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. 2010 May;12(5):366–375. doi: 10.1593/neo.91960

The rGel/BLyS Fusion Toxin Inhibits Diffuse Large B-cell Lymphoma Growth In Vitro and In Vivo1

Mi-Ae Lyu *, Deepak Rai †, Kwang Seok Ahn ‡, Bokyung Sung ‡, Lawrence H Cheung *, John W Marks *, Bharat B Aggarwal ‡, Ricardo CT Aguiar †, Varsha Gandhi ‡, Michael G Rosenblum *
PMCID: PMC2864474  PMID: 20454508

Abstract

Diffuse large B-cell lymphoma (DLBCL) is an aggressive subtype of B-cell non-Hodgkin lymphoma (NHL) and accounts for 30%to 40%of NHL. Molecules targeting nuclear factor-κB (NF-κB) are expected to be of therapeutic value in those tumors where NF-κB seems to play a unique survival role such as activated B-cell (ABC)-subtype DLBCL. We previously generated a rGel/BLyS fusion toxin for receptor-mediated delivery of the rGel toxin specifically to malignant B cells. In this study, we examined this fusion toxin for its ability to suppress DLBCL growth in vitro and in vivo. rGel/BLyS was specifically cytotoxic to DLBCL lines expressing all three BLyS receptors and constitutively active NF-κB. Treatment with rGel/BLyS induced down-regulation of the phosphorylation of inhibitory subunit of NF-κB (IκB-α), inhibition of NF-κB DNA-binding activity, and accumulation of IκB-α. In agreement with these results, we additionally found that rGel/BLyS downregulated levels of several NF-κB targets including Bcl-xL, Mcl-1, survivin, and x-chromosome linked inhibitor-of-apoptosis. Treatment also induced up-regulation of Bax and apoptosis through caspase-3 activation and poly ADP-ribose polymerase cleavage. Importantly, rGel/BLyS significantly inhibited tumor growth (P < .05) in a DLBCL xenograft model. Thus, our results indicate that rGel/BLyS is an excellent candidate for the treatment of aggressive NHLs that are both dependent on NF-κB and are resistant to conventional chemotherapeutic regimens.

Introduction

Diffuse large B-cell lymphomas (DLBCLs) are highly aggressive B-cell non-Hodgkin lymphoma (NHL) and account for 40% of NHL. On the basis of gene expression profiling studies, DLBCLs are subdivided into three groups: the germinal center B-cell-like (GC)-DLBCL, activated B-cell—like (ABC)-DLBCL, and primary mediastinal B-cell lymphoma [1,2]. It has been shown that GC-DLBCL responds favorably to chemotherapy, whereas ABC-DLBCL tends to be refractory to chemotherapeutic treatment [1,3,4]. Despite recent advances in the understanding of the molecular and cellular basis for their pathogenesis, these tumors are still associated with poor response to treatment and a fatal outcome [5]. It is now apparent that, to improve the cure rate in these malignancies, it will be necessary to translate this mechanistic knowledge into novel, rational, therapeutic modalities.

Nuclear factor-κB (NF-κB) is a transcription factor complex present in the cytoplasm as an inactive heterotrimer consisting of p50, p65, and IκB-α subunits. On activation, the IκB kinase phosphorylates IκB, thereby inducing IκB polyubiquitinylation and subsequent proteolytic degradation by the 26S proteasome. After IκB degradation, the p50–p65 heterodimer is released and then able to translocate into the nucleus and to bind to a specific consensus sequence in the DNA [6] and, subsequently, activate the NF-κB-regulated genes [7]. Several NF-βB-regulated proteins block programmed cell death, including members of the Bcl-2 family, the inhibitor-of-apoptosis (IAP) family, and GADD45β [8–11].

The NF-κB signaling pathway regulates the survival of normal and malignant B cells [12]. In particular, constitutive activation of the NF-κB signaling pathway is crucial for survival of ABC-DLBCL cells but not GC-DLBCL cells [13,14]. These results suggest that the NF-κB pathway may be an attractive therapeutic target for ABC-DLBCL expressing constitutively active NF-κB.

Successful development of tumor-targeted therapeutic agents is dependent, in part, on both site-specific delivery and on the intracellular activity of the delivered agent. Recombinant gelonin (rGel) is a type I ribosomal inactivating plant toxin, and it lacks the ability to bind to the cell surface and to enter intact cells [15]. However, the rGel toxin has impressive cytotoxic effects when delivered to target cells using growth factor ligand as delivery vehicle [16]. Therefore, we recently generated a fusion toxin rGel/BLyS containing rGel at the N-terminus followed by a glycine-glycine-glycine-glycine-serine (G4S) peptide tethered to the B-lymphocyte stimulator (BLyS) molecule for the specific delivery of rGel toxin to malignant B cells expressing BLyS receptors. In previous studies, we showed that rGel/BLyS had highly specific cytotoxic activity against mantle cell lymphoma lines and B chronic lymphocytic leukemia cells expressing the BLyS receptor B-cell-activating factor receptor (BAFF-R) [17,18].

As an extension of our previous studies, we examined this fusion toxin for its ability to suppress NHL growth in a DLBCL xenograft model. Our results show that rGel/BLyS targeting NF-κB signaling pathway is an excellent candidate for the treatment of aggressive ABC-DLBCL.

Materials and Methods

Materials

The following monoclonal and polyclonal antibodies were used: IκB-α, Bcl-xl, Bcl-2, Bax, Mcl-1, survivin, x-chromosome linked inhibitor-of-apoptosis (x-IAP), caspase-3, poly ADP-ribose polymerase (PARP), and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA); active caspase-3 (BD Biosciences, San Jose, CA); and phospho-IκB-α (Ser32/36; Cell Signaling, Beverly, MA). The biotin-conjugated anti-human transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), allophycocyanin-conjugated streptavidin, and phycoerythrin-conjugated anti-human BAFF-R antibodies were purchased from eBioscience (San Diego, CA). The fluorescein isothiocyanate (FITC)-conjugated anti-human B-cell maturation antibody (BCMA) antibody was purchased from Alexis Biochemicals (San Diego, CA). Cell proliferation kit II (XTT) and in situ cell death detection kit were purchased from Roche (Mannheim, Germany). The rGel/BLyS molecule was expressed in Escherichia coli and purified to homogeneity as previously described [17].

Cell Culture

The eight DLBCL cell lines (SUDHL-4, SUDHL-6, SUDHL-7, OCI-Ly1, OCI-Ly3, OCI-Ly4, OCI-Ly10, and OCI-Ly19) [1,13,19] were used for this study. Five GC-DLBCL lines (SUDHL-4, SUDHL-6, OCI-Ly1, OCI-Ly4, and OCI-Ly19) and one unclassified cell line (SUDHL-7) were grown in RPMI 1640 medium (ATCC, Manassas, VA) supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Two ABC-DLBCL lines (OCI-Ly3 and OCI-Ly10) were grown in 20%fetal bovine serum.

Cytotoxic Activity of rGel/BLyS against Diffuse Large B-cell Lymphoma Cells

To examine the comparative half maximal inhibitory concentration (IC50) values of rGel/BLyS against DLBCL cell lines, SUDHL-4, SUDHL-6, SUDHL-7, OCI-Ly1, OCI-Ly3, OCI-Ly4, OCI-Ly10, or OCI-Ly19 cells were seeded (1 x 104 cells per well) in flat-bottom 96-well microtiter plates (Becton Dickinson Labware, Franklin Lakes, NJ), and various concentrations of rGel/BLyS or rGel were added in quadruplicate wells. After 4 days, cell viability was assessed using the XTTassay as described previously [17]. Absorbance was measured at 450 nm using an ELISA reader (Bio-Tek Instruments, Winooski, VT).

For competitive inhibition assays, OCI-Ly3, OCI-Ly10, SUDHL-4, or SUDHL-6 cells were pretreated with 2 nMof Blys, 50 Nmof Blys, 10 µg/ml of BAFF-R:Fc, 10 µg/ml of TACI:Fc, or 10 µg/ml of BCMA:Fc for 2 hours, and then the cells were treated with various concentrations of free Rgel or Rgel/Blys. After 96 hours, cell viability was assessed using the XTT assay.

Cell Surface Expression of BAFF-R, TACI, and BCMA on DLBCL Cell Lines

The expression of BAFF-R, BCMA, and TACI in eight DLBCL cell lines was assessed by flow cytometry. The cells (1 x 106) were washed in phosphate-buffered saline (PBS), resuspended in ice-cold FACS buffer (PBS with 1% BSA and 0.01%s odium azide), and then incubated with the relevant antibodies (BAFF-R, BCMA, or TACI) or isotype control antibodies for 30 minutes at 4°C. The cells were washed and incubated with allophycocyanin-conjugated streptavidin for 30 minutes at 4°C, washed, and analyzed using FACS Aria instrument (BD Biosciences).

Electrophoretic Mobility Shift Assay

NF-κB-DNA binding were analyzed by electrophoretic mobility shift assay (EMSA) using a 32P-labeled probe as previously described [20]. Briefly, nuclear proteins were extracted from 1 x 106 cells after different treatments. EMSA was performed by incubating 4 µg of nuclear extract for 15 minutes at 37°C with 16 fmol of 32P-end-labeled 45-mer double-stranded oligonucleotide (15 µg of protein) containing the NF-κB binding site (5′-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3′) from the human immunodeficiency virus long terminal repeat. The DNA-protein complex formed was separated from free oligonucleotide on 6.6% native polyacrylamide gels, and the gel was then dried. The dried gels were visualized, and NF-κB DNA binding activity was quantitated by a Phosphor Imager Storm 820 (Amersham Biosciences, Piscataway, NJ) using Imagequant software.

Western Blot Analysis

To examine the effects of rGel/BLyS on the IκB-α, p-IκB-α, Bcl-xL, Bcl-2, Bax, Mcl-1, survivin, x-IAP, caspase-3, and PARP, OCI-Ly3, or OCI-Ly10 cells were seeded at 1 x 106 cells per 12-well plate and then treated with different concentration of rGel/BLyS for 24 hours. For competitive inhibition assay, OCI-Ly3 cells or OCI-Ly10 cells were seeded at 1 x 106 cells per 12-well plate and then pretreated with 1 µM of BLyS for 2 hours, and then the cells were treated with 1 nM of rGel/BLyS or medium for 24 hours. Whole-cell extracts or cytoplasmic extracts were prepared after wash with PBS and lysed on ice for 20 minutes in 0.3 ml of lysis buffer (10 mM Tris-HCl, pH 8, 60 mM KCl, 1 mM EDTA, 1 mM DTT, 0.2% NP-40). Cell lysates (50 µg) were separated by SDS-PAGE (8%–15%) and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Corporation, Bedford, MA) overnight at 4°C in transfer buffer (25 mM Tris-HCl pH 8.3, 190 mM glycine, 20% methanol). The PVDF membranes were blocked for 1 hour in Tris-buffered saline containing 5% nonfat milk and then probed with different primary antibodies for 1 hour at room temperature (RT). After three washes, the membranes were incubated with horseradish peroxidase-conjugated goat antimouse/antirabbit or bovine antigoat antibodies (Bio-Rad Laboratories, Hercules, CA) for 1 hour at RT. Detection of immunoreactive proteins was performed with ECL detection reagent (Amersham Pharmacia Biotech Inc., Piscataway, NJ). β-actin was used as a control for protein loading.

In Vivo Therapy in an ABC-DLBCL Xenograft Model

Animal procedures were performed according to a protocol by the University of Texas MD Anderson Cancer Center. Male severe combined immunodeficient (SCID) mice (CB17.SCID) were supplied by Taconic Farms at 3 to 4 weeks of age and were divided into groups (five mice per group). For in vivo efficacy, we used a DLBCL xenograft model. Four-week-old male SCID mice were injected with 1 x 107 OCI-Ly10 cells subcutaneously. After the tumors had become established (≈250 mm3; 7 weeks after tumor inoculation), the mice were treated with either PBS or rGel/BLyS twice per week for 2 weeks. The total dose of rGel/BLyS was 20 mg/kg. Tumor volume was calculated according to the formula: volume = L x W x H, where L = length, W = width, H = height.

Statistical Analysis

All statistical analyses were done with Microsoft Excel software (Microsoft, Redmond, WA). Data are presented as mean ± SD. P values were obtained using 2-tailed t test with 95% confidence interval for evaluation of the statistical significance compared with the controls. P < .05 was considered statistically significant.

Immunofluorescence Staining

A histologic study was performed in which groups of five mice bearing OCI-Ly10 tumors were given intraperitoneal injections of either PBS or rGel/BLyS (20 mg/kg). Twenty-four hours later, the mice were killed, and tumors were removed. Frozen sections were prepared.

To examine the presence of rGel/BLyS, OCI-Ly10 tumor frozen sections were dried and then fixed in 3.7%formaldehyde (Sigma, St. Louis, MO) for 20 minutes at RT and followed by a brief rinse with PBS. Cells were then permeabilized for 10 minutes in PBS containing 0.2% Triton X-100, washed three times with PBS, and blocked with PBS containing 3% bovine serum albumin for 1 hour at RT. Fixed cells were incubated with anti-rabbit rGel antibody for 2 hours at RT. The slides were washed three times with PBS and then incubated with anti-rabbit IgG-FITC-conjugated antibody for 1 hour at RT. After a final wash step, the slides were mounted in mounting medium and analyzed under fluorescence microscope.

Detection of Apoptosis

Apoptosis was detected by the TdT-mediated dUTP nick end labeling (TUNEL) assay. To assess apoptosis, OCI-Ly3 or OCI-Ly10 cells were seeded at 1 x 106 cells per 12-well plate, and the cells were then treated with 1 nM of rGel/BLyS or medium for 24 hours. For competitive inhibition assay, OCI-Ly10 cells were pretreated with 1 µM of BLyS for 2 hours, and then the cells were treated with 1 nM of rGel/BLyS or medium for 24 hours. Floating cells were then collected and affixed to slides using cytospin (Shandon, Pittsburgh, PA). The slides or OCI-Ly10 tumor frozen sections were dried and then fixed in 3.7% formaldehyde (Sigma) for 20 minutes at RT and followed by a brief rinse with PBS. The slides were then permeabilized for 10 minutes in PBS containing 0.2% Triton X-100 and 0.1% sodium citrate, washed three times with PBS, and blocked with PBS containing 3% bovine serum albumin for 1 hour at RT. Fixed cells were stained with In Situ Cell Death Detection Kit (Roche). After a final wash step, the slides were mounted in mounting medium and analyzed under fluorescence microscope. Apoptotic cells were counted in randomly selected fields (x 200) and expressed as a percentage.

Results

rGel/BLyS Is Selectively Toxic for DLBCL Cells Expressing the BLyS Receptors BAFF-R, BCMA, and TACI

To assess the efficacy of the fusion toxin rGel/BLyS in DLBCL, the cytotoxic effect of rGel/BLyS fusion toxin or free rGel and the expression of BLyS receptors were analyzed in eight DLBCL cell lines (Table 1). The ratio of IC50 values of rGel to rGel/BLyS (targeting index) was calculated for each cell line. This ratio represents the ability of the BLyS component of the rGel/BLyS to mediate delivery of the rGel toxin component to the target cell cytoplasm and normalizes for the inherent cellular sensitivity to the rGel toxin. Of all the tumor lines tested, two ABC-DLBCL lines (OCI-Ly3 and OCI-Ly10) were found to be the most sensitive to the rGel/BLyS fusion toxin (targeting index > 14,000) and expressed high levels of BAFF-R, BCMA, and TACI. Among five GC-DLBCL lines (SUDHL-4, SUDHL-6, OCI-Ly1, OCI-Ly4, and OCI-Ly19), two GC-DLBCL cell lines (SUDHL-4 and SUDHL-6) showed intermediate sensitivity to this fusion toxin (targeting index > 130) and expressed comparable amount of BAFF-R. However, three GC-DLBCL cell lines (OCI-Ly1, OCI-Ly4, and OCI-Ly19) showed less sensitivity to this fusion toxin. OCI-Ly1 cell line expressed relatively low levels of BAFF-R (targeting index = 11), whereas OCI-Ly4 and OCI-Ly19 did not express BAFF-R and TACI (targeting index = 1). The SUDHL-7 DLBCL cell line that has not been classified under either subgroup showed relatively lower sensitivity to this fusion toxin (targeting index = 0.7) and did not express either BAFF-R or TACI. Our results showed that rGel/BLyS was specifically cytotoxic to the ABC-DLBCL cells that express high levels of all three BLyS receptors (BAFF-R, BCMA, and TACI) but not GC-DLBCL cells that did not express BAFF-R and TACI.

Table 1.

Cell Surface Expression of BAFF-R, BCMA, TACI, and Comparative IC50 Values of the rGel/BLyS Fusion Toxin against Diffuse Large B-cell Lymphoma Cell Lines.

Cell Line Subgroup BAFF-R BCMA TACI IC50 (nM) Targeting Index*
rGel rGel/BLyS
OCI-Ly10 ABC ++ ++ +++ 35 0.0007 50,000
OCI-Ly3 ABC ++ +++ ++ 12 0.0008 15,000
SUDHL-4 GC ++ ++ − 350 0.1 3500
SUDHL 6 GC ++ + + 700 5 140
OCI-Ly1 GC + +++ + 700 65 11
OCI-Ly4 GC − +++ − 1900 1400 1
OCI-Ly19 GC − + − 15 11 1
SUDHL-7 U − ++ − 90 120 0.7
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The cell surface expression levels of BAFF-R, BCMA, and TACI were normalized to isotype control antibodies.

−, no expression; +, low expression; ++, intermediate expression; +++, high expression. ABC indicates activated B cell; GC, germinal center; U, unclassified.

*

Targeting index represents IC50 of rGel/IC50 of rGel/BLyS.

The Cytotoxic Effect of rGel/BLyS Is Mediated through Direct Binding to BLyS Receptors BAFF-R, BCMA, and TACI

The biological effects of BLyS are mediated by three cell surface receptors designated BAFF-R, TACI, and BCMA [21–23]. Therefore, we examined whether pretreatment with BLyS might partially block the rGel/BLyS-mediated cytotoxicity in four DLBCL lines (OCI-Ly3, OCI-Ly10, SUDHL-4, and SUDHL-6). We found that pretreatment of BLyS showed a shift in the dose-response curve in rGel/BLyS-treated DLBCL lines but not with treatment of rGel-treated cells (Figure 1A).

Figure 1.

Figure 1

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Competitive inhibition of rGel/BLyS binding to several DLBCL lines. For competitive inhibition assay, OCI-Ly3, OCI-Ly10, SUDHL-4, or SUDHL-6 cells were seeded (1 x 104 cells per well) in flat-bottom 96-well microtiter plates and pretreated with 2 nM of BLyS, 50 nM of BLyS (A), 10 µg/ml of BAFF-R:Fc, 10 µg/ml of TACI:Fc, or 10 µg/ml of BCMA:Fc (B) for 2 hours, and then rGel or rGel/BLyS was added in quadruplicate wells. After 96 hours, cell viability was assessed using the XTT assay. Absorbance was measured at 450 nm using an ELISA reader.

The constructs BAFF-R:Fc, TACI:Fc, or BCMA:Fc decoy receptors can bind to BLyS, prevent the binding of BLyS to their receptors, and inhibit BLyS-mediated B-cell activation. Therefore, we next examined whether pretreatment with various decoy receptors might partially block the rGel/BLyS-mediated cytotoxicity in two ABC-DLBCL lines (OCI-Ly3 and OCI-Ly10). We found that pretreatment of BAFF-R: Fc, TACI:Fc, or BCMA:Fc decoy receptors completely blocked the rGel/BLyS-mediated cytotoxicity in ABC-DLBCL lines (Figure 1B). These data demonstrate that the cytotoxic effects of rGel/BLyS seem to be mediated through direct binding of fusion toxin to cellular BLyS receptors BAFF-R, TACI, and BCMA.

rGel/BLyS Inhibits Constitutive NF-κB Activity in ABC-DLBCL Cells

NF-κB activity is critical for normal B-cell development and survival. In addition, BLyS signals have been shown to modulate the activity of this transcription factor complex. Therefore, we sought to determine the NF-κB binding activity in DLBCL cell lines and whether there was a correlation with rGel/BLyS-dependent cytotoxicity. As shown in Figure 2A, NF-κB was found to be constitutively activated in two ABC-DLBCL cell lines (OCI-Ly3 and OCI-Ly10) that are very sensitive to rGel/BLyS. However, five GC-DLBCL cell lines (OCI-Ly1, OCI-Ly4,OCI-Ly19, SUDHL-4, and SUDHL-6) and one unclassified DLBCL cell line (SUDHL-7) that are less sensitive to this fusion toxin showed low levels of NF-κB binding activity. Therefore, basal levels of NF-κB did not predict sensitivity to rGel/BLyS.

Figure 2.

Figure 2

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Effect of rGel/BLyS on NF-κB binding activity. (A) Constitutive NF-κB activation in DLBCL cell lines. Nuclear extracts from eight diffuse large B-cell lymphoma lines (OCI-Ly1, OCI-Ly3, OCI-Ly4, OCI-Ly10, OCI-Ly19, SUDHL-4, SUDHL-6, and SUDHL-7) were prepared and NF-κB binding activity was assessed. (B) Inhibition of NF-κB binding activity by rGel/BLyS. ABC-DLBCL cell line (OCI-Ly10) with the highest NF-κB binding activity was seeded (1 x 106 cells per well) in 12-well plates and incubated with different concentrations of rGel/BLyS for 24 hours. Nuclear extracts were prepared and subjected to EMSA to assess NF-κB binding activity.

To evaluate whether rGel/BLyS showed an inhibitory effect on constitutive NF-κB activation in DLBCL cells, we performed EMSA using nuclear extracts from rGel/BLyS-treated OCI-Ly10 cells, which demonstrated the highest NF-κB-DNA binding activity and which are very sensitive to rGel/BLyS. Data clearly showed that this fusion toxin suppressed constitutive NF-κB activation in a dose-dependent manner (Figure 2B).

rGel/BLyS Inhibits IκB-α Phosphorylation and IκB-α Degradation in ABC-DLBCL Cells

The degradation of IκB-α is required for NF-κB activation [7] and the proteolytic degradation of IκB-α is known to first require phosphorylation at serine residues 32 and 36. We next determined whether inhibition of NF-κB activation by rGel/BLyS was due to inhibition of IκB-α phosphorylation or IκB-α degradation. We found that rGel/BLyS treatment results in an inhibition in the levels of phospho IκB-α. As assessed by Western blot analysis, this led to a marked accumulation of IκB-α in the OCI-Ly10 cell line but only a very modest change in the IκB-α content of the OCI-Ly3 line (Figure 3).

Figure 3.

Figure 3

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Effects of rGel/BLyS on IκB-α phosphorylation. OCI-Ly3 or OCI-Ly10 cells were seeded (1 x 106 cells per well) in 12-well plates and incubated with different concentrations of rGel/BLyS for 24 hours. After treatment, the cells were collected, washed, and lysed in 0.2 ml of lysis buffer. Cytoplasmic extracts (50 µg) were fractionated by 8% to 15% SDS-PAGE and electrophoretically transferred to PVDF membranes. The membranes were blocked, and then probed with anti-IκB-α antibody, anti-phospho IκB-α (Ser32/36) antibody, or anti-β-actin antibody. Secondary antibodies conjugated with horseradish peroxidase were used to visualize immunoreactive proteins using ECL detection reagent. β-actin was used as a control for protein loading.

rGel/BLyS Suppresses NF-κB-Regulated Proteins in ABC-DLBCL Cells

NF-κB regulates the expression of several antiapoptotic proteins including IAP1, IAP2, Bcl-2, Bcl-xl, x-IAP, c-FLICE-like inhibitory protein, and survivin.Hence,we examined whether rGel/BLyS can modulate NF-κB target gene products by Western blot analysis in ABC-DLBCL cells. As shown in Figure 4, rGel/BLyS specifically inhibited the expression of Bcl-xl, Mcl-1, survivin, and x-IAP but not Bcl-2.We also found that rGel/BLyS induced the expression of Bax. Pretreatment of these ABC-DLBCL lines with BLyS for 2 hours partially blocked rGel/BLyS-mediated down-regulation of these NF-κB targets and up-regulation of Bax.

Figure 4.

Figure 4

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Effects of rGel/BLyS on NF-κB-regulated proteins. OCI-Ly3 or OCI-Ly10 cells were seeded (1 x 106 cells per well) in 12-well plates and pretreated with 1 µM of BLyS for 2 hours and then treated with 1 nM rGel/BLyS or medium for 24 hours. After treatment, the cells were collected, washed, and lysed in 0.2 ml of lysis buffer. Whole-cell extracts (50 µg) were fractionated by 8% to 15% SDS-PAGE and electrophoretically transferred to PVDF membranes. The membranes were blocked and then probed with various antibodies. Secondary antibodies conjugated with horseradish peroxidase were used to visualize immunoreactive proteins using ECL detection reagent. β-actin was used as a control for protein loading.

Treatment with rGel/BLyS Induces Apoptosis in ABC-DLBCL Cells

To determine whether the cytotoxic effect of rGel/BLyS was associated with apoptosis, two ABC-DLBCL lines (OCI-Ly3 and OCI-Ly10) were examined for apoptosis by TUNEL staining. As shown in Figure 5A, rGel/BLyS-treated OCI-Ly3 and OCI-Ly10 cells showed 34% and 37% of apoptotic cells, respectively. We also examined whether pretreatment with BLyS can block rGel/BLyS-induced apoptosis in OCI-Ly10 cells. In concert with the data in Figure 4, pretreatment with BLyS completely blocked the proapoptotic effect of rGel/BLyS fusion toxin (Figure 5B).

Figure 5.

Figure 5

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Effects of rGel/BLyS on apoptotic pathways. (A) Microscopic appearance of OCI-Ly3 or OCI-Ly10 cells after treatment. OCI-Ly3 or OCI-Ly10 cells were treated with 1 nM rGel/BLyS or media. After 24 hours, the cells were assayed for apoptosis by TUNEL staining. (B) Competitive inhibition of rGel/BLyS binding by pretreatment with BLyS. OCI-Ly10 cells were seeded (1 x 106 cells per well) in 12-well plates and pretreated with 1 µM of BLyS for 2 hours and then treated with 1 nM rGel/BLyS or medium for 24 hours. After 24 hours, the cells were assayed for apoptosis by TUNEL staining. Apoptotic cells were counted in randomly selected fields (x 200) and expressed as a percentage of total cells counted. (C) Effects of rGel/BLyS on apoptotic pathways. OCI-Ly3 or OCI-Ly10 cells were seeded at 1 x 106 cells per 12-well plate and treated with different concentrations of rGel/BLyS for 24 hours or pretreated with 1 µM of BLyS for 2 hours and then treated with 1 nM rGel/BLyS or medium for 24 hours. After treatment, the cells were collected, washed, and lysed in 0.2 ml of lysis buffer. Whole-cell extracts (50 µg) were fractionated by 8% to 15% SDS-PAGE and electrophoretically transferred to PVDF membranes. The membranes were blocked and then probed with various antibodies. Secondary antibodies conjugated with horseradish peroxidase were used to visualize immunoreactive proteins using ECL detection reagent. β-Actin was used as a control for protein loading.

The caspase proteins are known to be central mediators of the apoptotic effects of TNF and other cytokines. To determine whether caspase-3 was activated in ABC-DLBCL cells during rGel/BLyS-induced cell death, we examined the cleavage of caspase-3 and its substrate PARP. Exposure of cells to rGel/BLyS resulted in cleavage of caspase-3 and also induced PARP cleavage in ABC-DLBCL cell lines (OCI-Ly3 and OCI-Ly10), suggesting that the cytotoxic effects of this fusion toxin seemed to be mediated, at least in part, by caspase-3 and PARP cleavage. Pretreatment with BLyS partially blocked rGel/BLyS-induced cleavage of caspase-3 and PARP (Figure 5C).

rGel/BLyS Inhibits Tumor Growth in an ABC-DLBCL Xenograft Model

To investigate the potential in vivo efficacy of rGel/BLyS, we used a DLBCL xenograft model. SCID mice were subcutaneously inoculated with OCI-Ly10 cells and tumors were allowed to establish for 7 weeks before treatment (≈250 mm3). Tumor volume in the PBS-treated group increased from 258 to 1305 mm3 during the 13-day observation period, whereas tumor volume in the rGel/BLyS-treated group (20 mg/kg, intraperitoneally, twice weekly for 2 weeks) showed no significant increase on tumor growth during the course of the experiment (from 260 to 226 mm3). Compared with controls, treatment with rGel/BLyS was shown to result in an 83% reduction in tumor volume (1305 vs 226 mm3). The rGel/BLyS fusion toxin significantly inhibited tumor growth in SCID mice bearing ABC-DLBCL xenografts (P < .05; Figure 6).

Figure 6.

Figure 6

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In vivo efficacy of rGel/BLyS in a DLBCL xenograft model. For the ABC-DLBCL xenograft model, 4-week-old male SCID mice were injected with 1 x 107 OCI-Ly10 cells subcutaneously. After the tumors had become established (≈250 mm3; 7 weeks after tumor inoculation), the mice were treated with either PBS or rGel/BLyS twice per week for 2 weeks. The total dose of rGel/BLyS was 20 mg/kg. Points, mean tumor volume; bars, SD. X axis, arrows, treatment. *P < .05, Student's t test.

Internalized rGel/BLyS Induces Apoptosis in ABC-DLBCL Xenografts

We examined whether the rGel/BLyS fusion toxin can internalize into OCI-Ly10 tumors in vivo. As shown in Figure 7A, the rGel component of rGel/BLyS fusion toxin was shown to accumulate in OCI-Ly10 tumors 24 hours after the last intraperitoneal administration. This demonstrates that rGel/BLyS is capable of efficient localization in tumors. We next examined whether rGel/BLyS can induce apoptosis in OCI-Ly10 tumors. As shown in Figure 7B, there was a significant increase in apoptosis (as assessed by TUNEL staining) in tumors from mice treated with rGel/BLyS.

Figure 7.

Figure 7

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Localization of rGel/BLyS in tumor xenografts. (A) Detection of rGel/BLyS in ABC-DLBCL tumor. To examine the presence of rGel/BLyS, OCI-Ly10 tumor frozen sections were dried and then fixed in 3.7% formaldehyde for 20 minutes at RT and followed by a brief rinse with PBS. Tumor sections were then permeabilized, washed, blocked, and incubated with anti-rabbit rGel antibody for 2 hours at RT. The slides were washed three times with PBS and then incubated with anti-rabbit IgG-FITC-conjugated antibody for 1 hour at RT. After a final wash step, the slides were mounted in mounting medium and analyzed under fluorescence microscope. (B) Detection of apoptosis in ABC-DLBCL tumor. Apoptosis was detected by the TUNEL assay.

Discussion

The growth factor BLyS is crucial for B-cell survival, and the biological effects of BLyS are mediated by three cell surface receptors designated BAFF-R, TACI, and BCMA [21–23]. The BLyS ligand is expressed on multiple types of malignant B cells [24–26]. The BAFF-receptor (BAFF-R) is the most abundantly expressed in approximately 80% of mantle cell lymphoma and in approximately 44% of DLBCL [27]. Therefore, it has been suggested that targeting BLyS and its receptors may disrupt this important autocrine growth loop and may have value in designing therapeutic approaches for B-cell malignancies.

In this study, we evaluated the cytotoxic activity of rGel/BLyS against DLBCL lines. Of the eight DLBCL lines studied, two ABC-DLBCL lines (OCI-Ly3 and OCI-Ly10) expressing all three BLyS receptors were found to be the most sensitive to the fusion toxin (targeting index = 15,000 and 50,000), whereas nontargeted rGel itself showed no specific cytotoxic activity against DLBCL lines. As shown previously [16–18], this suggests that the rGel component has impressive cytotoxic effects when delivered to cells using growth factor ligands. Our results demonstrate that BLyS has the potential to serve as an excellent targeting ligand for the specific delivery of rGel toxin to DLBCL cells expressing all three BLyS receptors.

Novak et al. [26] reported that DLBCL cells expressed BAFF-R but not BCMA. Paterson et al. [28] reported that SUDHL-4 and SUDHL-6 lines expressed BAFF-R but not OCI-Ly3 and OCI-Ly10 lines. However, our results showed that BAFF-R was expressed in approximately 62.5% of DLBCL lines tested (OCI-Ly3, OCI-Ly10, SUDHL-4, and SUDHL-6), whereas BCMA and TACI were expressed in approximately 100% and 50% of the cell lines tested, respectively. We also observed that OCI-Ly3, OCI-Ly10, SUDHL-4, and SUDHL-6 cell line expressed similar levels of BAFF-R.

The transcription factor NF-κB has been shown to regulate the expression of a number of genes whose products are involved in tumorigenesis [7]. A recent gene profiling study has shown that the genes involved in TNF and NF-κB signaling pathways are overexpressed in ABC-DLBCL [1]. NF-κB is known to be responsible, in part, for mediating neoplastic B-cell growth and cell survival [29,30]. Dominant-negative inhibitors of the NF-κB pathway were shown to induce cell death of ABC-DLBCL cells but not GC-DLBCL cells [13]. The IκB kinase inhibitor PS-1145 was found to be selectively toxic for ABC-DLBCL cell lines but not for GC-DLBCL lines [14]. Transfection of DLBCL cells with siBLyS RNA was found to reduce NF-κB DNA binding activity [31]. These results suggest that inhibition of NF-κB is expected to be of therapeutic value in tumors such as ABC-DLBCL, where NF-κB seems to play a unique survival role.

The rGel toxin is an extremely potent inhibitor of protein synthesis, similar in action to ricin. Ricin has been shown to induce NF-κB activation and TNF production in human airway epithelial cells [32]. In contrast, treatment with the rGel/BLyS fusion toxin resulted in the inhibition of constitutive NF-κB activation in ABC-DLBCL lines, although rGel has similar enzymatic activity to ricin. Therefore, our results indicate that the rGel toxin is an ideal payload to target NHL expressing constitutively active NF-κB.

Accumulation of rGel/BLyS and apoptosis of tumor cells were observed 24 hours after the last intraperitoneal administration of rGel/BLyS to SCID mice bearing OCI-Ly10 cells. Importantly, rGel/BLyS was shown to significantly inhibit tumor growth (P < .05) in ABC-DLBCL (OCI-Ly10) xenograft model. We observed that the maximum tolerated dose of rGel/BLyS for intraperitoneal administration using a twice per week for 12 week schedule was more than 60 mg/kg, whereas the maximum tolerated dose of rGel/BLyS for intravenous administration was 7.5 mg/kg for twice per week for a 2-week schedule in Balb/c mice (data not shown).

Taken together, our results demonstrate that the rGel/BLyS fusion toxin was selectively toxic for ABC-DLBCL cells both in vitro and in vivo and indicate that this fusion toxin is an excellent candidate for the treatment of aggressive NHLs that are both dependent on NF-κB signals and resistant to conventional chemotherapeutic regimens.

Abbreviations

rGel/BLyS

a fusion toxin containing rGel at the N-terminus followed by a G4S peptide tethered to the BLyS molecule

G4S

glycine-glycine-glycine-glycine-serine

ABC-DLBCL

activated B-cell-like diffuse large B-cell lymphoma

GC-DLBCL

germinal center-like diffuse large B-cell lymphoma

NHL

non-Hodgkin lymphoma

NF-κB

nuclear factor-κB

BLyS

B-lymphocyte stimulator

IκB

inhibitory subunit of NF-κB

IAP

inhibitor-of-apoptosis protein

x-IAP

x-chromosome linked inhibitor-of-apoptosis

EMSA

electrophoretic mobility shift assay

PVDF

polyvinylidene difluoride

PARP

poly ADP-ribose polymerase

TUNEL

terminal deoxynucleotidyl transferase-mediated nick end labeling

Footnotes

1

This research was conducted, in part, by the Clayton Foundation for Research (M.G.R.), a Translational Research award (LLS 6234-07) by the Leukemia and Lymphoma Society (V.G. and M.G.R.), and Lymphoma SPORE CA136411, National Cancer Institute, National Institutes of Health, Department of Health and Human Services.

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