BLyS fusion toxin in in vitro and in vivo models of mantle cell lymphoma

Biochemical Pharmacology 84 (2012) 451–458

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The therapeutic effects of rGel/BLyS fusion toxin in in vitro and in vivo models of mantle cell lymphoma§ Mi-Ae Lyu a, Lan V. Pham b, Bokyung Sung c, Archito T. Tamayo b, Kwang Seok Ahn c, Walter N. Hittelman c, Lawrence H. Cheung a, John W. Marks a, Min-Jeong Cho d, Richard J. Ford b, Bharat B. Aggarwal c, Michael G. Rosenblum a,* a

Immunopharmacology and Targeted Therapy Laboratory, Department of Experimental Therapeutics, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77054, USA Department of Hematology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA c Department of Experimental Therapeutics, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77054, USA d The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 12 March 2012 Accepted 31 May 2012 Available online 9 June 2012

Mantle cell lymphoma (MCL) is an incurable, aggressive histo-type of B-cell non-Hodgkin lymphoma associated with both high relapsed rates and relatively short survival. Because MCL over-expresses receptors for B lymphocyte stimulator (BLyS) and displays constitutively active NF-kB, agents targeting these pathways may be of therapeutic relevance in this disease. To investigate the potential clinical use of the rGel/BLyS fusion toxin in combination with bortezomib, we evaluated this fusion toxin for its ability to inhibit MCL growth in severe combined immunodeficiency (SCID) xenograft model. Compared with PBStreated mice, mice treated with this fusion toxin prolonged both median (84 days vs. 125 days) and overall survival (0% vs. 40%) (p = 0.0027). Compared with bortezomib alone-treated mice, mice treated with rGel/ BLyS plus bortezomib showed significantly increased median (91 days vs. 158 days) and overall survival (0% vs. 20%) (p = 0.0127). Histopathologic analysis of peritoneal intestinal mesentery from MCL-SCID mice showed no demonstrable microscopic lymphomatous involvement at 225 days after treatment with rGel/ BLyS. Combination treatment resulted in a synergistic growth inhibition, down-regulation of NF-kB DNAbinding activity, inhibition of cyclin D1, Bcl-xL, p-Akt, Akt, p-mTOR, and p-Bad, up-regulation of Bax, and induction of cellular apoptosis. Our findings demonstrate that rGel/BLyS is an effective therapeutic agent for both primary and salvage treatment of aggressive MCL expressing constitutively active NF-kB and BLyS receptors and may be an excellent candidate for clinical development. ß 2012 Elsevier Inc. All rights reserved.

Keywords: Mantle cell lymphoma BLyS BLyS receptor Bortezomib Combination

1. Introduction Mantle cell lymphoma (MCL) is a histo-type of highly aggressive B-cell non-Hodgkin lymphoma characterized by activation of cyclin D1, a target molecule of nuclear factor-kB (NF-kB), resulting from the t(11;14)(q13;q32) translocation [1,2]. MCL are classified pathologically into at least two subtypes: (1) A classic form of MCL and (2) A blastoid form of MCL. Compared with the classic form of MCL, the blastoid MCL variant is more aggressive and demonstrates a comparatively poor prognosis

§ This research was conducted, in part, by a research funding from the Clayton Foundation for Research (MGR) and by a Translational Research award (LLS 623407) by the Leukemia and Lymphoma Society (MGR). * Corresponding author at: Immunopharmacology and Targeted Therapy Laboratory, Department of Experimental Therapeutics, The University of Texas M.D. Anderson Cancer Center, 1881 East Road, Unit 1950, Houston, TX 77054, USA. Tel.: +1 713 792 3554; fax: +1 713 794 4261. E-mail address: [email protected] (M.G. Rosenblum).

0006-2952/$ – see front matter ß 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bcp.2012.05.019

[3,4]. Studies by Argatoff et al. [4] have reported that the median overall survival of patients with blastoid MCL was 15 months whereas the median overall survival of patients with MCL was 45 months. Despite recent advances in the understanding of the molecular and cellular events driving the pathogenesis of MCL, these tumors are still associated with poor clinical response to treatment and fatal outcome [5,6]. It is now apparent that to improve the cure rate in these malignancies, it will be necessary to translate mechanistic knowledge into novel, rational therapeutic modalities. Both NF-kB and B lymphocyte stimulator (BLyS) are at least two factors which are constitutively activated in MCL cells [7,8]. The NF-kB signaling pathway regulates the survival of normal and malignant B cells [9]. The growth factor BLyS is crucial for B-cell survival and the biological effects of BLyS are mediated by three cell surface receptors designated B cell-activating factor receptor (BAFF-R), transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), and B-cell maturation antigen (BCMA) [10–12]. BAFF-R is expressed in about 80% of MCL [13].

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Therefore, molecules targeting NF-kB, BLyS and its receptor BAFF-R have significant potential to be of therapeutic value in MCL. Targeting and down-regulation of constitutive NF-kB by various agents such as proteasome inhibitor bortezomib [14,15], a specific p-IkB-a inhibitor designated BAY 11-7082 [7] or curcumin were found to induce apoptosis in MCL [16]. Selective inhibition of IkB kinase was shown to sensitize MCL cells to the cycotoxic effects of TRAIL by decreasing cellular FLIP levels [17]. These results suggest that the NF-kB pathway may be an attractive therapeutic target for MCL tumors which express constitutively activated NF-kB. Bortezomib (PS-341, Velcade) is a low molecular weight, watersoluble dipeptide boronic acid analog that targets the 26S proteasome [18], a multicatalyic enzyme that mediates many cellular regulatory signals by degrading proteins or their inhibitors. The proteasome pathway plays a central role in regulating multiple cellular process including cycle progression, apoptosis and cellular differentiation [19]. Although this agent appears to have excellent clinical activity alone [20–22], phase 2 clinical trials of bortezomib treatment for relapsed or refractory MCL demonstrated that 50–60% of the cases were not sensitive to this agent [23–25]. Therefore, combination strategies are needed to improve the overall survival in these patients. We recently generated a rGel/BLyS fusion toxin composed of rGel at the N-terminus followed by a G4S peptide tether to the growth factor BLyS for the specific delivery of rGel toxin to malignant B-cells expressing BLyS receptors. In previous studies [26], rGel/BLyS demonstrated highly specific cytotoxic activity against MCL lines expressing the BLyS receptor BAFF-R. To explore the potential clinical use of the rGel/BLyS fusion toxin in MCL, we evaluated this agent for its ability to inhibit MCL growth in severe combined immunodeficiency (SCID) xenograft model. For comparison, we included the well-known proteasome inhibitor bortezomib, which has been approved for the treatment of relapsed or refractory MCL [23–25]. In this report, we demonstrate that administration of rGel/ BLyS alone or combination of rGel/BLyS plus bortezomib significantly improved both median and overall survival of mice bearing MCL xenograft tumors. Our findings indicate that rGel/BLyS targeting NF-kB pathway is an effective therapeutic agent for both primary and salvage treatment of aggressive MCL that is refractory to conventional chemotherapeutic regimens and may be an excellent candidate for clinical development. 2. Materials and methods 2.1. Materials The following monoclonal and polyclonal antibodies were used: cyclin D1, Bcl-xL, p-Akt (Ser473), Akt, Bax, caspase-9, poly (ADP-ribose) polymerase (PARP), and b-actin (Santa Cruz Biotechnology, Santa Cruz, CA); cleaved caspase-3 (Cell Signaling, Danvers, MA); p-mTOR (Ser2448) and p-Bad (Ser136) (Millipore, Temecula, CA). The proteasome inhibitor bortezomib was kindly supplied by Dr. J. Adams (Millennium, Cambridge, MA). Cell proliferation kit II (XTT) was purchased from Roche (Mannheim, Germany).32P was purchased from MP Biochemicals (Solon, OH). 2.2. Cell culture The three MCL cell lines (Mino, JeKo-1, and SP53) were kindly provided by Dr. Hesham Amin (M.D. Anderson Cancer Center, Houston, TX) [27]. JeKo-1 cell line was cultured in RPMI 1640 medium (HyClone Laboratories, Logan, UT) supplemented with 10% heat-inactivated fetal bovine serum (Atlanta Biologicals Inc., Lawrenceville, GA), 100 units/ml penicillin (Gibco, Grand Island, NY) and 100 mg/ml streptomycin (Gibco). Mino and SP53 cells were

grown in RPMI 1640 medium (HyClone Laboratories) containing 20% heat-inactivated fetal bovine serum (Atlanta Biologicals Inc.), 100 units/ml penicillin, and 100 mg/ml streptomycin (Gibco). 2.3. In vitro combination studies of rGel/BLyS with bortezomib on MCL cell lines Mino, JeKo-1, or Sp53 cells were seeded (5  105 cells/well) in flat bottom 12 well plates (Becton Dickinson Labware, Franklin Lakes, NJ) and various concentrations of rGel/BLyS in the absence or presence of bortezomib (Millennium) were added. After 2 days, cell viability was assessed using the XTT assay kit (Roche) as described previously [26]. Absorbance was measured at 450 nm using an ELISA reader (Bio-Tek Instruments, Winooski, VT). To analyze the cellular interaction between the two agents, for tested combination of the two agents combination index (CI) values were calculated as proposed by Chou and Talalay [28] utilizing CalcuSyn software (Biosoft, Cambridge, United Kingdom): CI = (D)1/(Dx)1 + (D)2/(Dx)2 + aD1D2/(Dx)1(Dx)2, where (D)1 and (D)2 in combination kill x% of cells, and (Dx)1 and (Dx)2 are the estimated dose of the drug alone capable of producing the same effect of the combined drugs. CI < 1, CI = 1, and CI > 1 indicate synergism, additive effect, and antagonism, respectively. 2.4. Electrophoretic mobility shift assay To determine the status of active NF-kB in three MCL cell lines under study, we performed electrophoretic mobility shift assay (EMSA) using a 32P-labeled probe as described previously [29]. To determine the effects of rGel/BLyS alone on the NF-kB DNA-binding, Mino cells were seeded at 5  105 cells per 12-well plate (Becton Dickinson) and then treated with different concentrations of rGel/ BLyS for 8 h or 10 nM rGel/BLyS for different times. For combination studies, Mino, JeKo-1, or SP53 cells were seeded at 5  105 cells per 12-well plate (Becton Dickinson) and then treated with 10 nM rGel/ BLyS plus 5 nM bortezomib (Millennium) for 48 h. Nuclear proteins were extracted from three MCL cells after different treatments. EMSA was performed by incubating 4 mg of nuclear extract for 15 min at 37 8C with 16 fmol of 32P-end-labeled 45-mer doublestranded oligo nucleotide (Life Technologies, Grand Island, NY) (15 mg of protein) containing the NF-kB binding site (50 TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-30 ) from the human immunodeficiency virus long terminal repeat. The DNA-protein complex formed was separated from free oligonucleotide on 6.6% native polyacrylamide (Bio-Rad Laboratories, Hercules, CA) gels and the gel was then dried. A double-stranded oligonucleotide (Life Technologies) with mutated NF-kB sites (50 -TTGTTACAACTCACTTTCCGCTGCTCACTTTCCAGGGAGGCGTGG-30 ) was used to examine the specificity of binding of NF-kB to the DNA. The specificity of binding was also examined by competition with the unlabeled oligonucleotide. The dried gels were visualized and NF-kB activation was quantitated by a PhosphoImager Storm 820 (Amersham Biosciences, Picastaway, NJ) using Imagequant software. 2.5. Western blot analysis To examine the effects of rGel/BLyS, bortezomib (Millennium) or combination (rGel/BLyS plus bortezomib) on the cyclin D1, BclxL, p-Akt (Ser473), Akt, p-mTOR (Ser2448), p-Bad (Ser136), Bax, caspase-9, cleaved caspase-3, and PARP, Mino, JeKo-1, or SP53 cells were seeded at 1  106 cells/12-well plate (Becton Dickinson), and then treated with 10 nM rGel/BLyS, 5 nM bortezomib (Millennium), or combination for 48 h. Whole cell extracts were prepared after wash with PBS and lysed on ice for 20 min in 0.2 ml of lysis buffer (Promega). Cell lysates (50 mg) were separated by SDS-PAGE (8–15%) and electrophoretically transferred to polyvinylidene

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2.8. Microscopy

difluoride (PVDF) membranes (Millipore Corporation, Bedford, MA) overnight at 4 8C in transfer buffer [25 mM Tris–HCl (pH 8.3), 190 mM Glycine, 20% methanol]. The PVDF membranes were blocked for 1 h in Tris-buffered saline containing 5% nonfat dry milk (LabScientific Inc., Livingston, NJ) and then probed with different primary antibodies at a 1:500 dilution for 1 h at room temperature (RT). After three washes, the membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse/anti-rabbit (Bio-Rad Laboratories) or bovine antigoat antibodies (Santa Cruz Biotechnology) at a 1:2000 dilution for 1 h at RT. Detection of immunoreactive proteins was performed with ECL detection reagent (Amersham Pharmacia Biotech Inc., Piscataway, NJ). b-actin was used as a control for protein loading.

H&E stained sections were examined using an Olympus BX41 microscope (Olympus, Melville, NY) equipped with a 40/0.65 or 40/1.25 oil-immersion Olympus Uplan F1 objective lens. Images were captured with an Olympus Q color 5 camera, acquired with Olympus Q Capture software version 2.7, and processed with Adobe Photoshop CS2 software (Adobe Systems, San Jose, CA). 2.9. Statistical analysis Survival curves were plotted using Kaplan–Meier method. All statistical analyses were done with GraphPad Prism 4 software (GraphPad Software, La Jolla, CA). p-Values <0.05 were considered statistically significant.

2.6. In vivo therapy in a MCL xenograft model Three-week-old female severe combined immunodeficiency (SCID) mice were purchased from Taconic (Hudson, NY). The mice were housed five per cage and maintained under specific pathogen-free conditions at the SCID Mouse Barrier Facility at M.D. Anderson Cancer Center. The experimental protocol was reviewed and approved by the M.D. Anderson Institutional Animal Care and Use Committee. Mino cells (5  107) were injected intraperitoneally into the mice using a 27-gauge needle (Becton Dickinson). One week after inoculation, mice were pooled and randomized into five treatment groups (n = 5/group). The mice were treated intraperitoneally with PBS (control), bortezomib (Millennium), rGel/BLyS, or rGel/BLyS plus bortezomib every 3–4 days for 35 days (Days 7, 10, 14, 17, 24, 28, 31. and 35). The total dose of bortezomib (Millennium) was 167 mg/kg and the total dose of rGel/BLyS was 20 mg/kg or 40 mg/kg. At 225 days post tumor inoculation and treatments the remaining mice were euthanized and subjected to necropsy.

3. Results 3.1. rGel/BLyS interacts synergistically with bortezomib in MCL cell lines The proteasome inhibitor bortezomib has been approved for the treatment of relapsed and/or refractory MCL [23–25]. In previous study [26], we observed that the rGel/BLyS fusion toxin had highly specific cytotoxic activity against MCL lines expressing the BLyS receptors BAFF-R, TACI, and BCMA. To examine the potential clinical use of rGel/BLyS in combination with bortezomib, we investigated in vitro and in vivo efficacy of combination treatment. First, we evaluated whether rGel/BLyS can induce a synergistic or an additive effect in three MCL lines. As shown in Fig. 1, combination of rGel/BLyS plus bortezomib exhibited significant synergistic cytotoxicity against Mino, JeKo-1, or SP53 cells (CI < 1). We also observed that rGel/BLyS in combination with bortezomib exhibited a significant additive cytotoxicity against Z-138 (blastoid form) cells in vitro (data not shown).

2.7. Morphologic and immunohistochemical analysis

3.2. Treatment with rGel/BLyS plus bortezomib inhibits constitutive NF-kB activation in MCL cells The hallmark of MCL is the t(11;14) (q13;32) translocation that has been shown to result in constitutive over-expression of cyclin D1. Cyclin D1 is an NF-kB target molecule and NF-kB activity is

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Fig. 1. Synergistic effect of rGel/BLyS and bortezomib in MCL cell lines. To assess the cytotoxic activity of rGel/BLyS in combination with bortezomib against MCL lines, Mino, JeKo-1, or SP53 cells (5  105 cells/well) were seeded in 12-well plates and treated with various concentrations of rGel/BLyS, bortezomib, or rGel/BLyS plus bortezomib. After 48 h, cell viability was assessed using the XTT assay. Absorbance was measured at 450 nm using an ELISA reader. Normalized isobolograms were then generated using the CalcuSyn software, depicting CI values (no. 1–3) of combination drug studies with 4 nM (1), 5 nM (2), or 6 nM (3) bortezomib and 10 nM rGel/BLyS. CI < 1, CI = 1, and CI > 1 indicate synergism, additive interaction, and antagonism, respectively.

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critical for cell growth and survival in normal B cell development and also regulates growth and survival in malignant B cells [30]. To investigate in vitro mechanistic studies of rGel/BLyS in combination with bortezomib, we sought to determine the status of active NF-kB in three MCL cell lines under study. As shown in Fig. 2A, NF-kB (p65) was found to be constitutively activated in JeKo-1, SP53, and Mino lines. The Mino cell line was found to express the highest level of constitutive NF-kB DNA binding activity. To evaluate whether rGel/BLyS treatment exerts an inhibitory effect on NF-kB activation in MCL cells, we performed EMSA with nuclear extracts from control and rGel/BLyS-treated Mino cells. As shown in Fig. 2B, rGel/BLyS was found to suppress constitutive NF-kB activation in both a dose- and time-dependent manner. Treatment of Mino, JeKo-1, or SP53 cells with rGel/BLyS in combination with bortezomib down-regulated NF-kB DNA binding activity in three MCL cells (Fig. 2C).

apoptosis by stimulating the transactivation potential of the RelA/ p65 subunit of NF-kB [36]. As shown in Fig. 3B, combination treatment inhibited the expression levels of p-Akt (Ser473) and total Akt in Mino, JeKo-1, and SP53 cells. Mammalian target of rapamycin (mTOR) is a direct target of Akt signaling pathway in mitogen-stimulated cells [37] and mTOR inhibitor temsirolimus (CCI-779) has been used in phase II trial for relapsed mantle cell lymphoma [38]. We found that treatment of three MCL cells (Mino, JeKo-1, or SP53) with rGel/BLyS plus bortezomib down-regulated phosphorylation of mTOR at Ser2448 (Fig. 3B). Bad is a proapoptotic member of the Bcl-2 family that promotes cell death by displacing Bax from binding to Bcl-2 and Bcl-xL and phosphorylation of Ser136 by Akt can block the Bad-induced death of primary neurons in a site-specific manner [39]. Our results demonstrated that combination treatment induced dephosphorylation of Bad at Ser136 (Fig. 3B).

3.3. Treatment with rGel/BLyS plus bortezomib supresses Akt/NF-kB dependent intracellular targets

3.4. Treatment with rGel/BLyS plus bortezomib induces apoptosis

The canonical NF-kB pathway directly influences cellular proliferation and downstream targets include cyclin D1, c-myc, and IL-6 [31–33]. In addition, NF-kB also regulates the expression of several anti-apoptotic proteins including IAP1, IAP2, Bcl-2, BclxL, x-IAP, c-FLIP, and survivin [34]. As shown in Fig. 3A, treatment of three MCL cells (Mino, JeKo-1, or SP53) with rGel/BLyS plus bortezomib inhibited the expression of NF-kB-target molecules such as cyclin D1 and Bcl-xL. Both bortezomib and rGel/BLyS alone also showed an inhibitory activity on cyclin D1 expression. Akt has been shown to regulate cell survival through activation of NF-kB-mediated Bcl-xL expression [35] and can also suppress

Bax, a pro-apoptotic protein member, participates in the induction of apoptosis in response to many apoptotic signals. We next examined whether combination treatment can modulate Bax expression and found that combination treatment resulted in induction of Bax after 48 h exposure (Fig. 4A). The caspase proteins are known to be central mediators of the apoptotic effects of TNF and other cytokines. To determine whether caspases were activated in MCL cells during combination-induced cell death, we examined the cleavage of caspase-9, caspase-3, and its substrate PARP at 48 h. As shown in Fig. 4B, treatment with rGel/BLyS in combination with bortezomib or

Fig. 2. Treatment with rGel/BLyS in combination with bortezomib inhibits constitutive NF-kB activation in MCL lines. (A) Constitutive activation of NF-kB in MCL lines. Nuclear extracts from three mantle cell lymphoma lines (JeKo-1, SP53, or Mino) were prepared. (B) Dose-dependent and time-dependent inhibition of NF-kB DNA binding activity by rGel/BLyS. Mino cells were seeded (1  106 cells/well) in 12 well plates and incubated with different concentrations of rGel/BLyS for 8 h or 10 nM rGel/BLyS for different times and then collected nuclear extracts. (C) Inhibition of NF-kB DNA binding activity by rGel/BLyS plus bortezomib. Mino, JeKo-1, or SP53 cells were seeded (1  106 cells/well) in 12 well plates and incubated with 10 nM rGel/BLyS plus 5 nM bortezomib for 48 h and then collected nuclear extracts. Nuclear extracts were subjected to EMSA to assess NF-kB binding activity.

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Fig. 3. Treatment with rGel/BLyS plus bortezomib suppresses Akt/NF-kB-regulated proteins in MCL lines. To examine the effect of rGel/BLyS in combination with bortezomib on cyclin D1 (A, left panel), Bcl-xL (A, right panel), p-Akt (Ser473) (B), Akt (B), p-mTOR (Ser2448) (B), or p-Bad (Ser136) (B), Mino, JeKo-1, or SP53 cells were seeded at 1  106 cells/12-well plate, and then treated with 10 nM rGel/BLyS, 5 nM bortezomib, or combination for 48 h. Whole cell extracts (50 mg) were fractionated by 8–15% SDS-PAGE and electrophoretically transferred to polyvinylidene fluoride membranes. Membranes were blocked, and then probed with anti-cyclin D1, anti-Bcl-xL, anti-p-Akt (Ser473), antiAkt, anti-p-mTOR (Ser2448), or anti-p-Bad (Ser136) antibodies. Secondary antibodies conjugated with horseradish peroxidase were used to visualize immunoreactive proteins using ECL detection reagent. b-actin was used as a control for protein loading.

single treatment (bortezomib or rGel/BLyS) was found to induce cleavage of caspase-9, caspase-3, and PARP in three MCL cells suggesting that the cytotoxic effects of rGel/BLyS in combination with bortezomib appeared to be mediated, at least in part, by caspase activation. 3.5. Treatment with rGel/BLyS plus bortezomib improves both median and overall survival in a MCL xenograft model

Fig. 4. Treatment with rGel/BLyS plus bortezomib induces apoptosis in MCL lines. To examine the effect of rGel/BLyS in combination with bortezomib on Bax (A), caspase-9 (B), caspase-3 (B), or PARP (B) activation, Mino, JeKo-1, or SP53 cells were seeded at 1  106 cells/12-well plate, and then treated with 10 nM rGel/BLyS, 5 nM bortezomib, or combination for 48 h. Whole cell extracts (50 mg) were fractionated by 8–15% SDSPAGE and electrophoretically transferred to polyvinylidene fluoride membranes. Membranes were blocked, and then probed with anti-Bax, anti-caspapse-9, anticleaved caspase-3, or anti-PARP antibodies. Secondary antibodies conjugated with horseradish peroxidase were used to visualize immunoreactive proteins using ECL detection reagent. b-actin was used as a control for protein loading.

To investigate the potential in vivo efficacy of rGel/BLyS in combination with bortezomib, we utilized a MCL xenograft model that we had developed. SCID mice were inoculated (i.p.) with Mino cells and various experimental groups were treated i.p. with PBS (control), bortezomib (167 mg/kg), rGel/BLyS (20 mg/kg or 40 mg/ kg) or rGel/BLyS (40 mg/kg) plus bortezomib (167 mg/kg). Compared with PBS-treated mice, mice treated with 40 mg/kg of rGel/ BLyS significantly prolonged both median (84 days vs. 125 days) and overall survival (0% vs. 40%) (p = 0.0027) whereas mice treated with bortezomib (167 mg/kg) had no significant effect on both median (84 days vs. 91 days) and overall survival (0% vs. 0%). Interestingly, mice treated with 20 mg/kg of rGel/BLyS only improved median survival (84 days vs. 106 days) (p = 0.0027). Compared with mice treated bortezomib alone, mice treated with rGel/BLyS (40 mg/kg) plus bortezomib (167 mg/kg) showed significantly increased median (91 days vs. 158 days) and overall survival (0% vs. 20%) (p = 0.0127) and mice treated with rGel/BLyS (40 mg/kg) also significantly improved both median (91 days vs. 125 days) and overall survival (0% vs. 40%) (p = 0.0018) during the 225 day observation period (Fig. 5).

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Bortezomib vs. 20 mg/kg rGel/BLyS ( P=0.0551 ) Bortezomib vs. 40 mg/kg rGel/BLyS ( P=0.0018 ) Bortezomib vs. Bortezomib plus 40 rGel/BLyS ( P=0.0127 ) 40 mg/kg rGel/BLyS vs. B plus 40 mg/kg rGel/BLyS ( P=0.8677 )

Fig. 5. In vivo therapy in a MCL xenograft model. For the MCL xenograft model, 3-week-old female SCID mice were inoculated (i.p.) with Mino cells (5  107). After 7 days of post tumor inoculation, the mice were treated with PBS (control), bortezomib (167 mg/kg), rGel/BLyS (20 mg/kg or 40 mg/kg), or rGel/BLyS (40 mg/kg) plus bortezomib (167 mg/kg) every 3–4 days for 35 days (Days 7, 10, 14, 17, 24, 28, 31, and 35). The total dose of bortezomib was 167 mg/kg and the total dose of rGel/BLyS was 20 mg/kg or 40 mg/kg. Survival was monitored till 225 days. X-Axis, arrows, treatment.

3.6. Necropsy studies At 225 days post tumor inoculation, we performed necropsy studies and histopathological analysis. As shown in Fig. 6A, photomicrographs of representative necropsy sections showed peritoneal MCL tumor development in PBS-treated mice which were sacrificed at 10 weeks – when tumor size reached maximum allowed size. Bortezomib-treated mice which expired after 15 weeks of treatment showed tumor infiltration and partial organ replacement with MCL cells (Fig. 6B). In contrast, histopathologic section of peritoneal intestinal mesentery from the necropsy of a MCL-SCID mice at >200 days after treatment with rGel/BLyS (40 mg/kg) showed no demonstrable microscopic lymphomatous involvement (Fig. 6C) whereas analysis of representative MCLSCID mice at >200 days after treatment with combination showed only a micro-aggregate of typical MCL cells in the intestinal mesentery but no gross tumor involvement in the peritoneum or organ involvement (Fig. 6D). 4. Discussion Mantle cell lymphoma (MCL) is an incurable aggressive histotype of B-cell NHL and it accounts for 5–10% of all NHL [40]. MCL is associated with high relapsed rates [41] but recent studies have identified new treatment approaches for relapsed MCL including proteasome inhibitors [23–25], mammalian target of rapamycin (mTOR) inhibitors [38], a cycline-dependent kinase inhibitor, flavopiridol [42], and thalidomide in combination with rituximab [43]. However, MCL is still associated with poor response to treatment and has the worst prognosis among lymphomas, with a median survival of approximately 3 years. It is now apparent that novel therapeutic agents with unique mechanisms of action and combination strategies are needed to improve the overall survival in these patients. The transcription factor NF-kB has been shown regulate the expression of a number of genes whose products are involved in tumorigenesis [44] and abnormal regulation of the NF-kB pathway has been associated with chemotherapy resistance in both solid tumors and hematological malignancies [45]. A recent gene array

profiling study has shown that the genes involved in TNF and NF-

kB signaling pathways are over-expressed in MCL [46] and the NFkB pathway is a central mediator in the growth and survival of MCL [7,8,30]. These results suggest that inhibition of NF-kB is expected to be of therapeutic value in MCL. Bortezomib is a potent and reversible inhibitor of 26S proteasome [18] and inhibits tumor cell growth by inhibiting NF-kB activation, particularly in MCL constitutively expressing this transcription factor. MCL frequently fails to respond to treatment with bortezomib or subsequently becomes refractory to bortezomib [23–25], suggesting that combination strategies are required. In previous studies [26], rGel/BLyS, a fusion toxin composed of rGel and BLyS, demonstrated highly specific cytotoxic activity against MCL lines expressing the BLyS receptor BAFF-R. To improve the efficacy of bortezomib, we examined the feasibility of combining rGel/BLyS with bortezomib to treat MCL. Before examining the in vivo efficacy of rGel/BLyS in combination with bortezomib in a SCID xenograft model of MCL, we performed in vitro combination studies using Mino, JeKo-1, or SP53 cells. We found that rGel/BLyS plus bortezomib provided significant synergistic cytotoxic activity in Mino, JeKo-1, or SP53 cells (Fig. 1). In our in vivo studies, we also found that compared with PBS-treated or bortezomib alone-treated group, administration of rGel/BLyS (40 mg/kg) or combination of Gel/BLyS (40 mg/kg) plus bortezomib (167 mg/kg) significantly improved both median and overall survival during a 225 day observation period in MCL xenografts (Fig. 5). However, there was no significant median or overall survival differences between rGel/BLyS (40 mg/kg) plus bortezomib and rGel/BLyS (40 mg/kg) alone. This result suggests that the optimal doses, schedule, or sequence of administration maybe needed for optimal treatment. Our results indicate that combination of rGel/BLyS and bortezomib has potential therapeutic efficacy against MCL and may be an excellent candidate for clinical development. The B-cell differentiation antigen CD20 is an integral transmembrane protein expressed primarily in B lineage cells [47] and plays a role in B-cell activation, the regulation of cell growth, and transmembrane calcium flux [48]. The chimeric anti-CD20 antibody rituximab C2B8 (Rituxan; Genentech/Biogen-IDEC) has

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Fig. 6. Clinical appearance of MCL tumor in SCID mice. (A) MCL-SCID mouse treated with PBS. Gross necropsy photo of PBS-treated mouse (A.1). Infiltrating peritoneal adipose tissue showed peritoneal MCL tumor growth (inset) (A.2). Peri-intestinal MCL infiltration from GI mesentery and peritoneal compartments with MCL tumor cells with multiple mitotic figures (inset) (A.3). (B) MCL-SCID mouse treated with bortezomib (167 mg/kg). Gross necropsy photo of bortezomib-treated mouse (B.1). Peritoneal MCL growth involving pancreas and peri-pancreatic connective tissues, showing infiltration and partial organ replacement with MCL cells (inset) (B.2). Hepatic microscopic involvement, showing multiple micro-aggregates of MCL cells, showing typical MCL morphology (inset) (B.3). (C) MCL-SCID mouse treated with rGel/BLyS (40 mg/kg). Gross necropsy photo of rGel/BLyS-treated mouse (C.1). Hepatic microscopic involvement with sparse micro-aggregates of typical MCL tumor cells, showing the usual pattern of liver parenchymal involvement (C.2). Representative histopathologic random section of peritoneal intestinal mesentery, showing the lack of demonstrable microscopic lymphomatous involvement at >200 days after treatment with rGel/BLyS (C.3). (D) MCL-SCID mouse treated with rGel/BLyS (40 mg/kg) plus bortezomib (167 mg/kg). Gross necropsy photo of rGel/BLyS plus bortezomib-treated mouse (D.1). Hepatic microscopic involvement with focal, characteristic MCL tumor cell mico-aggregates, after combined treatments with both rGel/BLyS and bortezomib, necropsied after >200 days (inset) (D.2). Focal small MCL aggregates of typical MCL cells in the intestinal mesentery, but no gross tumor involvement in the peritoneum or organ involvement (inset) (D.3). Higher magnification (200 inset) shows typical MCL morphology.

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