, 10% FBS, and 1% penicillin/streptomycin. HS-5 green fluorescent protein (GFP) stromal cells were developed by stably expressing enhanced green fluorescent protein under hygromycin (Invitrogen, Carlsbad, CA) selection (50 μg/mL).

Compounds. Tipifarnib was kindly provided by David End (Johnson & Johnson Pharmaceutical Research and Development, LLC, Titusville, NJ). Tipifarnib was dissolved in 100% DMSO (Sigma Chemical) and sonicated for 10 minutes at room temperature. Bortezomib (Millennium Pharmaceuticals, Cambridge, MA) was also dissolved in 100% DMSO, and both compounds were stored at −20°C before use.

Patient samples. Multiple myeloma and AML patient samples were collected under two Institutional Review Board–approved protocols (MCC# 13715 and MCC# 13947/13355). After obtaining informed consent for bone marrow aspiration, mononuclear cells were isolated by Ficoll-Hypaque gradient purification as per the manufacturer's instructions (Amersham Biosciences, Piscataway, NJ). Primary isolates were exposed to tipifarnib and bortezomib and analyzed as described below.

To establish patient bone marrow stromal cells, multiple myeloma patient specimens with <20% myeloma cells were cultured continuously in MEM-α medium (Invitrogen) supplemented with 15% FBS and 1% penicillin with streptomycin until an adherent layer of stromal cells predominated. AML patient specimens were processed by CD33+ selection using CD33 microbeads and the AutoMacs magnetic cell sorter (Miltenyi Biotec, Inc., Auburn, CA). CD33 populations were cultured continuously as above. For coculture experiments, stromal cells were seeded to near confluence and incubated overnight at 37°C. Cell lines were adhered, exposed to tipifarnib and bortezomib, and analyzed as described below.

Combination index analysis. The dose-effect relationship between tipifarnib and bortezomib was analyzed using CalcuSyn software (Biosoft, Ferguson, MO). The combination index equation is based on the following multiple drug effect equation of Chou-Talalay (11): combination index = (D)1 / (Dx)1 + (D)2 / (Dx)2 + (D)1(D)2 / (Dx)1 / (Dx)2. Combination index = 1, >1, or <1 is considered additive, antagonistic, or synergistic, respectively. Drug combination studies were based on the fraction of cells affected relative to untreated controls. The mean and SD of the combination index were calculated using the Monte Carlo algorithm. To evaluate the relative contribution of each agent, 8226 and U937 cells were seeded at 1 × 103 per well and exposed to five concentrations of tipifarnib, bortezomib, and the combination (constant molar ratio, 100:1). After 72 hours at 37°C, cytotoxicity was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as previously described (12).

Fibronectin adhesion and cell death analysis. Adhesion of primary isolates and tumor cell lines to fibronectin was done as previously described (12). For cell lines, adhered tumor cells were incubated overnight at 37°C, and then control-supplemented media or bortezomib ± tipifarnib was added for an additional 24 hours. In primary isolates, control-supplemented media or bortezomib ± tipifarnib was added for 24 hours after 2 hours of adhesion. In parallel, primary isolates or tumor cell lines were cultured in 0.1% bovine serum albumin–coated plates (Boehringer-Mannheim, Indianapolis, IN; multiple myeloma) or 0.1% poly-hema (Sigma, St. Louis, MO)–coated plates (AML) and exposed to control media or bortezomib ± tipifarnib as above. Cell death was determined by flow cytometry after Annexin V/FITC (Biovision, Mountain View, CA) and propidium iodide (Biovision) or 7-amino actinomycin-D (BD PharMingen, San Jose, CA) staining as described previously (13). In primary isolates, samples were also stained with anti CD138 (BD PharMingen, San Jose, CA; myeloma cells) or anti CD33 (BD PharMingen; leukemic cells) antibodies to identify tumor cell populations.

Adhesion assays. Adhesion assays were done similar to previously described (12) using the cell tracker 5-chloromethylfluorescein diacetate (Molecular Probes, Eugene, OR). For pre-adhesion drug treatment, tumor cells were incubated with tipifarnib (5 μmol/L), bortezomib (5 nmol/L), or the combination for 2 hours before adhesion to fibronectin. After 2 hours of adhesion, wells were washed and fluorescence was measured at 490 nm on a Wallac Victor-2 1420 Multilabel Counter. For post-adhesion drug treatment, tumor cells were stained with 5-chloromethylfluorescein diacetate (as above) and then adhered to fibronectin for 2 hours followed by exposure to tipifarnib (5 μmol/L), bortezomib (5 nmol/L), or the combination for an additional 2 hours. Wells were then washed, and absorbance was read as described.

Adhesion to bone marrow stroma and cell death analysis. In coculture experiments, HS-5 GFP stromal cells were seeded to near confluence and incubated overnight at 37°C. The next morning, stromal cells were washed once with serum-free medium, and primary isolates or tumor cell lines were allowed to adhere for 2 hours in either serum-free MEM (Alpha MEM, Invitrogen) or RPMI 1640, respectively. Nonadhered cells were removed, and samples were then exposed to control supplemented media or bortezomib ± tipifarnib for 24 to 36 hours (see figure legends). Percent cell death was determined by flow cytometry as described above. For coculture experiments involving cell lines, gating on GFP populations identified tumor cells. In primary isolates, additional staining with anti-CD138 (myeloma cells) or anti-CD33 (leukemic cells) antibodies distinguished tumor cells from background mononuclear cells. For our experiments, we also determined the number of live cells per sample using flow cytometry as previously described (13).

Transwell analysis. 8226 myeloma and U937 leukemia cells were either adhered (as described above) or separated from HS-5 GFP stromal cells by a Transwell insert (costar, 0.4-μm mesh, 12-mm diameter; Corning, Corning, NY). Cells were treated with either control-supplemented media or the tipifarnib (5 μmol/L) and bortezomib (5 nmol/L) combination for 36 hours. Cell death was determined by flow cytometry after staining with Annexin V/FITC and 7-amino actinomycin-D. Cell death of multiple myeloma and AML cells was determined in coculture by gating on GFP populations.

Western blotting. Western blotting was done as described previously (13). Antibodies were purchased from the following vendors: GADD153 and anti-K-Ras-2B (Santa Cruz Biotechnology, Santa Cruz, CA), β-actin (Sigma Chemical), anti-HDJ-2 (NeoMarkers, Freemont, CA). Densitometry was done by scanning radiographic images, and bands were quantitated using Alpha Ease image analysis software (Alpha Innotech Corp., San Leandro, CA).

Proteasome assay. 8226/S myeloma cells (4 × 106) were exposed to control media, tipifarnib, bortezomib, or the combination for 2 hours at 37°C. Cells were harvested, washed twice with ice-cold PBS, and then lysed in 50 μL of TE buffer [10 mmol/L Tris, 1 mmol/L EDTA (pH 7.9)]. Total protein was quantitated using a Bio-Rad protein assay kit (Hercules, CA), and 10 μg of protein were analyzed for proteasome activity using a 20S proteasome activity assay as per the manufacturer's instructions (Chemicon International, Temecula, CA).

Statistical analysis. Unless otherwise stated, statistical data are expressed as mean ± SE.

Tipifarnib and bortezomib are synergistic in multiple myeloma and AML cell lines. It has previously been reported that FTIs and proteasome inhibitors induce apoptosis in multiple myeloma (7, 1316) and leukemic (17, 18) cell lines. By analyzing the activity of tipifarnib and bortezomib as single agents in several representative lines, we defined concentrations with low toxicity for combination studies (data not shown). We first tested tipifarnib and bortezomib on a diverse group of myeloma lines maintained in suspension culture (Fig. 1A-C). Evidence for greater than additive cell death was observed and was most pronounced in H929 and MM1s cells (Fig. 1A and C). Interestingly, the MM1s line was relatively resistant to single-agent tipifarnib, yet the combination maintained its activity. We also tested KG-1 leukemia cells and found that they were sensitive although to a lesser degree when compared with our myeloma lines (Fig. 1D). To confirm that tipifarnib and bortezomib were synergistic in vitro, 8226/S myeloma cells and U937 leukemia cells were treated with single-agent tipifarnib, bortezomib, and the combination in cell suspension. Combination index analysis revealed synergistic activity between the two compounds at several dose combinations tested (Fig. 2A and B). These results indicate that tipifarnib combined with bortezomib is an active regimen in diverse multiple myeloma and AML cell lines.

Fig. 1.

Tipifarnib combined with bortezomib induces cell death in diverse multiple myeloma and AML cell lines. H929 (A), U266 (B), MM1s (C), or KG-1 (D) cell lines were treated with the indicated concentrations of tipifarnib, bortezomib, or the combination for 24 hours in cell suspension. Cell death was determined by flow cytometry after Annexin V/FITC and propidium iodide staining. Specific cell death was calculated relative to untreated controls. Three independent experiments. To identify evidence for greater than additive effect, a simple linear regression (no intercept model) was used that included drug effect for each individual drug and the interaction effect of the combination. SAS software was used in the calculations using Proc REG (n = 9 observations for each cell line). Ps are provided. Columns, mean; bars, SE.

Fig. 1.

Tipifarnib combined with bortezomib induces cell death in diverse multiple myeloma and AML cell lines. H929 (A), U266 (B), MM1s (C), or KG-1 (D) cell lines were treated with the indicated concentrations of tipifarnib, bortezomib, or the combination for 24 hours in cell suspension. Cell death was determined by flow cytometry after Annexin V/FITC and propidium iodide staining. Specific cell death was calculated relative to untreated controls. Three independent experiments. To identify evidence for greater than additive effect, a simple linear regression (no intercept model) was used that included drug effect for each individual drug and the interaction effect of the combination. SAS software was used in the calculations using Proc REG (n = 9 observations for each cell line). Ps are provided. Columns, mean; bars, SE.

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Fig. 2.

Tipifarnib and bortezomib are synergistic in cytotoxicity assays. 8226/S (A) and U937 (B) cells were treated with tipifarnib, bortezomib, or a constant molar ratio (100:1) of the combination for 72 hours. Cytotoxicity was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays (as described in Materials and Methods). The resulting data was used to generate combination index plots (CI) for various dose combinations. The dashed line indicates additive affect (CI = 1). Antagonism (above dashed line) and synergism (below dashed line). Tables are included to provide the drug concentrations tested. Points, average of quadruplicate values of three independent experiments; bars, SD.

Fig. 2.

Tipifarnib and bortezomib are synergistic in cytotoxicity assays. 8226/S (A) and U937 (B) cells were treated with tipifarnib, bortezomib, or a constant molar ratio (100:1) of the combination for 72 hours. Cytotoxicity was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays (as described in Materials and Methods). The resulting data was used to generate combination index plots (CI) for various dose combinations. The dashed line indicates additive affect (CI = 1). Antagonism (above dashed line) and synergism (below dashed line). Tables are included to provide the drug concentrations tested. Points, average of quadruplicate values of three independent experiments; bars, SD.

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Tipifarnib combined with bortezomib overcomes CAM-DR. It has previously been established that adherence of multiple myeloma and AML cells to the extracellular matrix component fibronectin results in resistance to chemotherapeutic agents, including melphalan, doxorubicin, and cytosine arabinoside (3, 5). To determine whether tipifarnib and bortezomib overcome CAM-DR, 8226/S myeloma (Fig. 3A and B) and U937 leukemia cells (Fig. 3C and D) were adhered to fibronectin and evaluated for sensitivity to single-agent tipifarnib and bortezomib. In both lines and with both compounds, a dose-dependent increase in cell death was observed in suspension and fibronectin-adhered tumor cells. Importantly, fibronectin adherence did not protect tumor cells from tipifarnib- or bortezomib-induced apoptosis. Furthermore, the tipifarnib and bortezomib combination consistently induced cell death more efficiently in adhered 8226/S and U937 cells when compared with cells treated in suspension (Fig. 3E). Evaluation of three multiple myeloma and five AML patient samples also revealed that fibronectin-adhered primary tumor isolates were not protected (Fig. 3E). These results indicate that tipifarnib combined with bortezomib overcomes the CAM-DR phenotype.

Fig. 3.

Tipifarnib and bortezomib induce cell death in fibronectin adhered multiple myeloma (MM) and AML cells. 8226/S myeloma cells (A and B) and U937 leukemic cells (C and D) were treated with the indicated concentrations of tipifarnib or bortezomib for 24 hours either in cell suspension or after adhesion to fibronectin. Cell death was determined by flow cytometry after Annexin V/FITC and propidium iodide staining. Combined from three independent experiments (A-D). E, 8226/S and U937 cell lines along with mononuclear cells from three multiple myeloma and five AML patients were exposed to 5 μmol/L tipifarnib and 5 nmol/L bortezomib for 24 hours either in cell suspension or after adhesion to fibronectin. Cell death was determined as in (A-D). In primary isolates, tumor cells were identified by staining with anti-CD138 (myeloma cells) or anti-CD33 (leukemic cells) antibodies. For cell lines, five (8226/S) and seven (U937) independent experiments were compared using a paired t test with Ps. Columns/points, mean; bars, SE.

Fig. 3.

Tipifarnib and bortezomib induce cell death in fibronectin adhered multiple myeloma (MM) and AML cells. 8226/S myeloma cells (A and B) and U937 leukemic cells (C and D) were treated with the indicated concentrations of tipifarnib or bortezomib for 24 hours either in cell suspension or after adhesion to fibronectin. Cell death was determined by flow cytometry after Annexin V/FITC and propidium iodide staining. Combined from three independent experiments (A-D). E, 8226/S and U937 cell lines along with mononuclear cells from three multiple myeloma and five AML patients were exposed to 5 μmol/L tipifarnib and 5 nmol/L bortezomib for 24 hours either in cell suspension or after adhesion to fibronectin. Cell death was determined as in (A-D). In primary isolates, tumor cells were identified by staining with anti-CD138 (myeloma cells) or anti-CD33 (leukemic cells) antibodies. For cell lines, five (8226/S) and seven (U937) independent experiments were compared using a paired t test with Ps. Columns/points, mean; bars, SE.

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Reversal of CAM-DR is not related to decreased tumor adherence. Because it has been reported that bortezomib can alter the expression of adhesion molecules (19), we addressed whether the activity of tipifarnib and bortezomib was related to decreased adherence of tumor cells to fibronectin. 8226/S myeloma cells (Fig. 4A) and U937 leukemia cells (Fig. 4C) were exposed to tipifarnib, bortezomib, or the combination for 2 hours before adhesion to fibronectin. Tumor cell attachment was not prevented in either line, and pre-adhesion drug exposure for up to 8 hours revealed no significant decrease in attachment relative to controls (data not shown). To verify that tipifarnib and bortezomib did not reverse cell adhesion in attached tumor cells, 8226/S (Fig. 4B) and U937 (Fig. 4D) cells were exposed to tipifarnib, bortezomib, or the combination for 2 hours after being adhered to fibronectin. Once again, we detected no reduction in tumor cell adhesion that could explain sensitivity to the tipifarnib and bortezomib combination.

Fig. 4.

Adhesion to fibronectin is not disrupted by tipifarnib and bortezomib. 8226/S myeloma cells (A and B) and U937 leukemic cells (C and D) were stained for 30 minutes with the cell tracker 5-chloromethylfluorescein diacetate (see Materials and Methods). A and C, cells were washed and then treated with the indicated concentrations of tipifarnib, bortezomib, or the combination for 2 hours (pre-adhesion exposure). After drug treatment, cells were adhered to fibronectin for an additional 2 hours and washed, and fluorescence was analyzed on a fluorescence plate reader. B and D, cells were adhered to fibronectin for 2 hours and then exposed to the indicated concentrations of tipifarnib, bortezomib, or the combination for an additional 2 hours (post-adhesion exposure). Samples were washed, and fluorescence was measured as in (C and D). Combined from three independent experiments. Percent adhesion to fibronectin is relative to cells treated with control media only. Columns, mean; bars, SE.

Fig. 4.

Adhesion to fibronectin is not disrupted by tipifarnib and bortezomib. 8226/S myeloma cells (A and B) and U937 leukemic cells (C and D) were stained for 30 minutes with the cell tracker 5-chloromethylfluorescein diacetate (see Materials and Methods). A and C, cells were washed and then treated with the indicated concentrations of tipifarnib, bortezomib, or the combination for 2 hours (pre-adhesion exposure). After drug treatment, cells were adhered to fibronectin for an additional 2 hours and washed, and fluorescence was analyzed on a fluorescence plate reader. B and D, cells were adhered to fibronectin for 2 hours and then exposed to the indicated concentrations of tipifarnib, bortezomib, or the combination for an additional 2 hours (post-adhesion exposure). Samples were washed, and fluorescence was measured as in (C and D). Combined from three independent experiments. Percent adhesion to fibronectin is relative to cells treated with control media only. Columns, mean; bars, SE.

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Activation of endoplasmic reticulum stress correlates with reversal of CAM-DR. It has been reported that the antitumor activity of isoprenoid inhibitors (such as FTIs and geranylgeranyl transferase inhibitors) may in part be related to inhibition of the proteasome proteolytic pathway.4

4

E.T. Efuet and K. Keyomarsi, AACR abstract #1681, April 2005.

We therefore evaluated proteasome proteolysis in tumor cells after tipifarnib and bortezomib treatment. We found that tipifarnib had little to no effect on endogenous proteasome activity, and the combination was no more active than bortezomib alone (Fig. 5A). Similarly, inhibition of FTase (using HDJ-2 prenylation as a surrogate marker) was also not enhanced (Fig. 5B). Inhibition of K-ras farnesylation was not observed after treatment with tipifarnib and/or bortezomib, consistent with our prior report of a Ras-independent mechanism of cell death (20). These results indicate that enhanced activity at the purported targets of tipifarnib and bortezomib is not responsible for drug synergy; rather, events downstream of the 26S proteosome and FTase likely cooperate to activate intrinsic proapoptotic cascades.

Fig. 5.

Reversal of CAM-DR correlates with activation of the ER stress response. A, 8226 myeloma cells were treated with the indicated concentrations of tipifarnib and bortezomib in cell suspension for 2 hours. Proteasome activity was measured as described in Materials and Methods. Percent proteasome (PS) activity is relative to cells treated with control media. B, 8226/S myeloma cells were treated with control media (lane 1), 5 μmol/L tipifarnib (lane 2), 5 nmol/L bortezomib (lane 3), or the combination (lane 4) for 24 hours in cell suspension. Cell lysates were harvested and analyzed by Western blotting using the indicated antibodies. u, unprocessed HDJ-2; p, processed HDJ-2 or K-Ras. C, 8226 myeloma cells were treated for 12 hours in cell suspension or after adherence to fibronectin as follows: suspension, control media (lane 1), 5 μmol/L tipifarnib (lane 2), 5 nmol/L bortezomib (lane 3), 10 nmol/L bortezomib (lane 4), 5 μmol/L tipifarnib and 5 nmol/L bortezomib (lane 5), 5 μmol/L tipifarnib and 10 nmol/L bortezomib (lane 6); fibronectin, control media (lane 7), 5 μmol/L tipifarnib (lane 8), 5 nmol/L bortezomib (lane 9), 10 nmol/L bortezomib (lane 10), 5 μmol/L tipifarnib and 5 nmol/L bortezomib (lane 11), 5 μmol/L tipifarnib and 10 nmol/L bortezomib (lane 12). As a control for ER stress, U266 cells were either treated with control media (lane 13) or 25 μmol/L tunicamycin (lane 14) for 12 hours. Cell lysates were harvested and analyzed by Western blotting using the indicated antibodies. Densitometric evaluation of bands. A, combined from three independent experiments. B and C, representative of two independent experiments. Columns, mean; bars, SE.

Fig. 5.

Reversal of CAM-DR correlates with activation of the ER stress response. A, 8226 myeloma cells were treated with the indicated concentrations of tipifarnib and bortezomib in cell suspension for 2 hours. Proteasome activity was measured as described in Materials and Methods. Percent proteasome (PS) activity is relative to cells treated with control media. B, 8226/S myeloma cells were treated with control media (lane 1), 5 μmol/L tipifarnib (lane 2), 5 nmol/L bortezomib (lane 3), or the combination (lane 4) for 24 hours in cell suspension. Cell lysates were harvested and analyzed by Western blotting using the indicated antibodies. u, unprocessed HDJ-2; p, processed HDJ-2 or K-Ras. C, 8226 myeloma cells were treated for 12 hours in cell suspension or after adherence to fibronectin as follows: suspension, control media (lane 1), 5 μmol/L tipifarnib (lane 2), 5 nmol/L bortezomib (lane 3), 10 nmol/L bortezomib (lane 4), 5 μmol/L tipifarnib and 5 nmol/L bortezomib (lane 5), 5 μmol/L tipifarnib and 10 nmol/L bortezomib (lane 6); fibronectin, control media (lane 7), 5 μmol/L tipifarnib (lane 8), 5 nmol/L bortezomib (lane 9), 10 nmol/L bortezomib (lane 10), 5 μmol/L tipifarnib and 5 nmol/L bortezomib (lane 11), 5 μmol/L tipifarnib and 10 nmol/L bortezomib (lane 12). As a control for ER stress, U266 cells were either treated with control media (lane 13) or 25 μmol/L tunicamycin (lane 14) for 12 hours. Cell lysates were harvested and analyzed by Western blotting using the indicated antibodies. Densitometric evaluation of bands. A, combined from three independent experiments. B and C, representative of two independent experiments. Columns, mean; bars, SE.

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It has been shown that proteasome inhibitors induce apoptosis in myeloma cells via triggering of endoplasmic reticulum (ER) stress with subsequent disruption of the unfolded protein response (21). It has also been observed that proteasome inhibitors combined with other compounds that activate ER stress (such as tunicamycin) induce cell death in synergy (21). Of relevance, we found that tipifarnib activates the ER stress response in multiple myeloma cell lines (20). Based on these observations, we surmised that apoptosis induced by tipifarnib and bortezomib may be related to activation of the ER stress response. Therefore, 8226/S myeloma cells were exposed to tipifarnib, bortezomib, or the combination for 24 hours either in suspension culture or after being adhered to fibronectin (Fig. 5C). Activation of ER stress was determined by monitoring the expression of GADD153, a well-established ER stress marker (22). We found that both tipifarnib and bortezomib increased GADD153 expression under suspension and adhered conditions consistent with their ability to activate ER stress–related cascades. Importantly, the combination enhanced the ER stress response particularly under fibronectin-adhered conditions. These results imply a link between reversal of CAM-DR and activation of the ER stress response.

Stroma-adhered tumor cells are sensitive to tipifarnib and bortezomib. Due to the fact that soluble factors may also participate in environment-mediated drug resistance, we tested tipifarnib and bortezomib in coculture models of the bone marrow microenvironment. 8226/S myeloma cells were adhered to HS-5 bone marrow stromal cells and then exposed to increasing concentrations of bortezomib (Fig. 6A) or tipifarnib (Fig. 6B) for 24 hours. The percentage of live cells decreased in a dose-dependent manner after treatment with both compounds, and importantly, stroma-adhered 8226/S cells were nearly as sensitive as suspension cells. Also of significance was the fact that high concentrations of bortezomib and tipifarnib were not cytotoxic to cocultured HS-5 stromal cells (Fig. 6A and B). Combined activity was maintained in both stroma-adhered and suspension 8226/S cells after treatment with the tipifarnib and bortezomib combination (Fig. 6C). Once again, however, stroma-adhered tumor cells were partially protected. A similar trend was observed in U937 leukemia cells (Fig. 6D). To determine whether HS-5 stromal cells could also protect primary tumor isolates, primary multiple myeloma and AML cells were adhered to HS-5 stroma and treated with the tipifarnib and bortezomib combination (Fig. 7A and B). Similar to our observations in tumor cell lines, stroma-adhered primary isolates were partially protected relative to suspension cells, whereas fibronectin-adhered tumor cells seemed more sensitive. Stromal cells derived from a multiple myeloma and two AML patients were also able to prevent apoptosis of adhered 8226/S and U937 cells, respectively (Fig. 7C-E).

Fig. 6.

Bone marrow stroma partially protects multiple myeloma and AML cell lines from tipifarnib- and bortezomib-induced cell death. 8226/S myeloma cells were treated with the indicated concentrations of bortezomib (A), tipifarnib (B), or the combination (C) for 24 hours. Cells were treated either in suspension culture (suspension) or after adhesion to HS-5 stromal cells (adhered). The HS-5 stroma line (stroma) expresses GFP in a stable fashion. Myeloma cells were distinguished from stromal cells in coculture by using flow cytometry with gating on GFP+ and GFP populations. D, U937 leukemia cells were treated and analyzed as in (C). Cell death was determined by staining with Annexin V/FITC and 7-amino actinomycin-D as described in Materials and Methods. Percent live cells are relative to tumor cells treated with control media only. Combined from three independent experiments (A-D). Columns/points, mean; bars, SE.

Fig. 6.

Bone marrow stroma partially protects multiple myeloma and AML cell lines from tipifarnib- and bortezomib-induced cell death. 8226/S myeloma cells were treated with the indicated concentrations of bortezomib (A), tipifarnib (B), or the combination (C) for 24 hours. Cells were treated either in suspension culture (suspension) or after adhesion to HS-5 stromal cells (adhered). The HS-5 stroma line (stroma) expresses GFP in a stable fashion. Myeloma cells were distinguished from stromal cells in coculture by using flow cytometry with gating on GFP+ and GFP populations. D, U937 leukemia cells were treated and analyzed as in (C). Cell death was determined by staining with Annexin V/FITC and 7-amino actinomycin-D as described in Materials and Methods. Percent live cells are relative to tumor cells treated with control media only. Combined from three independent experiments (A-D). Columns/points, mean; bars, SE.

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Fig. 7.

Stromal cells partially protect primary isolates from the tipifarnib and bortezomib combination. After obtaining informed consent for bone marrow aspiration, mononuclear cells from a multiple myeloma (A) and an AML (B) patient were isolated by Ficoll-Hypaque gradient purification. Cells were treated with the combination of 5 μmol/L tipifarnib and 5 nmol/L bortezomib for 36 hours either in suspension or after adhesion to fibronectin or HS-5 bone marrow stromal cells. Cell death was determined by flow cytometry after Annexin V/FITC and 7-amino actinomycin-D staining. Tumor cells were identified in coculture by staining with anti-CD138 (myeloma cells) or anti-CD33 (leukemic cells) antibodies. 8226/S myeloma cells (C) and U937 leukemic cells (D and E) were adhered to HS-5 stroma and bone marrow stromal cells derived from a patient with multiple myeloma (C) and AML (D and E), respectively. Tumor cells were exposed to 5 μmol/L tipifarnib and 5 nmol/L bortezomib for 36 hours, and cell death was determined as described above. Cell death in adhered samples was compared with tumor cell lines treated in suspension culture. Columns, mean; bars, SE.

Fig. 7.

Stromal cells partially protect primary isolates from the tipifarnib and bortezomib combination. After obtaining informed consent for bone marrow aspiration, mononuclear cells from a multiple myeloma (A) and an AML (B) patient were isolated by Ficoll-Hypaque gradient purification. Cells were treated with the combination of 5 μmol/L tipifarnib and 5 nmol/L bortezomib for 36 hours either in suspension or after adhesion to fibronectin or HS-5 bone marrow stromal cells. Cell death was determined by flow cytometry after Annexin V/FITC and 7-amino actinomycin-D staining. Tumor cells were identified in coculture by staining with anti-CD138 (myeloma cells) or anti-CD33 (leukemic cells) antibodies. 8226/S myeloma cells (C) and U937 leukemic cells (D and E) were adhered to HS-5 stroma and bone marrow stromal cells derived from a patient with multiple myeloma (C) and AML (D and E), respectively. Tumor cells were exposed to 5 μmol/L tipifarnib and 5 nmol/L bortezomib for 36 hours, and cell death was determined as described above. Cell death in adhered samples was compared with tumor cell lines treated in suspension culture. Columns, mean; bars, SE.

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HS-5 bone marrow stromal cells secrete a protective soluble factor. Partial resistance provided by bone marrow stromal cells could be explained by either CAM-DR or by the participation of protective soluble factors. To address these two possibilities, 8226/S myeloma cells (Fig. 8A) and U937 leukemia cells (Fig. 8B) were treated with the combination of tipifarnib and bortezomib either in suspension culture (suspension), after adhesion to HS-5 stromal cells (coculture), or with separation between the two populations using a transwell insert (transwell). As described previously, stroma-adhered tumor cells were less sensitive to the drug combination when compared with cells treated in suspension (36-hour incubation). Interestingly, tumor cells separated from stromal cells using a transwell insert remained protected. These results imply that HS-5 cells produce a soluble factor(s) that can attenuate the toxicity of tipifarnib and bortezomib. To determine whether tumor-stroma contact enhanced the production of this factor(s), tumor cells were adhered to HS-5 stromal cells and additional tumor cells were separated from coculture by a transwell insert (coculture/transwell). The nonadhered tumor cells were protected similarly to tumor cells separated by the transwell insert (no coculture), suggesting that HS-5 cells constitutively secrete a protective soluble factor(s), and its production is not enhanced by tumor-stroma contact.

Fig. 8.

HS-5 stromal cells secrete a soluble factor(s) that protects multiple myeloma and AML cell lines. 8226/S myeloma (A) and U937 leukemia cells (B) were treated with 5 μmol/L tipifarnib and 5 nmol/L bortezomib for 36 hours. Tumor cells were maintained as follows: in suspension culture (suspension), adhered to HS-5 GFP stromal cells (coculture), separated from HS-5 GFP stromal cells by a transwell insert (transwell), or adhered to HS-5 GFP stromal cells with additional tumor cells separated by a transwell insert (coculture/transwell). In the latter sample, nonadhered tumor cells were harvested for evaluation. Cell death was determined by flow cytometry after staining with Annexin V/FITC and 7-amino actinomycin-D. In coculture, death of myeloma and leukemia cells was determined by gating on GFP cells. Combined from three independent experiments (A and B). Individual conditions were compared using a paired t test with Ps. Columns, mean; bars, SE.

Fig. 8.

HS-5 stromal cells secrete a soluble factor(s) that protects multiple myeloma and AML cell lines. 8226/S myeloma (A) and U937 leukemia cells (B) were treated with 5 μmol/L tipifarnib and 5 nmol/L bortezomib for 36 hours. Tumor cells were maintained as follows: in suspension culture (suspension), adhered to HS-5 GFP stromal cells (coculture), separated from HS-5 GFP stromal cells by a transwell insert (transwell), or adhered to HS-5 GFP stromal cells with additional tumor cells separated by a transwell insert (coculture/transwell). In the latter sample, nonadhered tumor cells were harvested for evaluation. Cell death was determined by flow cytometry after staining with Annexin V/FITC and 7-amino actinomycin-D. In coculture, death of myeloma and leukemia cells was determined by gating on GFP cells. Combined from three independent experiments (A and B). Individual conditions were compared using a paired t test with Ps. Columns, mean; bars, SE.

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The emergence of drug resistance continues to be a major obstacle to the successful treatment of patients with multiple myeloma and AML. Acquired drug resistance is contingent upon the survival of tumor cells during their initial exposure to cancer chemotherapy (de novo drug resistance). The interaction between tumor cells and components of their microenvironment remains critical during this early phase of treatment. Environment-mediated resistance is a result of both physical contact between tumor cells and environmental components as well as the exposure of tumor cells to soluble factors. Both of these interactions participate in prosurvival processes that allow tumor cells to resist chemotherapy and acquire multidrug resistance (1, 4). Novel agents or drug combinations that overcome de novo drug resistance are eagerly sought.

Tipifarnib is a FTI that has clinical activity in multiple myeloma and AML (9, 10). Interestingly, tipifarnib accumulates in bone marrow (10), a desirable property in hematopoietic malignancies that are dependent on the bone marrow microenvironment. We observed that as a single-agent tipifarnib can overcome the CAM-DR phenotype in multiple myeloma and AML cell lines (Fig. 3A and C) and primary isolates (data not shown). It had previously been reported that bortezomib shares similar activity in fibronectin-adhered (7) and stroma-adhered (16) MM1s myeloma cells, and consistent with this, we found that bortezomib efficiently induced cell death in stroma-adhered 8226/S myeloma cells (Fig. 6A). Because bortezomib has been shown to sensitize fibronectin-adhered myeloma cells to chemotherapy-mediated apoptosis (7), we set out to discern whether low doses of bortezomib (5 nmol/L) could enhance the activity of tipifarnib in microenvironment models of multiple myeloma and AML.

Our data reveal that this combination has activity not only in multiple myeloma and AML cells maintained in suspension culture (Fig. 1) but also in tumor cells adhered to the extracellular matrix component fibronectin (Fig. 3), indicating that tipifarnib combined with bortezomib effectively overcomes CAM-DR. It has been previously reported that bortezomib decreases the adhesion of myeloma cells to bone marrow stromal cells (16). Conversely, tipifarnib did not reduce the adhesion of AML blasts to either primary bone marrow stroma or human umbilical endothelial cells (23). In our models, tipifarnib, bortezomib, nor the combination decreased tumor cell adhesion to fibronectin implying that cell death was not associated with the loss of tumor-microenvironment contact (Fig. 4). Stroma-adhered tumor cells were also sensitive to the combination, although they were partially protected relative to cells maintained in suspension culture (Figs. 6 and 7). The fact that tipifarnib and bortezomib were particularly active in fibronectin-adhered tumor cells leads us to consider soluble factors as a possible source of stroma-mediated resistance. In experiments where tumor cells were physically separated from stromal cells using a transwell insert, protection persisted confirming that a soluble factor(s) could partially suppress the activity of the drug combination (Fig. 8).

Several key cytokines serve as autocrine and paracrine growth factors for multiple myeloma and AML cells. IL-6 is known to be a major growth and survival factor in multiple myeloma (24). IL-6 is secreted by HS-5 bone marrow stromal cells (25) and protects multiple myeloma cells from dexamethasone-mediated apoptosis (26). Of importance, it has been reported that IL-6 is incapable of protecting myeloma cells from bortezomib-induced cell death (16). It therefore remains possible that other cytokines and/or growth factors are involved. HS-5 stromal cells also secrete IL-1β that can contribute to autocrine and paracrine growth loops in multiple myeloma (27, 28) and AML (29, 30). However, we previously reported that an FTI inhibits the expression of IL-1β in leukemic cell lines (17), implying that IL-1β is not the protective cytokine in our models. Vascular endothelial growth factor has also been shown to promote the growth of multiple myeloma (31) and AML cells (32), and its mRNA is expressed in HS-5 stromal cells (25, 33). It remains to be seen whether any of the abovementioned cytokines are relevant to the observed stromal-mediated resistance, but experiments are under way to define the cytokine(s) and/or growth factor(s) involved. Our findings will have clinical relevance for antagonists to all three of these factors, as well as other soluble proteins are being clinically tested.

With respect to the mechanism of action of this drug combination, as single agents, both tipifarnib and bortezomib have been shown to induce ER stress–related apoptosis (20, 21, 34, 35). We found that the combination enhanced activation of the ER stress marker GADD153 (Fig. 5C), and this correlated with apoptosis and reversal of CAM-DR. We have previously observed that tipifarnib increases the expression and activity of Bax (20) and Bim5

5

Unpublished observation.

in myeloma cell lines. Interestingly, it has been reported that the expression of Bax and Bim can be regulated via a proteasome-mediated pathway (36, 37). It therefore remains possible that tipifarnib and bortezomib cooperate to enhance the activity of these proapoptotic proteins. Bax and Bim are known to target the ER, leading to ER calcium release (38), activation of caspase-12 (39), and apoptosis in a mitochondria-independent fashion (40). Mitochondrial dysfunction also occurs after tipifarnib treatment (20) and is likely the result of localization of Bax and Bim to mitochondria. It remains possible, however, that intracytoplasmic calcium participates in mitochondrial membrane depolarization by opening of the mitochondrial permeability transition pore (34, 41). Importantly, tipifarnib and bortezomib are active when tumor cells are adhered to fibronectin. Bim expression is known to decrease in adhered myeloma cells (5), and it remains possible that the combination reverses this effect. Current investigations are determining the role of Bax and Bim in reversal of the CAM-DR phenotype. Delineation of the ER stress–related mechanisms responsible for tipifarnib and bortezomib combined activity may ultimately lead to treatment strategies that specifically target environment-mediated drug resistance.

In conclusion, in this study, we provide the preclinical rationale for clinical trials testing tipifarnib and bortezomib in patients with multiple myeloma and AML. Future trials may also include a cytokine and/or growth factor neutralization strategy once protective soluble factors are identified. In theory, such a regimen would eradicate tumor cells protected by the microenvironment compartment, leaving sensitive tumor cell populations for standard or high-dose chemotherapy.

Grant support: NIH Clinical Scholars in Oncology grant 5K12 CA 87989-02 (D.M. Beaupre and L.E. Perez), Moffitt Aging and Cancer Pilot Research grant program P20 CA103676 (D.M. Beaupre), Leukemia Research Foundation New Investigator Award (D.M. Beaupre), and Translational Research and Flow Cytometry Core Facilities at the H. Lee Moffitt Cancer Center and Research Institute.

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.

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