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Published OnlineFirst January 19, 2012; DOI: 10.1158/1535-7163.MCT-11-0635

Molecular Cancer Therapeutic Discovery Therapeutics

Galiximab Signals B-NHL Cells and Inhibits the Activities of NF-kB–Induced YY1- and Snail-Resistant Factors: Mechanism of Sensitization to Apoptosis by Chemoimmunotherapeutic Drugs

Melisa A. Martinez-Paniagua1,2, Mario I. Vega1,3, Sara Huerta-Yepez1,4, Stavroula Baritaki1, Gabriel G. Vega3, Kandasamy Hariharan5, and Benjamin Bonavida1

Abstract Galiximab (anti-CD80 monoclonal antibody) is a primatized (human IgG1 constant regions and cynomologus macaque variable regions) monoclonal antibody that is currently in clinical trials. Galiximab inhibits tumor cell proliferation through possibly cell signaling–mediated effects. Thus, we hypothesized that galiximab may signal the tumor cells and modify intracellular survival/antiapoptotic pathways such as the þ NF-kB pathway. This hypothesis was tested using various CD80 Burkitt B-NHL (non–Hodgkin lymphomas) cell lines as models. Treatment of B-NHL cells with galiximab (25–100 mg/mL) resulted in significant inhibition of NF-kB activity and its target resistant factors such as YY1, Snail, and Bcl-2/Bcl-XL. Treatment of B-NHL cells with galiximab sensitized the tumor cells to both cis-diamminedichloroplatinum(II) (CDDP)- and TRAIL- induced apoptosis. The important roles of YY1- and Snail-induced inhibition by galiximab in the sensitization to CCDP and TRAIL were corroborated following transfection of Raji cells with YY1 or Snail short interfering RNA. The transfected cells were shown to become sensitive to both CCDP- and TRAIL-induced apoptosis in the absence of galiximab. Furthermore, knockdown of YY1 or Snail inhibited Bcl-XL. The involvement of Bcl-XL inhibition in sensitization was corroborated by the use of the pan-Bcl-2 inhibitor 2MAM-3 whereby the treated cells were sensitive to both CDDP- and TRAIL-induced apoptosis. These findings show that galiximab inhibits the NF-kB/Snail/YY1/Bcl-XL circuit that regulates drug resistance in B-NHL and in combination with cytotoxic drugs results in apoptosis. The findings also support the therapeutic application of the combination of galiximab and cytotoxic drugs in the treatment of drug-resistant CD80-positive B-cell malignancies. Mol Cancer Ther; 11(3); 572–81. 2012 AACR.

Introduction relapse and resistance to common chemotherapy are Non–Hodgkin lymphomas (NHL) are a heterogeneous usually observed (1–4). Monoclonal antibodies have rev- group of malignancies of the lymphoid system. Current olutionized the treatment of malignances such as NHL. treatments for NHL are not optimally effective as both Rituximab is an anti-CD20 monoclonal antibody that has been approved by the U.S. Food and Drug Admin- þ istration for the treatment of CD20 B-NHL (5, 6). It induces cell lysis through antibody-dependent cell- Authors' Afﬁliations: 1Department of Microbiology, Immunology & Molec- ular Genetics, David Geffen School of Medicine, Jonsson Comprehensive mediated cytotoxicity, complement-dependent cytotox- Cancer Center, University of California, Los Angeles, California; 2Unidad de icity, and apoptosis (7, 8). There has not been an increase Investigacion Medica en Inmunología e Infectología, Hospital de Infecto- in overall survival (5, 6). Approximately 50% of treated logía, CMN "La Raza", IMSS; 3Unidad de Investigacion Medica en Enfer- medades Oncologicas, Hospital de Oncología CMN Siglo XXI IMSS, patients are refractory or develop resistance during the Mexico; 4Unidad de Investigacion en Enfermedades Oncologicas Hospital course of prolonged treatment with rituximab as single Infantil de Mexico S.S.A, Mexico; and 5BiogenIdec, Inc., Research & agent or in combination with cyclophosphamide-Adria- Corporate, San Diego, California mycin-vincristine-prednisone (CHOP; ref. 9). Because Note: M.-A. Martinez-Paniagua and M.I. Vega contributed equally to the resistance to rituximab occurs, there is a need for new study. therapies (10). Corresponding Author: Benjamin Bonavida, Department of Microbiology, CD80 is a membrane-bound immune costimulatory Immunology & Molecular Genetics, David Geffen School of Medicine, Jonsson Comprehensive Cancer Center, University of California, Los molecule involved in the regulation of the activation of Angeles, CA 90095. Phone: 310-825-2233; Fax: 310-206-3865; E-mail: T cells (11). It is a member of the B7 family of costimu- [email protected] latory molecules (12). CD80 isexpressedonthesurface doi: 10.1158/1535-7163.MCT-11-0635 of normal activated B cells, antigen-presenting cells, and 2012 American Association for Cancer Research. T cells (13 and on the surface of a variety of lymphoid

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malignances (14–17). Preclinical studies have showed of resistant factors such as YY1, Snail, and Bcl-XL and that anti-CD80 antibodies can inhibit cell proliferation what is the direct involvement of each of these factors in of lymphoma and induce antibody-dependent cell- sensitization? The findings reported herein support the mediated cytotoxicity (18) in vivo.Conjugatedanti- above hypothesis and establish a novel mechanism of CD80 antibodies with immunotoxins exerted antitumor galiximab-induced cell signaling resulting in the inhibi- þ activity in vitro using CD80 Burkitt lymphoma (Raji) tion of the NF-kB/Snail/YY1/Bcl-XL dysregulated resis- and Hodgkin lymphoma (L428) cell lines (19). Galix- tant circuit that controls the resistance in B-NHL and imab delays progression and prolongs survival in a leading to sensitization of B-NHL cells to drug-induced human lymphoma xenograft/severe combined immu- apoptosis. nodeficient (SCID) mouse model (20). Previous findings showed that treatment with a monoclonal antibody for Materials and Methods CD80 (16-10A1) on B cells resulted in a significant Cell lines and reagents decrease in tumor cell proliferation, upregulation of The human B-NHL cell lines Raji, Ramos, DHL4, and proapoptotic molecules, and downregulation of the Daudi were purchased from the American Type Culture expression of antiapoptotic proteins such as Bcl-XL, Collection. The AIDS-related lymphoma B cell line 2F7 thus, resulting in tumor cell apoptosis (21). was kindly provided by Dr. O. Martinez-Maza, University Galiximab (IDEC-11) is a high-affinity primatized anti- of California, Los Angeles, CA. The cell lines were cul- CD80 (IgG1l) monoclonal antibody that contains variable tured as described previously (27). All cells used in this regions of primate (cynomologus macaque) origin and study were within 15 passages after resuscitation. The constant regions of human origin (22). A phase I/II clinical cells were check routinely by morphology and tested for trial involving patients with relapsed or refractory follic- Mycoplasma contamination with CELLshipper Myco- ular lymphoma resulted in an overall response rate of 11% plasma Detection Kit (Bionique Testing Laboratories). and tumor regression in 49% of the patients (23). Preclin- Galiximab was obtained from BiogenIdec, Inc. Soluble ical data suggest the synergy between rituximab and recombinant human TRAIL was purchased from Pepro- galiximab in patients with relapsed or refractory follicular Tech Inc. The cis-Diamminedichloroplatinum(II) (CDDP) lymphoma (23, 24). was purchased from Sigma and was diluted in dimethyl Galiximab serves as a counter receptor that transduces sulfoxide. The NF-kB inhibitor DHMEQ was provided by distinct signaling to B cells upon engagement. Several Dr. K. Umezawa (Keio University, Yokohama, Japan) and studies have reported the signaling on T cells through the diluted in dimethyl sulfoxide (34). The phycoerythrin CD80 ligand (CD28) via interaction with CD80-expressing (PE)-labeled anti-CD80 antibody and the fluorescein cells (25). However, little is known about the direct sig- isothiocyanate (FITC)-labeled antiactive caspase-3 anti- naling by galiximab of cells expressing CD80. These bodies, as well as the corresponding IgG1 isotype controls results indicated that CD80 can mediate signal transduc- were obtained from BD Pharmingen. The Bcl-2 family tion and regulate B-cell function (21). inhibitor 2MAM-A3 was purchased from BIOMOL. The We and others have reported that rituximab treatment following antibodies were obtained from Santa Cruz affects apoptotic signaling in lymphoma cell lines via Biotechnology: Bcl-XL, Bcl-2, p50, p65, phospho-p65 upstream inhibition of constitutively activated survival Ser-536, IkBa, phospho-IkBa Ser-32, IKK, phosphor- signaling pathways such as the p38MAPK and NF-kB (26, IKK-a/b Ser-176, AKT, phospho-AKT Thr-308, YY1, and 27) and downstream inhibition of Bcl-2 and Bcl-XL expres- Snail. sion and in the reversal of resistance (28). In addition, treatment of B-NHL cell lines with rituximab resulted in potentiation of apoptosis induced by Fas-L and TRAIL, Viability assay respectively, through the inhibition of the transcriptional Cell viability was assessed by either the trypan blue repressor YY1 (29, 30). Furthermore, NF-kB activates dye exclusion assay by microscopy or by the 2,3—bis(2- the transcription of resistant gene products such as Snail methoxy-4-nitro-S-sulfophenynl)H-tetrazolium-5 car- (31–33). boxanilide inner salt (XTT) dye absorbance according Galiximab interferes with the apoptotic pathways by to the manufacturer’s instruction (Roche Diagnostic targeting gene products regulated by NF-kB. Therefore, GmbH) and as previously described (35). The viability we hypothesized that galiximab may inhibit the consti- of the untreated cells was set at 100%. Furthermore, total tutively activated NF-kB pathway and may also sensitize cell recovery was also recorded. Each experimental resistant B-NHL cell lines to apoptosis by cytotoxic drugs. condition was conducted in triplicate and the SD was The following were examined to test this hypothesis: (i) calculated. þ Does treatment of CD80 Burkitt B-NHL cell lines inhibit the constitutively activated NF-kB and AKT pathways; (ii) Western blot analysis for protein expression Does treatment of B-NHL cell lines with galiximab sen- One million of Raji cells per milliliter were incubated sitize the cells to apoptosis by chemotherapeutic and with or without the indicated concentrations of galiximab immunotherapeutic drugs?; and (iii) Does galiximab- at 37C for 18 hours. Western blot analysis was conducted mediated sensitization to apoptosis result from inhibition as previously described (35).

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Electrophoretic mobility shift assay to high (Raji, Daudi, 2F7) as shown in the histogram in Raji cells (106) were incubated with or without the Fig. 1A. The mean fluorescence intensity (MFI) for all indicated concentrations of galiximab at 37C for 18 hours. the cell lines is shown in a table format depicted in Fig. 1A. Ten microliters of nuclear proteins was mixed with the The resistant B-NHL cell lines were tested for galixi- biotin probe for analysis of the transcription factors NF- mab-mediated sensitization to apoptosis by measuring kB, Snail, and YY1, using the EMSA kits from Panomics, the activation of caspase-3 by flow cytometry following following the manufacturer’s instructions and as previ- the addition of suboptimal concentrations of CDDP and ously described (35). TRAIL. As shown in Fig. 1B (left), single-cell treatment with galiximab (20 mg/mL) had no significant apoptotic Surface expression of CD80 effect on the cell lines; however, with the combination of Cells were stained with the PE-conjugated specific anti- galiximab and drug, there was significant sensitization to CD80 for 1 hour at 4C according to the manufacturer’s both CDDP and TRAIL apoptosis in all of the B-NHL cell instruction. Analysis was conducted using Flow Epics XL- lines tested. The single-cell treatment with CDDP or MCL (Coulter) equipment. The mean fluorescence inten- TRAIL had no significant apoptosis. The level of apoptosis sity was recorded using the System II Software. achieved was different for each cell line tested. 2F7, Raji, and Daudi cells were significantly sensitized by both Apoptosis determination CDDP and TRAIL. Ramos, however, was not sensitized Apoptosis was assessed in tumor cells as previously to CDDP, although it was sensitized to TRAIL. The DHL4 described for activated caspase-3 by flow cytometry (35). cell line was not sensitized to CDDP in combination with In addition, we also used the Annexin-V method to galiximab treatment but was sensitized to TRAIL (data no corroborate the caspase-3 method. shown). The apoptotic effect assessed by the activation of caspase-3 was corroborated by analysis of apoptosis using Transfection with short interfering RNA Annexin-V as shown in Fig. 1B (right). Transfection was conducted using the Lipofectamine We have chosen Raji cells as a model for further analysis transfection reagent (Invitrogen Life Technology). Scram- of the underlying mechanism of galiximab-mediated sen- bled RNA, Snail, and YY1 short interfering RNAs (siRNA) sitization. Treatment of Raji cells with various concentra- were obtained from Santa Cruz Biotechnology. Raji cells tions of galiximab resulted in a concentration-dependent were cultured at a density of 2.5 105/mL in RPMI-1640 inhibition of cell survival. Concentrations of galiximab devoid of antibiotics for 24 hours. Cells were then trans- 25 mg/mL resulted in a decrease of viability and a fected with 50 nmol/L siRNA in a final volume of 100 mL plateau (75%–80% viability) was reached at concentra- of medium in the presence of 10 mL of Lipofectamine 2000 tions 25 mg/mL. In addition, there was inhibition of cell in Opti-MEM. To determine YY1 or Snail siRNA-induced proliferation by galiximab concentrations of 10 mg/mL sensitization to CDDP- or to TRAIL-induced apoptosis, 48 as determined by total cell recovery (Fig. 1C). The effects hours following treatments, untransfected cells and cells of various galiximab concentrations as well as different transfected with scrambled siRNA, YY1 siRNA, or Snail concentrations of CDDP and TRAIL were examined for were treated with CDDP or TRAIL for 24 hours and sensitization to apoptosis. Raji cells were pretreated with apoptosis was measured by FITC-labeled antiactive cas- various concentrations of galiximab (10–100 mg/mL) for pase-3 antibody using flow cytometry. 18 hours and then treated for an additional 24 hours with CDDP (5–20 mg/mL) or TRAIL and apoptosis was deter- Isobologram analysis for synergy determination mined. The findings show that there was significant The isobologram analysis was used to evaluate the effect sensitization by the combination treatment and the level of the galiximab/CDDP combination as described (36). of apoptosis was a function of the concentrations used for both galiximab and CDDP (Fig. 1D, left). The combination Statistical analysis treatment was synergistic as determined by isobologram All results were expressed as the mean SD of data analysis. Likewise, the combination of galiximab and obtained from 3 triplicate independent and separate TRAIL resulted in apoptosis and the level of apoptosis experiments. The statistical significance of differences was a function of the increased concentration of TRAIL between group means was determined using one-way (Fig. 1D, right). ANOVA to compare variance. Significant differences were considered for probabilities <5% (P < 0.05). Mechanism by which galiximab sensitizes Raji cells to apoptosis by CDDP and TRAIL Results Inhibition of NF-kB activity. We examined whether Galiximab-mediated inhibition of cell proliferation galiximab, due to its antiproliferative activity, inhibited the and sensitization of B-NHL cells to apoptosis by constitutively activated NF-kB pathway in Raji cells. Raji CDDP and TRAIL cells were treated with several concentrations of galiximab Several B-NHL cell lines were first examined for surface (25, 50, and 100 mg/mL) for 18 hours, and cell lysates CD80 expression by flow cytometry. There was differen- were prepared for analysis for the expression of several tial CD80 expression ranging from low (Ramos, DHL4) gene products of the NF-kB pathway. Western blot and

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A C

120 Cell line MFI Viability Cells recovery 6 12 24 6 12 24 Ramos 4.7 Time (h) Time (h) % cells recovery DHL4 5.3 150 100 80 Ramos Daudi Raji 141 100 DHL4 Raji 2F7 60 Events 40 Daudi 205 50 20 % viability 2F7 505 0 0 0 5 10 25 50 100 0 100 101 102 103 104 Galiximab (μg/mL) FL2-Height

B 50 Medium ** Galiximab (20 μg/mL) 45 ** ** Galiximab + CDDP (5 μg/mL) Galiximab + TRAIL (5 ng/mL) 40 60 Medium (20 μg/mL) * μ 35 * ** Galiximab (5 g/mL) * * 50 ** Galiximab + CDDP 30 Galiximab + TRAIL (5 ng/mL) 25 40 20 30

caspase-3) 15 * 10 20 * 5

Apoptosis (% of active Apoptosis (% of active 10

0 V/7-AAD-positivity) 2F7 Raji Ramos Daudi 0 Apoptosis (% of Annexin- Raji Ramos D 1 Control 40 60 Galiximab 10 *** Control ** 0.8 Galiximab 25 35 (μg/mL) Galiximab (20 μg/mL) ** 50 Galiximab 50 30 0.6 Galiximab 100 ** 0.4 40 * 25 * * 20 0.2 30 * * FIC Raji Galiximab * 15

0 20 caspase-3)

0 0.2 0.4 0.6 0.8 1 caspase-3) 10 FIC Raji CDDP 10 5 Apoptosis (% of active Apoptosis (% of active Apoptosis (% of active Apoptosis (% of active 0 0 0 51020 0 2.5 5 10 TRAIL (ng/mL) CDDP (μg/mL)

Figure 1. Galiximab inhibits cell proliferation of B-NHL cell lines and sensitizes B-NHL cells to apoptosis by CDDP and TRAIL. A, surface expression of CD80 on B-NHL cell lines. The surface expression of CD80 was analyzed by flow cytometry. A, representative histogram is shown for the various B-NHL cell lines and an isotype control is shown. In addition, the mean fluorescence intensity (MFI) is represented in the table. B, sensitization to apoptosis. B-NHL cell lines were treated with galiximab (20 mg/mL) for 18 hours and followed by treatment with CDDP (5 mg/mL) or TRAIL (5 ng/mL) for an additional 18 hours and apoptosis was determined by activation of caspase-3 (left) as described. , P < 0.05; , P < 0.01. Apoptosis was also determined by Annexin-V as described (right). C, galiximab-induced inhibition of Raji cell viability and cell recovery. The B-NHL cell line Raji was treated with various concentrations of galiximab and incubated for different time periods (6–24 hours), and cell viability was determined by trypan blue dye exclusion and total cell recovery was recorded. Raji cells that were not treated with galiximab represented 100% viability. The data represent the mean SD from 3 independent experiments , P < 0.05. D, galiximab sensitizes resistant B-NHL cell lines to apoptosis by CDDP and TRAIL. Sensitization of B-NHL Raji cells by galiximab to apoptosis by CDDP or TRAIL is synergistic. Raji cells were treated with various concentrations of galiximab (10–100 mg/mL) for 18 hours and then treated with either CDDP (5, 10, and 20 mg/mL; left) or only one concentration of galiximab (20 mg/mL) and various concentrations of TRAIL (2.5, 5, and 10 ng/mL; right) for an additional 18 hours and apoptosis was determined. The data represent the mean SD from 3 independent experiments. , P < 0.05; , P < 0.01. In addition, the data were analyzed for synergy by isobologram analysis as described in Materials and Methods. The isobologram is represented left of D. AAD, aminoactinomycin D. densitometric analyses revealed that following galiximab resistance, the tumor cells were treated with the specific treatment, there was some inhibition of p50 but there were NF-kB inhibitor, DHMEQ, and tested for sensitivity to significant inhibition of p65, phospho-p65, and both phos- apoptosis induced by CDDP and TRAIL. Treatment with pho- and total IkBa expression levels in a concentration- DHMEQ sensitized the tumor cells to apoptosis by CDDP dependent manner (Fig. 2A, left) and quantified by densi- and TRAIL and, thus, mimicking the treatment with galix- tometric analysis (Fig. 2A, right). The inhibition of NF-kB imab (Fig. 2C). The effect of galiximab treatment on the DNA-binding activity by galiximab and by the specific NF- AKT pathway was examined by Western blot analysis. kB inhibitor DHMEQ was corroborated by electrophoretic Galiximab inhibited phospho-AKT and phospho-IKKa/b mobility shift assay (EMSA; Fig. 2B). To show the involve- but not the unphosphorylated forms as shown by Western ment of NF-kB inhibition by galiximab in the reversal of blot analysis and densitometry (Fig. 2D).

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A Galiximab (μg/mL) 0 25 50 100 p50 0 25 50 100 2.50 Galiximab (μg/mL) p65 2.00 1.50 phospho-p65 1.00

IκB-α Arbitrary units 0.50 0.00 κ α κ α phospho-IκB-α p50 p65 phospho-p65 I B- phospho-I B- C Actin 60 Control μ * B 50 Galiximab (20 g/mL) DHMEQ (10 μg/mL) * * 40 *

Galiximab 30 No nuclear extracts No nuclear –+ DHMEQ Cold probe Anti-p65 20 Super shift 10 NF-κB Apoptosis (% of active caspase-3) Apoptosis (% of active 0 Control CDDP TRAIL (5 μg/mL) (5 μg/mL)

0 25 50 100 Free probe μ 2.00 Galiximab ( g/mL) μ D Galiximab ( g/mL) 1.50 0 25 50 100 1.00 AKT 0.50 Arbitrary units

phospho-AKT 0.00 α/β IKK-α/β AKT IKK-α/β phospho-AKT phospho-IKK-α/β phospho-IKK-

Actin

Figure 2. Galiximab inhibits NF-kB activity in Raji cells and the role of NF-kB inhibition in the sensitization of Raji to apoptosis by CDDP and TRAIL. Raji cells were treated with different concentrations of galiximab (25, 50, and 100 mg/mL) for 18 hours, and aliquots were used to prepare both nuclear and total cell lysates as described in Materials and Methods. A, Western blot analysis for NF-kB expression. Total cell lysates were tested for various gene products of the NF-kB pathway. b-Actin was used as a loading control. Densitometric analysis is also shown and intensity of the bands was normalized to b-actin bands. B, inhibition of NF-kB DNA-binding activity by galiximab. Raji cells were treated with galiximab (25 mg/mL). Nuclear lysates were tested for NF-kB DNA- binding activity by EMSA as described. The NF-kB inhibitor DHMEQ (10 mg/mL) was used as a positive control and cold probes as competitors. For the supershift assay, the nuclear proteins were incubated with anti-p65 antibody overnight at 4C before the analysis by EMSA. C, galiximab-induced inhibition of NF-kB in the sensitization to apoptosis by CDDP and TRAIL. Raji cells were treated with galiximab (20 mg/mL) for 18 hours or with the NF-kB inhibitor DHMEQ (10 mg/mL) for 18 hours and the cells were subsequently treated with either CDDP (5 mg/mL) or TRAIL (5 mg/mL) for an additional 24 hours and apoptosis was determined. The data represent the mean SD from 3 independent experiments. , P < 0.01. D, inhibition of the AKT pathway by galiximab. Total cell lysates were tested for various gene products of the AKT pathway. b-Actin was used as a loading control. Densitometric analysis is also shown.

Inhibition of the resistant factors YY1 and Snail by Snail (31) and YY1 (32) and, in addition, Snail transcription galiximab; inhibition of the expression and activity of is also regulated by YY1 (42). Therefore, we examined both YY1 and Snail by galiximab. We and others have whether galiximab-induced inhibition of NF-kB also reported that several antiapoptotic gene products regu- inhibited the expression and activity of both YY1 and lated by NF-kB participate in the acquisition of tumor cell Snail. Raji cells were treated with various concentrations resistance to apoptotic stimuli (37–39). For instance, the of galiximab (25, 50, and 100 mg/mL) for 18 hours and both transcriptional repressor YY1 was shown to inhibit the nuclear and total cell lysates were prepared. Western blot response to TRAIL apoptosis via the TRAIL receptor DR5 analysis revealed that galiximab signiﬁcantly inhibited (40). Furthermore, the transcription repressor Snail was both YY1 and Snail expressions in a concentration-depen- also reported to participate in the antiapoptotic activity of dent manner and quantiﬁed by densitometric analyses tumor cells (41). NF-kB regulates the transcription of both (Fig. 3A). The inhibition of the DNA-binding activities of

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ABGaliximab (μg/mL) 0 25 50 100

YY1 Galiximab Snail –+ Cold probe extracts No nuclear Actin

YY1 0 25 50 100 Galiximab (μg/mL) 1.5

1.0 Snail 0.5

Arbitrary units 0.0 YY1 Snail

Figure 3. Galiximab inhibits the expression and the activity of the transcription factors YY1 and Snail in Raji cells. A, inhibition of YY1 and Snail by galiximab. Raji cells were treated with various concentrations of galiximab (25, 50, and 100 mg/mL) for 18 hours and total cell lysates were prepared for Western blot analysis. b-Actin was used as a loading control. The Western blots analyses were also analyzed by densitometry and is shown below the Western blot analysis ﬁgure. B, galiximab inhibits the DNA-binding activity of YY1 and Snail. Raji cells were treated with galiximab (25 mg/mL) for 18 hours and nuclear lysates were tested for DNA-binding activities for YY1 and Snail as described in Materials and Methods. The speciﬁcity of DNA-binding activity was determined by the use of a corresponding competitive cold probe and in the absence of nuclear extracts in the assay.

YY1 and Snail by galiximab was determined by EMSA in the regulation of Bcl-XL expression and their role in (Fig. 3B). sensitization was tested following treatment of Raji cells The direct role each of YY1- and Snail-induced inhibi- with the pan-Bcl-2 family inhibitor 2MAM-A3. Such treat- tion by galiximab in the sensitization of Raji tumor cells ment reversed the resistance of Raji cells to apoptosis by to apoptosis by CDDP and TRAIL. Raji cells were trea- both CDDP and TRAIL (Fig. 4E). ted with YY1 siRNA or control siRNA and Western blot Altogether, the above findings show that galiximab analysis was conducted. Treatment with YY1 siRNA inhibits NF-kB activity and downstream both YY1 and inhibited YY1, Snail, and p-p65. Treatment with Snail Snail. Individually, the inhibitions of either one of those siRNA inhibited Snail, p-p65, and p-AKT (Fig. 4A). The factors reverse the resistance. In addition, those factors siRNA-transfected cells were then treated with CDDP or have in common the regulation of the antiapoptotic TRAIL and examined for apoptosis. Whereas treatment gene product Bcl-XL whose inhibition reverses the with control siRNA did not reverse resistance, treatment resistance. with YY1 siRNA significantly sensitized the cells to apoptosis by both CDDP and TRAIL (Fig. 4B, top). Treatment Discussion of Raji cells with Snail siRNA, unlike control siRNA, Galiximab (anti-CD80 monoclonal antibody) is current- significantly sensitized the tumor cells to apoptosis by ly being investigated as a novel therapeutic against B- both CDDP and TRAIL (Fig. 4B, bottom). NHL and is currently in phase III clinical trials (43). A Mechanism of YY1- and Snail-induced inhibition by randomized, double-blind study of galiximab in combi- galiximab in the reversal of resistance: the role of Bcl-XL– nation with rituximab was compared with rituximab in induced inhibition by galiximab, YY1 and Snail in the combination with placebo for the treatment of subjects sensitization to apoptosis by CCDP and TRAIL. The with relapsed or refractory, follicular NHL. The results antiapoptotic Bcl-2/Bcl-XL factors in tumor cell resistance show that the addition of galiximab to rituximab reduced are well established (26, 27). Both Bcl-2 and Bcl-XL expres- the hazard for disease progression or death by 26% com- sions were inhibited by galiximab in a concentration- pared with the rituximab þ placebo group. Galiximab has dependent manner (Fig. 4C). We examined whether the shown some clinical benefits; however, the mechanism by sensitization was mediated by the inhibition of Bcl-2/Bcl- which galiximab mediates its antitumor effects is not XL gene products by YY1 and Snail in Raji cells. Treatment clear. The present in vitro findings show that galiximab with YY1 siRNA, and not with control siRNA, resulted in exerts an antiproliferative response on Burkitt’s B-NHL the specific inhibition of both Bcl-2 and Bcl-XL (Fig. 4D). cell lines and also sensitizes the resistant tumor cells to However, treatment with Snail siRNA inhibited signifi- apoptosis by both chemotherapeutic and immunothera- cantly Bcl-XL expression and modestly Bcl-2 (Fig. 4D). peutic drugs. Galiximab signals the cells via the CD80 These findings showed that both Snail and YY1 participate receptor and inhibits intracellular survival/antiapoptotic

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A B 60 * Control * TRAIL (5 ng/mL) 50 CDDP (5 μg/mL)

20 C μ 10 Galiximab ( g/mL) No transfection Control siRNA YY1 siRNA Snail siRNA

Apoptosis (% of active caspase-3) Apoptosis (% of active 02550 100 YY1 0 Control siRNA YY1 siRNA 70 Bcl-2 Snail Control * 60 TRAIL (5 ng/mL) * p65 CDDP (5 μg/mL) Bcl-XL phospho-p65 50 40 AKT Actin phospho-AKT 30 Actin 20 10

Apoptosis (% of active caspase-3) Apoptosis (% of active 0 Control siRNA Snail siRNA DE 60 Control * 50 2MAM-A3 (5 μg/mL) * 40 No transfection Control siRNA YY1 siRNA Snail siRNA Bcl-XL 30

Bcl-2 20 Actin 10

Apoptosis (% of active caspase-3) Apoptosis (% of active 0 Control CDDP TRAIL (5 μg/mL) (5 ng/mL)

Figure 4. The involvement of YY1- and Snail-induced inhibition by galiximab and downstream inhibition of Bcl-XL in the sensitization of Raji cells to apoptosis by both CDDP and TRAIL. A, transfection with YY1 siRNA and Snail siRNA and sensitization. Raji cells were transfected with YY1 siRNA, Snail siRNA, or control siRNA. Treatment with YY1 siRNA but not control siRNA inhibited the expression of YY1, Snail, and phospho-p65. Treatment with Snail siRNA inhibited the expression of Snail, phospho-p65, and phospho-AKT. b-Actin was used as a loading control. B, the direct role of YY1 and Snail in sensitization. Sensitization of Raji cells to TRAIL- and CDDP-induced apoptosis following treatment with YY1 siRNA (top) or with Snail siRNA (bottom). , P < 0.05. C, inhibition of Bcl-2 and Bcl-XL by galiximab. Raji cells were treated with various concentrations of galiximab (25, 50, and 100 mg/mL) and cell lysates were tested for Bcl-2 and Bcl-XL expression. b-Actin was used as loading control. D, inhibition of Bcl-XL expression by knocking down YY1 and/or Snail. Raji cells were transfected with either YY1 siRNA, Snail siRNA, or control siRNA and lysates were examined for Bcl-XL and Bcl-2 expression by Western blot analysis. b-Actin was used as a loading control. E, the role of Bcl-2 inhibition in the sensitization to CDDP and TRAIL. Raji cells were treated with the pan-Bcl-2 family inhibitor 2MAM-A3 for 18 hours and then treated with either CDDP or TRAIL for an additional 18 hours and apoptosis was determined. , P < 0.05.

pathways such as the NF-kB and AKT pathways and their binding activity. It is not clear that how galiximab inhibits targets, namely, the antiapoptotic transcription factors NF-kB activity. CD80 is a transmembrane immune costi- YY1 and Snail and each leading to inhibition of the anti- mulatory glycoprotein involved in the regulation of T-cell apoptotic gene product Bcl-XL. Individually, inhibition activation (44). CD80 also serves as a receptor that trans- by galiximab of NF-kB, YY1, Snail, or Bcl-XL in B-NHL duces distinct signals to the cells expressing CD80 upon cells results in the sensitization to both CDDP- and engagement by CD28 (45). It is possible that it translocates TRAIL-mediated apoptosis. These ﬁndings establish the the CD80 receptor into lipid rafts and inhibits Src kinases presence of a dysregulated NF-kB/Snail/YY1/Bcl-XL upstream of NF-kB as we and others have previously resistant circuit in B-NHL cells and its inhibition by shown following treatment of B-NHL cells with rituximab galiximab leads to the reversal of drug resistance. (26, 27). The involvement of galiximab-induced inhibition Galiximab inhibited the expression of phospho-p65, a of NF-kB in sensitization was corroborated by the use of member of the NF-kB pathway, as well as NF-kB DNA- the NF-kB–speciﬁc inhibitor DHMEQ which mimicked

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Galiximab Reverses YY1/NF-kB/Snail Loop Resistance

galiximab in sensitizing Raji cells to apoptosis by both Galiximab CDDP and TRAIL. We report here a mechanism by which galiximab-mediated inhibition of NF-kB and AKT resulted in the reversal CD80 of resistance. We show the presence of a dysregulated NF- kB/YY1/Snail/Bcl-XL resistant circuit and whose inhibition by galiximab reverses resistance. Each of the gene AKT P products individually participated in the reversal of resistance following their inhibition by galiximab. Galiximab IKKα/βP inhibited the expression of the transcription factor YY1 NF-κBP that is regulated, in part, by NF-kB (40). We show here the direct role of YY1 inhibition by galiximab in the reversal of resistance using YY1 siRNA in agreement with our pre- Snail YY1 vious findings (40, 46). The involvement of YY1 in the regulation of the apoptotic pathway is shown here by its Bcl-2/Bcl-XL ability to regulate the expression of Bcl-XL and Bcl-2 as analysis of the promoters revealed the presence of putative YY1-binding sites. Resistance to CDDP- or TRAIL- Sensitization to CDDP- or TRAIL- Galiximab inhibited Snail expression and Snail is mediated apoptosis mediated apoptosis involved in the regulation of tumor cell response to CDDP and TRAIL as shown here in cells transfected with Snail Figure 5. Schematic diagram representing the mechanism by which siRNA. Like YY1, treatment with Snail siRNA also inhib- galiximab sensitizes B-NHL cells to both chemo- and immunocytotoxic ited Bcl-XL expression. The mechanism by which Snail drugs. Raji cells exhibit constitutively activated NF-kB and AKT activities inhibits Bcl-XL is not clear. Because Snail is primarily a and resulting downstream in the expression of Snail, YY1, and Bcl-2/Bcl- XL. These resistant gene products regulate the resistance to apoptotic repressor and there are no putative binding sites of Snail stimuli. However, treatment with galiximab inhibits NF-kB and AKT on the Bcl-XL promoter, we envisaged that Snail inhibits activities and downstream in the inhibition of Snail, YY1, and Bcl-2/Bcl- the regulation of Bcl-XL indirectly (47). Studies through XL expressions. Each of these gene products, namely, Snail, YY1, or Bcl- activation of the mitogen-activated protein kinase 2/Bcl-XL, inhibited by galiximab result in the reversal of resistance and sensitization to both CDDP and TRAIL apoptosis. Hence, the tumor cells (MAPK) and PI3K/AKT pathways showed that Snail- exhibit a constitutively dysregulated NF-kB/Snail/YY1/Bcl-2-Bcl-XL expressing cells show hyperactivation of MAPK and resistant circuit whose inhibition by galiximab results in the reversal of PI3K/AKT activities. Both pathways can modulate the resistance when used in combination with cytotoxic drugs. upregulation of Bcl-XL expression (48). We show here that the treatment with Snail siRNA inhibited phospho-AKT. Also, Snail negatively regulates Wnt, which encodes a regulates tumor cell resistance. These findings offer a secreted Wnt family of proteins that negatively regulate potential therapeutic approach using the combination of NF-kB activity and downstream Bcl-XL (49). The involve- galiximab and either subtoxic chemoimmunotherapeutic ment of galiximab-induced inhibition of Snail and YY1 drugs or specific inhibitors of the resistant factors in the and their regulation of Bcl-2 and Bcl-XL leading to sen- circuit in the treatment of drug-resistant CD80-expressing sitization was corroborated in experiments showing that hematologic malignancies. the pan-Bcl-2 inhibitor 2MAM-A3 sensitized the tumor cells to CDDP and TRAIL apoptosis. Disclosure of Potential Conflicts of Interest CD80 is expressed in pediatric B-cell lymphoblastic No potential conflicts of interests were disclosed. lymphoma, Burkitt’s lymphoma, and diffuse large B-cell Acknowledgments lymphoma (50). Therefore, our findings here with the The authors thank the assistance of Kerry Choy, Daphne Liang, and Burkitt’s lymphoma cell line, Raji, may be consistent with Melissa Cao in preparation of the manuscript and Dr. Kazuo Umezawa for the NF-kB inhibitor. They also thank Programa de Posgrado; Doctorado en non-Burkitt’s CD80-expressing lymphomas. Ciencias Biomedicas, Facultad de Medicina UNAM, and Dr. Otoniel Our findings in this report are schematically dia- Martinez-Maza for valuable input and support. grammed in Fig. 5 and can be summarized as follows: the constitutively activated NF-kB and AKT pathways in Grant Support Raji cells lead to the expression of YY1 and Snail and to the This study was supported, in part by, academic support CONACYT, Mexico (165639 to M.A. Martinez-Paniagua), Jonsson Comprehensive overexpression of antiapoptotic gene products such as Cancer Center (B. Bonavida and M.I. Vega), UCLA AIDS Institute (M.I. Bcl-2 and Bcl-XL. Treatment with galiximab inhibits NF- Vega), and Fogarty International Center Fellowship (D43 TW00013-14; M. k I. Vega & S. Huerta-Yepez). B and AKT activities and downstream the expression of The costs of publication of this article were defrayed in part by the YY1 and Snail, leading to inhibition of Bcl-2 and Bcl-XL payment of page charges. This article must therefore be hereby marked and reversal of resistance. The present findings support advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. the existence of a dysregulated NF-kB/YY1/Snail/Bcl-2/ Bcl-XL resistance circuit as each of the gene products in Received August 16, 2011; revised December 21, 2011; accepted this circuit is inhibited by galiximab and each directly December 22, 2011; published OnlineFirst January 19, 2012.

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Martinez-Paniagua et al.

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Galiximab Signals B-NHL Cells and Inhibits the Activities of NF-κB− Induced YY1- and Snail-Resistant Factors: Mechanism of Sensitization to Apoptosis by Chemoimmunotherapeutic Drugs

Melisa A. Martinez-Paniagua, Mario I. Vega, Sara Huerta-Yepez, et al.

Mol Cancer Ther 2012;11:572-581. Published OnlineFirst January 19, 2012.

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