Introduction

Mantle cell lymphoma (MCL) is associated with poor clinical outcomes and inevitable relapses. It is a well-defined lymphoid neoplasm genetically characterized by the t(11;14)(q13;q32) translocation, with constitutive overexpression of cyclin D1.^{1, 2} Relapsed MCL responds poorly to standard antilymphoma therapies, and the median failure-free survival duration is approximately 18 months without a plateau in the survival curve. The median overall survival duration is 3–4 years.^{3, 4, 5} Therefore, novel and more effective agents or regimens are urgently needed to improve the treatment of this disease.

Bortezomib (BTZ) is a potent, selective and reversible inhibitor of the 26S proteasome. BTZ inhibits cell cycle progression, induces cell apoptosis, blocks NF-B activity and inhibits angiogenesis.^{6, 7, 8} After it was shown to have clinical efficacy in relapsed and refractory MCL, BTZ was approved by the US Food and Drug Administration (FDA) for the treatment of patients with MCL who have received at least one prior therapy. Objective response is achieved with BTZ treatment in 29–50% of MCL patients; however, complete remission (CR) rates are low (about 20%) and duration of response is short (6–9 months).^{9, 10, 11} Moreover, BTZ treatment is limited by severe toxic events in patients with relapsed or refractory MCL.^{12, 13} These limitations, however, may be overcome by strategically combining BTZ with other agents.

Rituximab (RTX), a chimeric anti-CD20 monoclonal antibody, has been tested as a single agent for the treatment of both previously untreated and relapsed MCL, with a response rate of 27–38%.^{14, 15, 16} RTX, as a single agent, is generally not adequate for MCL treatment, and is commonly used in combinations of R-HyperCVAD/R-Methotrexate-Cytarabine. Also, cyclophosphamide (CTX) is the most frequently used agent in treating MCL and is a major agent in HyperCVAD and CHOP.

To improve the treatment of relapsed MCL, we have explored combinational therapies using BTZ and other chemotherapy drugs. In this study, pre-clinical studies were performed to examine the effects of a combined BTZ, RTX and CTX (BRC) regimen on MCL cells. Our results showed that the BRC regimen, compared with each of the single agents, not only had more potent growth inhibition activity and induced apoptosis in larger populations of cultured MCL cell lines and primary tumor cells freshly isolated from patients in vitro, but also eradicated subcutaneous tumors in the majority of MCL-bearing SCID mice. Hence, this study provides the basis for future use of this novel BRC regimen in clinical trials.

Materials and methods

Cell lines

MCL cell lines SP53, MINO, Grant 519 and Jeko-1 were positive for t(11;14)(q13; q32) translocation. Cell lines (0.3–0.5 10⁶ cells ml^-1) were cultured in RPMI-1640 (Life Technologies, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum, penicillin (10 000 U ml^-1) (Sigma-Aldrich, St Louis, MO, USA), streptomycin (10 mg ml^-1; Sigma-Aldrich), gentamicin (50 mg ml^-1; Sigma-Aldrich) and L-glutamine (29.2 mg ml^-1; Life Technologies).

Patients

The study was approved by the Institutional Review Board at The University of Texas M. D. Anderson Cancer Center. Bone marrow aspirates and peripheral blood samples were obtained from patients with relapsed MCL. Three patients with a tissue diagnosis of MCL, including cyclin D1 positivity, were included in the study and gave informed consent. Mononuclear cells were separated by Ficoll-Hypaque density centrifugation, and MCL cells were isolated using anti-CD20 magnetic microbeads (Miltenyi Biotec, Auburn, CA, USA). Purity of isolated tumor cells (CD19⁺and CD5⁺ and light chain restriction) was confirmed to be more 95% by flow cytometric analysis.

Reagents and antibodies

BTZ (Millennium Pharmaceuticals Inc., Cambridge, MA, USA) and RTX (Genentech Inc., South San Francisco, CA, USA) were obtained from the pharmacy at M. D Anderson Cancer Center and solubilized in phosphate-buffered saline (PBS; Herndon, VA, USA) as a stock solution. CTX was purchased from Sigma-Aldrich and dissolved in water as a stock solution. Pan-caspase inhibitor Z-VAD-FMK was obtained from Alexis Corp (San Diego, CA, USA), dissolved in dimethyl sulfoxide (DMSO) and used at a final concentration of 100 M. FITC-conjugated Annexin-V was purchased from Caltag Laboratories (Burlingame, CA, USA). Propidium iodide (PI) was purchased from Sigma-Aldrich. Antibodies to caspases-8, -9 and -3 were purchased from Cell Signaling Technology (Beverly, MA, USA); poly (ADP-ribose) polymerase (PARP) was obtained from BD Bioscience (San Jose, CA, USA) and -actin was obtained from Santa Cruz Biotech (Santa Cruz, CA, USA). 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium (MTS) was purchased from Promega (Madison, WI, USA). Ficoll-Hypaque were purchased from Amersham Pharmacia Biotech (Piscataway, NY, USA).

Immunophenotyping

PE- or FITC-conjugated monoclonal antibodies were added to cell pellets, incubated for 30 min on ice and washed three times before analysis. Samples were analyzed using a Becton Dickinson FACScan flow cytometer using CellQuest software (BD Biosciences) and analyzed using CellQuest Pro software (BD Biosciences).

Cell proliferation assays

Cell growth was assessed with a non-radioactive cell proliferation MTS assay using CellTiter 96 Aqueous One Solution Reagent. Briefly, cells were plated in 96-well plates at a concentration of 5 10⁴ cells per well and incubated for 48 h at 37 °C in triplicate with various doses of BTZ (0–80 nM), RTX (0–10 g ml^-1) and CTX (0–20 mM) individually, or with increasing doses of BTZ (0–80 nM) and a fixed dose of RTX (10 g ml^-1) and CTX (10 mM; BRC regimen). Cells in suspension (80 l) were added to 20 l of the reagents and incubated in 96-well plates for 3 h at 37 °C in 5% CO₂, and the light absorbance of formazan was measured at 495 nm on a universal microplate reader equipped with KC4 software (Biotek Instruments, Winooski, VT, USA).

Dose–response curves of the four MCL cell lines were developed based on growth inhibition assays of cells treated with BTZ, RTX or CTX alone or with the BRC regimen. The dose–response curves were then normalized to the control.

Apoptosis assays

An Annexin-V binding assay was used to detect cell apoptosis. Annexin-V and PI were used in combination as markers for both early and late apoptotic changes. Annexin-V has a high affinity for the phospholipid phosphatidylserine, which is translocated to the extracellular leaflet of the cell membrane early in the apoptotic process. Following exposure to a single agent (10 nM BTZ, 10 g ml^-1 RTX or 10 mM CTX), RC (RTX 10 g ml^-1 and CTX 10 mM), or BRC in the same concentrations for 24 h, MCL cells were washed and incubated with 5 l of Annexin-V and 5 l of PI and resuspended in 100 l binding buffer at a concentration of 1 10⁶ cells ml^-1. The tube was gently vortexed and incubated for 15 min at room temperature in dark. At the end of incubation, 300 l binding buffer was added. Samples were analyzed using a FACScan flow cytometer. Apoptotic cells were determined as Annexin V-positive cells.

Western blot analysis

MCL cells were cultured with 10 nM BTZ, 10 g ml^-1 RTX and 10 mM CTX individually, or with the combinational BRC regimen. Cells were harvested, washed twice with cold PBS and lysed with lysis buffer (Cell Signaling, Danvers, MA, USA). Cell lysates were kept on ice for 30 min and then centrifuged at 24 000 rpm for 20 min at 4 °C. Supernatants were collected, and the protein content of each fraction was determined by Bradford assay (Bio-Rad, Hercules, CA, USA). Samples were boiled in loading buffer and separated by 10% SDS–PAGE. After electrophoresis, proteins were transferred onto a nitrocellulose membrane (Bio-Rad), which was incubated with blocking solution (5% non-fat dry milk in PBS containing 0.05% Tween-20) for 2 h and immunoblotted with anti-PARP or anti-caspase-3, caspase-8, caspase-9, or -actin antibodies. The membrane was visualized following incubation with reagents from a chemiluminescence western blot kit (Pierce Biotechnology, Rockford, IL, USA).

In vivo xenograft models

In our animal research facility, 6- to 8-week-old male CB-17 SCID mice (Harlan, Indianapolis, IN, USA) were housed and monitored. All experimental procedures and protocols were approved by the Institutional Animal Care and Use Committee at M. D. Anderson Cancer Center. SCID mice were subcutaneously inoculated in the right flank with 8 10⁶ Jeko-1 cells suspended in 50 l PBS. Tumor burdens were assessed using the two largest perpendicular axes measured with standard calipers. When palpable tumors (5 mm in diameter) developed, mice were separated into five treatment groups of 10 mice each and treated with intraperitoneal injections of PBS, BTZ (1 mg kg^-1), RTX (10 mg kg^-1), CTX (40 mg kg^-1), RC or BRC regimen at the same concentrations on days 1, 4, 7 and 10. Tumor size and body weight were measured daily for the duration of the experiment. Animals were killed when the one-dimensional tumor diameter reached 15 mm or after the loss of >10% of their body weight, in accordance with the institutional guidelines.

Statistical analysis

All assays were performed in triplicate, and data are expressed as mean valuess.d. Statistical significance of differences observed between experimental groups was determined using the Student's t-test. In mouse experiments, overall survival was measured using the Kaplan–Meier method. P-values <0.05 were considered significant.

Results

Drug combination displays synergistic effects of growth inhibition on MCL cells

The effects of BTZ, RTX and CTX alone and in the BRC regimen on the growth of four MCL cell lines were evaluated using the MTS assay. Incubation of the cells with BTZ (Figure 1a) or CTX (Figure 1c) alone resulted in significant and dose-dependent growth inhibition on SP53, MINO, Grant 519 and Jeko-1 cells. RTX alone did not affect proliferation of except MINO cells (Figure 1b). BTZ demonstrated significant cytotoxicity toward the four MCL cell lines (P<0.01) with an IC50 (inhibitory concentration at 50%) between 10 and 20 nM. CTX inhibited the growth of the four MCL cell lines (P<0.01) with an IC50 between 5 and 20 mM. BRC regimen with increasing doses of BTZ in combination with a fixed dose of RTX (10 g ml^-1) and CTX (10 mM) resulted in synergistic growth inhibition, with the growth curves steeper and shifted to the left on the four MCL cell lines (Figure 1d). The BRC regimen induced growth inhibition (P<0.01) with an IC50 between 5 and 10 nM BTZ in combination with RTX and CTX. Taken together, these data clearly demonstrate the capacity of the BRC regimen to synergistically inhibit the growth of MCL cells in vitro.

Figure 1.

Growth inhibitory effects of BTZ, CTX and RTX individually or in combination of three (the BRC regimen) on four MCL cell lines. (a) BTZ at concentrations of 0–80 nM, (b) RTX at concentrations of 0–10 g ml^-1, (c) CTX at concentrations of 0–20 mM and (d) BRC regimen with different concentrations of BTZ combined with a fixed concentration of RTX (10 g ml^-1) and CTX (10 mM). BRC dose–response curves in the four MCL cell lines are steeper and shifted to the left, suggesting synergistic growth inhibition effects. Cell growth was measured in a 48-h culture using MTS assay. Results of three independent experiments are shown.

Full figure and legend (199K)

In vitro induction of apoptosis in MCL cells

Induction of apoptosis by BTZ was analyzed in four MCL cell lines and in primary tumor cells isolated from three MCL patients. The cells were exposed to BTZ at doses ranging from 5 to 80 nM for 24 h, and induction of cell apoptosis was examined by Annexin-V binding assay. As shown by the representative histograms of Annexin-V Positive Grant 519 (Figure 2a), BTZ induced a dose-dependent response of apoptosis in the cells. Indeed, similar responses of apoptosis induction were demonstrated in all the four MCL cell lines (Figure 2b) and primary MCL cells from three patients (Figure 2c). BTZ at concentrations of 10 and 20 nM induced significant apoptosis in primary tumor cells and MCL cell lines (P<0.05, compared with medium controls), although the response to BTZ varied among different cell lines. For example, Grant 519 cells had high sensitivity to BTZ (82% apoptosis with 10 nM BTZ), whereas MINO cells showed a much lower apoptosis rate (23% apoptosis with 10 nM BTZ). Primary tumor cells from three MCL patients were all sensitive to BTZ treatment (53, 74 and 64% apoptosis at 10 nM BTZ) (Figure 2c).

Figure 2.

Dose-dependent apoptosis induced by BTZ. (a) Representative histograms showing Annexin-V positive apoptotic cells (Grant 519) in cultures with addition of different concentrations of BTZ, (b) BTZ-induced apoptosis in four MCL cell lines and (c) BTZ-induced apoptosis in primary tumor cells from three MCL patients. Cells were exposed to various concentrations of BTZ for 24 h and analyzed by Annexin-V-binding assay. Results of three independent experiments are shown.

Full figure and legend (167K)

To compare the effects of the three drugs as single agents or RC combination with the BRC regimen, apoptosis was measured in the four MCL cell lines and in primary tumor cells from three MCL patients using Annexin-V binding assay after 24 h of incubation with 10 nM BTZ, 10 g ml^-1 RTX or 10 mM CTX, RC combination or the BRC regimen at the same concentrations. The BRC regimen induced an average 69.7% of apoptosis in the MCL cell lines (P<0.05 and P<0.01, compared with BTZ, RTX, CTX and RC, respectively) (Figure 3a) and an average 92.6% of apoptosis in primary MCL cells (P<0.05 and P<0.01, compared with the single agents or RC, respectively) (Figure 3b). These data suggest that the BRC regimen markedly enhances apoptosis in MCL cell lines and primary cells compared with single-agent or RC treatment.

Figure 3.

Apoptosis of MCL cells induced by BTZ, RTX, CTX, RC or BRC. Shown are apoptotic cells in (a) four MCL cell lines and (b) freshly isolated primary MCL cells from three patients (PT1–PT3). Cells were treated with 10 nM BTZ, 10 g ml^-1 RTX, 10 mM CTX, RC or BRC at the same concentrations for 24 h, and apoptosis was analyzed by Annexin-V binding assay. Results of three independent experiments are shown.

Full figure and legend (107K)

Activation of caspase pathways in MCL cell apoptosis

To elucidate the molecular mechanisms by which BTZ, RTX, CTX, RC combinations and the BRC regimen induce apoptosis in MCL cells, the activation and cleavage of caspases and PARP were analyzed by western blotting assays. Jeko-1 and Grant 519 MCL cells were incubated with 10 nM BTZ, 10 g ml^-1 RTX, 10 mM CTX, the RC or BRC regimens at the same concentrations for various times. The results show that caspases-8, -9 and -3, and PARP were activated or cleaved in MCL cells treated with BTZ, CTX, RC or the BRC regimens. Furthermore, the BRC regimen appears to have induced apoptosis earlier and stronger than the single-agent treatments (Figure 4a). To confirm the importance of caspase activation in apoptosis of the cells, the effect of caspase inhibition was analyzed. Jeko-1 and Grant 519 cells were incubated for 2 h with 100 M of the pan-caspase inhibitor Z-VAD-FMK before BTZ, RTX, CTX and BRC regimen were added to the cells. Cell apoptosis, detected by Annexin-V binding assay, was completely inhibited by the inhibitor (Figure 4b). These results indicate that the BRC regimen was able to activate caspase-8, -9 and -3 and cleave PARP, and induce apoptosis at earlier time points in larger populations of MCL cells as compared with the single agents.

Figure 4.

Caspase activation and cleavage by BTZ, RTX, CTX, RC and BRC-treated MCL cells. (a) Activation and cleavage of caspases-8, -9 and -3 and PARP in Jeko-1 and Grant 519 cells for different culture times. Whole cell lysates were prepared and subjected to western blot analysis. (b) Caspase inhibition assay on Jeko-1 and Grant 519 cells. Shown is the inhibition of apoptosis by the pan-caspase inhibitor Z-VAD-fmk (ZVAD) in MCL cells treated with BTZ, CTX, RTX, or BRC. Representative results of three experiments performed are shown.

Full figure and legend (145K)

In vivo effects of the drugs on established MCL tumors

To examine the in vivo anti-MCL effects of the BRC regimen in comparison with single agents or RC combination, an MCL SCID mouse model was established. Jeko-1 cells were subcutaneously inoculated into the right flank of the SCID mice, and MCL-bearing mice were treated with BTZ, RTX, CTX, RC or BRC regimens. As shown in Figure 5, mice receiving the BRC regimen had significantly smaller tumor burdens compared with mice treated with single agents, RC or control mice (Figure 5a, P<0.01). In addition, treatment with the BRC regimen significantly improved the long-term event-free survival of tumor-bearing mice (Figure 5b, P<0.05, compared with single-agent or RC treated and PBS control groups). All tumor-bearing mice in control and single or RC treated groups died of aggressive MCL within 5 weeks, while 70% of the mice receiving the BRC regimen survived without or with minimal tumor burdens at the end of the study (10 weeks). Thus, these data clearly demonstrate the superior in vivo anti-MCL activity of the BRC regimen as compared with the single agents or the RC combination.

Figure 5.

In vivo therapeutic effects of BTZ, CTX, RTX, RC and BRC on established MCL in SCID mouse model. (a) Tumor volumes and (b) survival of tumor-bearing SCID mice. CB-17 SCID mice were inoculated subcutaneously in the right flank with 8 10⁶ Jeko-1 cells. Four weeks later, when palpable tumors (5 mm in diameter) developed, mice (10 per group) were treated with intraperitoneal injections of BTZ (1.0 mg kg^-1 day^-1), RTX (10 mg kg^-1 day^-1) or CTX (40 mg kg^-1 day^-1) on days 1, 4, 7 and 10. RC and BRC regimen-treated cohorts received the drugs at the same concentrations on the same time points. Mice receiving injection of PBS on the same day were used as controls.

Full figure and legend (135K)

Discussion

Recent insights into the biology of MCL and the discovery of effective combinational chemotherapy and novel agents have made MCL an inviting target for new agents. BTZ is a first-in-class proteasome inhibitor approved in the United States and the European Union for treatment of multiple myeloma for patients who have received at least one prior therapy. The antineoplastic effect of BTZ involves several different potential mechanisms, including inhibition of cell-cycle progression, induction of apoptosis, NF-B blockade and inhibition of angiogenesis, all of which suggest that BTZ should be active in MCL. Recent results from clinical trials using single-agent BTZ have shown an overall response rate (ORR) of 41% with CR of 21%, partial response (PR) of 21% and failure-free duration of 6–9 months in MCL patients.¹⁰ However, grade 3–4 toxicities were common.^{9, 10, 12, 17} Based on our in vitro data, it is likely that the response to BTZ may be diverse, depending on individual sensitivity. The response of different MCL cell lines to 10 nM BTZ varied from 22.8 to 81.9% apoptotic cells, and the response of primary tumor cells from MCL patients varied from 48.2 to 73.7%. BTZ was also approved by FDA for relapsed or refractory MCL. Because its response rate is still low, a rationally combinational therapy would be the next step. Similarly, Weigert et al.¹⁸ performed in vitro studies and showed a sequence-dependent synergy between BTZ and cytarabine in MCL. Their data suggest that pretreatment with cytarabine followed by proteasome inhibition may be a preferred approach. We exploited the benefits of commonly used cytoreductive agent CTX and the benefits of the new proteasome inhibitor BTZ, together with another established single agent RTX. We chose the BRC combination based on the effects of its single-agent activity and potential synergistic effect with other agents.^{18, 19, 20}

RTX has been used in clinical trials in newly-diagnosed and relapsed or refractory MCL, with ORR of 37%.²¹ At M. D. Anderson Cancer Center, our upfront therapy for MCL is R-HyperCVAD/R-Methotrexate-Cytarabine. Its ORR was 97%, CR 87% and 3-year progression-free and overall survival rates were 64 and 82%, respectively. Our trials were mainly with new agents alone or in combination, such as CCI-779, RTX and lenalidomide. These therapies often do not provide enough cytoreductive power and adequate rate of response in the relapsed setting. However, using western blot analysis, we showed that the BRC regimen was able to activate caspases-8, -9 and -3 and cleave PARP earlier and stronger in MCL cell lines as compared with the single agents. BRC showed significantly stronger activity than the single-agent or RC treatment groups in vitro. These results suggest that the BRC regimen may offer new opportunities for integrating novel targeted therapies with conventional chemotherapy. Perez-Galan et al.²² elucidated the molecular mechanism by which BTZ induces apoptosis in MCL, and showed that BTZ-induced apoptosis was associated with caspase activation, triggered by alteration of mitochondrial membrane potential and conformational changes of Bax and Bak proteins. However, they also showed that ROS generation played a critical role in BTZ-induced apoptosis, since the disruption of mitochondrial function and induction of apoptosis could be completely rescued by ROS scavengers but only partly rescued by the pan-caspase inhibitor Z-VAD.²² In contrast, other researchers found that ROS generation is upstream of caspase activation in BTZ-induced apoptosis, and Z-VAD completely prevented BTZ-induced apoptosis, although it did not alter BTZ-induced ROS generation.^{23, 24} Our data are consistent with these results and also indicate that BTZ-induced apoptosis is caspase-dependent. It is yet unclear what caused the discrepancy between these studies. Further investigation into the roles of caspases and ROS in BTZ-induced apoptosis in various tumor cells may be needed.

CTX was commonly used in MCL in combination with other drugs (CHOP, HyperCVAD). It has no accumulative cardiotoxicity as found with doxorubicin. Based on our in vivo data, the BRC regimen achieved CR rates of 70% and prolonged the duration of response up to three months. CTX did not have overlapping toxicity with BTZ. The mechanism of action of BTZ and its non-overlapping toxicity profile observed in our study make this drug very appealing for combination with other chemotherapeutic or biologic agents. Such combinations may enhance the effectiveness of both BTZ and the standard agents.

In conclusion, combining the cytoreductive agent CTX with the proteasome inhibitor and anti-CD20 monoclonal antibody may represent a unique synergistic strategy for sensitizing MCL cells to treatment with less damaging conventional agents. Clearly, our data support further preclinical and/or clinical evaluation of the BRC regimen in relapsed MCL. As a result, a clinical trial based on these preclinical data is being planned.

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Acknowledgements

This work was supported by institutional start-up funds from The University of Texas M. D. Anderson Cancer Center and funds from the Crutchfield family and the Kimmel family philanthropic foundations. We would like to thank Ms Alison Woo for reviewing and editing this paper.