Galiximab Signals B-NHL Cells and Inhibits the Activitiesof NF-kB–Induced YY1- and Snail-Resistant Factors:Mechanism of Sensitization to Apoptosis byChemoimmunotherapeutic Drugs
Melisa A. Martinez-Paniagua1,2, Mario I. Vega1,3, Sara Huerta-Yepez1,4, Stavroula Baritaki1,Gabriel G. Vega3, Kandasamy Hariharan5, and Benjamin Bonavida1
AbstractGaliximab (anti-CD80 monoclonal antibody) is a primatized (human IgG1 constant regions and cynomo-
logus 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
NF-kB pathway. This hypothesis was tested using various CD80þ Burkitt B-NHL (non–Hodgkin lymphomas)
cell lines asmodels. Treatment of B-NHL cellswith galiximab (25–100 mg/mL) resulted in significant inhibition
ofNF-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 cellswere shown tobecomesensitive tobothCCDP- andTRAIL-inducedapoptosis in the
absence of galiximab. Furthermore, knockdown of YY1 or Snail inhibited Bcl-XL. The involvement of Bcl-XL
inhibition in sensitizationwas corroborated by the use of the pan-Bcl-2 inhibitor 2MAM-3whereby 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.
IntroductionNon–Hodgkin lymphomas (NHL) are a heterogeneous
group of malignancies of the lymphoid system. Currenttreatments for NHL are not optimally effective as both
relapse and resistance to common chemotherapy areusually observed (1–4). Monoclonal antibodies have rev-olutionized the treatment of malignances such as NHL.
Rituximab is an anti-CD20 monoclonal antibody thathas been approved by the U.S. Food and Drug Admin-istration for the treatment of CD20þ B-NHL (5, 6). Itinduces cell lysis through antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotox-icity, and apoptosis (7, 8). There has not been an increasein overall survival (5, 6). Approximately 50% of treatedpatients are refractory or develop resistance during thecourse of prolonged treatment with rituximab as singleagent or in combination with cyclophosphamide-Adria-mycin-vincristine-prednisone (CHOP; ref. 9). Becauseresistance to rituximab occurs, there is a need for newtherapies (10).
CD80 is a membrane-bound immune costimulatorymolecule involved in the regulation of the activation ofT cells (11). It is a member of the B7 family of costimu-latory molecules (12). CD80 is expressed on the surfaceof normal activated B cells, antigen-presenting cells, andT cells (13 and on the surface of a variety of lymphoid
Authors' Affiliations: 1Department ofMicrobiology, Immunology &Molec-ular Genetics, David Geffen School of Medicine, Jonsson ComprehensiveCancer Center, University of California, LosAngeles, California; 2Unidad deInvestigaci�on Medica en Inmunología e Infectología, Hospital de Infecto-logía, CMN "La Raza", IMSS; 3Unidad de Investigaci�on M�edica en Enfer-medades Oncol�ogicas, Hospital de Oncología CMN Siglo XXI IMSS,M�exico; 4Unidad de Investigaci�on en Enfermedades Oncol�ogicas HospitalInfantil de M�exico S.S.A, M�exico; and 5BiogenIdec, Inc., Research &Corporate, San Diego, California
Note: M.-A. Martinez-Paniagua and M.I. Vega contributed equally to thestudy.
Corresponding Author:Benjamin Bonavida, Department ofMicrobiology,Immunology & Molecular Genetics, David Geffen School of Medicine,Jonsson Comprehensive Cancer Center, University of California, LosAngeles, CA 90095. Phone: 310-825-2233; Fax: 310-206-3865; E-mail:[email protected]
malignances (14–17). Preclinical studies have showedthat anti-CD80 antibodies can inhibit cell proliferationof lymphoma and induce antibody-dependent cell-mediated cytotoxicity (18) in vivo. Conjugated anti-CD80 antibodies with immunotoxins exerted antitumoractivity in vitro using CD80þ Burkitt lymphoma (Raji)and Hodgkin lymphoma (L428) cell lines (19). Galix-imab delays progression and prolongs survival in ahuman lymphoma xenograft/severe combined immu-nodeficient (SCID) mouse model (20). Previous findingsshowed that treatment with a monoclonal antibody forCD80 (16-10A1) on B cells resulted in a significantdecrease in tumor cell proliferation, upregulation ofproapoptotic molecules, and downregulation of theexpression of antiapoptotic proteins such as Bcl-XL,thus, resulting in tumor cell apoptosis (21).Galiximab (IDEC-11) is a high-affinity primatized anti-
CD80 (IgG1l) monoclonal antibody that contains variableregions of primate (cynomologus macaque) origin andconstant regions of humanorigin (22).Aphase I/II clinicaltrial involving patients with relapsed or refractory follic-ular lymphoma resulted in an overall response rate of 11%and tumor regression in 49% of the patients (23). Preclin-ical data suggest the synergy between rituximab andgaliximab in patientswith relapsed or refractory follicularlymphoma (23, 24).Galiximab serves as a counter receptor that transduces
distinct signaling to B cells upon engagement. Severalstudies have reported the signaling on T cells through theCD80 ligand (CD28) via interactionwithCD80-expressingcells (25). However, little is known about the direct sig-naling by galiximab of cells expressing CD80. Theseresults indicated that CD80 can mediate signal transduc-tion and regulate B-cell function (21).We and others have reported that rituximab treatment
affects apoptotic signaling in lymphoma cell lines viaupstream inhibition of constitutively activated survivalsignaling pathways such as the p38MAPK andNF-kB (26,27) anddownstream inhibitionof Bcl-2 andBcl-XL expres-sion and in the reversal of resistance (28). In addition,treatment of B-NHL cell lines with rituximab resulted inpotentiation of apoptosis induced by Fas-L and TRAIL,respectively, through the inhibition of the transcriptionalrepressor YY1 (29, 30). Furthermore, NF-kB activatesthe transcription of resistant gene products such as Snail(31–33).Galiximab interferes with the apoptotic pathways by
targeting gene products regulated by NF-kB. Therefore,we hypothesized that galiximab may inhibit the consti-tutively activated NF-kB pathway and may also sensitizeresistant B-NHL cell lines to apoptosis by cytotoxic drugs.The following were examined to test this hypothesis: (i)Does treatment of CD80þ Burkitt B-NHL cell lines inhibitthe constitutively activatedNF-kBandAKTpathways; (ii)Does treatment of B-NHL cell lines with galiximab sen-sitize the cells to apoptosis by chemotherapeutic andimmunotherapeutic drugs?; and (iii) Does galiximab-mediated sensitization to apoptosis result from inhibition
of resistant factors such as YY1, Snail, and Bcl-XL andwhat is the direct involvement of each of these factors insensitization? The findings reported herein support theabove hypothesis and establish a novel mechanism ofgaliximab-induced cell signaling resulting in the inhibi-tion of the NF-kB/Snail/YY1/Bcl-XL dysregulated resis-tant circuit that controls the resistance in B-NHL andleading to sensitization of B-NHL cells to drug-inducedapoptosis.
Materials and MethodsCell lines and reagents
The human B-NHL cell lines Raji, Ramos, DHL4, andDaudi were purchased from the American Type CultureCollection. The AIDS-related lymphoma B cell line 2F7was kindlyprovidedbyDr.O.Martinez-Maza,Universityof California, Los Angeles, CA. The cell lines were cul-tured as described previously (27). All cells used in thisstudy were within 15 passages after resuscitation. Thecells were check routinely by morphology and tested forMycoplasma contamination with CELLshipper Myco-plasma Detection Kit (Bionique Testing Laboratories).Galiximab was obtained from BiogenIdec, Inc. Solublerecombinant human TRAIL was purchased from Pepro-Tech Inc. The cis-Diamminedichloroplatinum(II) (CDDP)was purchased from Sigma and was diluted in dimethylsulfoxide. The NF-kB inhibitor DHMEQwas provided byDr. K. Umezawa (Keio University, Yokohama, Japan) anddiluted in dimethyl sulfoxide (34). The phycoerythrin(PE)-labeled anti-CD80 antibody and the fluoresceinisothiocyanate (FITC)-labeled antiactive caspase-3 anti-bodies, aswell as the corresponding IgG1 isotype controlswere obtained from BD Pharmingen. The Bcl-2 familyinhibitor 2MAM-A3 was purchased from BIOMOL. Thefollowing antibodies were obtained from Santa CruzBiotechnology: Bcl-XL, Bcl-2, p50, p65, phospho-p65Ser-536, IkBa, phospho-IkBa Ser-32, IKK, phosphor-IKK-a/b Ser-176, AKT, phospho-AKT Thr-308, YY1, andSnail.
Viability assayCell viability was assessed by either the trypan blue
dye exclusion assay by microscopy or by the 2,3—bis(2-methoxy-4-nitro-S-sulfophenynl)H-tetrazolium-5 car-boxanilide inner salt (XTT) dye absorbance accordingto the manufacturer’s instruction (Roche DiagnosticGmbH) and as previously described (35). The viabilityof the untreated cells was set at 100%. Furthermore, totalcell recovery was also recorded. Each experimentalcondition was conducted in triplicate and the SD wascalculated.
Western blot analysis for protein expressionOne million of Raji cells per milliliter were incubated
with or without the indicated concentrations of galiximabat 37�C for 18 hours.Western blot analysis was conductedas previously described (35).
Electrophoretic mobility shift assayRaji cells (106) were incubated with or without the
indicated concentrations of galiximabat 37�Cfor 18hours.Ten microliters of nuclear proteins was mixed with thebiotin probe for analysis of the transcription factors NF-kB, Snail, and YY1, using the EMSA kits from Panomics,following the manufacturer’s instructions and as previ-ously described (35).
Surface expression of CD80Cells were stainedwith the PE-conjugated specific anti-
CD80 for 1 hour at 4�C according to the manufacturer’sinstruction.Analysiswas conductedusingFlowEpicsXL-MCL (Coulter) equipment. The mean fluorescence inten-sity was recorded using the System II Software.
Apoptosis determinationApoptosis was assessed in tumor cells as previously
described for activated caspase-3 by flow cytometry (35).In addition, we also used the Annexin-V method tocorroborate the caspase-3 method.
Transfection with short interfering RNATransfection was conducted using the Lipofectamine
transfection reagent (Invitrogen Life Technology). Scram-bledRNA, Snail, andYY1 short interferingRNAs (siRNA)were obtained from Santa Cruz Biotechnology. Raji cellswere cultured at a density of 2.5 � 105/mL in RPMI-1640devoid of antibiotics for 24 hours. Cells were then trans-fected with 50 nmol/L siRNA in a final volume of 100 mLof medium in the presence of 10 mL of Lipofectamine 2000in Opti-MEM. To determine YY1 or Snail siRNA-inducedsensitization toCDDP- or to TRAIL-induced apoptosis, 48hours following treatments, untransfected cells and cellstransfected with scrambled siRNA, YY1 siRNA, or Snailwere treated with CDDP or TRAIL for 24 hours andapoptosis was measured by FITC-labeled antiactive cas-pase-3 antibody using flow cytometry.
Isobologram analysis for synergy determinationThe isobologramanalysiswasused toevaluate theeffect
of the galiximab/CDDP combination as described (36).
Statistical analysisAll results were expressed as the mean � SD of data
obtained from 3 triplicate independent and separateexperiments. The statistical significance of differencesbetween group means was determined using one-wayANOVA to compare variance. Significant differenceswere considered for probabilities <5% (P < 0.05).
ResultsGaliximab-mediated inhibition of cell proliferationand sensitization of B-NHL cells to apoptosis byCDDP and TRAIL
Several B-NHL cell lineswere first examined for surfaceCD80 expression by flow cytometry. There was differen-tial CD80 expression ranging from low (Ramos, DHL4)
to high (Raji, Daudi, 2F7) as shown in the histogram inFig. 1A. The mean fluorescence intensity (MFI) for allthe cell lines is shown in a table format depicted in Fig. 1A.
The resistant B-NHL cell lines were tested for galixi-mab-mediated sensitization to apoptosis by measuringthe activation of caspase-3 by flow cytometry followingthe addition of suboptimal concentrations of CDDP andTRAIL. As shown in Fig. 1B (left), single-cell treatmentwith galiximab (20 mg/mL) had no significant apoptoticeffect on the cell lines; however, with the combination ofgaliximab and drug, there was significant sensitization toboth CDDP and TRAIL apoptosis in all of the B-NHL celllines tested. The single-cell treatment with CDDP orTRAILhadno significant apoptosis. The level of apoptosisachieved was different for each cell line tested. 2F7, Raji,and Daudi cells were significantly sensitized by bothCDDP and TRAIL. Ramos, however, was not sensitizedto CDDP, although it was sensitized to TRAIL. The DHL4cell line was not sensitized to CDDP in combination withgaliximab treatment but was sensitized to TRAIL (data noshown). The apoptotic effect assessed by the activation ofcaspase-3was corroborated by analysis of apoptosis usingAnnexin-V as shown in Fig. 1B (right).
Wehave chosenRaji cells as amodel for further analysisof the underlyingmechanism of galiximab-mediated sen-sitization. Treatment of Raji cells with various concentra-tions of galiximab resulted in a concentration-dependentinhibition of cell survival. Concentrations of galiximab�25 mg/mL resulted in a decrease of viability and aplateau (75%–80% viability) was reached at concentra-tions �25 mg/mL. In addition, there was inhibition of cellproliferation by galiximab concentrations of �10 mg/mLas determined by total cell recovery (Fig. 1C). The effectsof various galiximab concentrations as well as differentconcentrations of CDDP and TRAIL were examined forsensitization to apoptosis. Raji cells were pretreated withvarious concentrations of galiximab (10–100 mg/mL) for18 hours and then treated for an additional 24 hours withCDDP (5–20 mg/mL) or TRAIL and apoptosis was deter-mined. The findings show that there was significantsensitization by the combination treatment and the levelof apoptosis was a function of the concentrations used forboth galiximab andCDDP (Fig. 1D, left). The combinationtreatment was synergistic as determined by isobologramanalysis. Likewise, the combination of galiximab andTRAIL resulted in apoptosis and the level of apoptosiswas a function of the increased concentration of TRAIL(Fig. 1D, right).
Mechanism by which galiximab sensitizes Raji cellsto apoptosis by CDDP and TRAIL
Inhibition of NF-kB activity. We examined whethergaliximab, due to its antiproliferative activity, inhibited theconstitutively activated NF-kB pathway in Raji cells. Rajicells were treatedwith several concentrations of galiximab(25, 50, and 100 mg/mL) for 18 hours, and cell lysateswere prepared for analysis for the expression of severalgene products of the NF-kB pathway. Western blot and
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densitometric analyses revealed that following galiximabtreatment, there was some inhibition of p50 but there weresignificant inhibition of p65, phospho-p65, and both phos-pho- and total IkBa expression levels in a concentration-dependent manner (Fig. 2A, left) and quantified by densi-tometric analysis (Fig. 2A, right). The inhibition of NF-kBDNA-binding activity bygaliximab andby the specificNF-kB inhibitor DHMEQwas corroborated by electrophoreticmobility shift assay (EMSA; Fig. 2B). To show the involve-ment of NF-kB inhibition by galiximab in the reversal of
resistance, the tumor cells were treated with the specificNF-kB inhibitor, DHMEQ, and tested for sensitivity toapoptosis induced by CDDP and TRAIL. Treatment withDHMEQ sensitized the tumor cells to apoptosis by CDDPand TRAIL and, thus, mimicking the treatment with galix-imab (Fig. 2C). The effect of galiximab treatment on theAKT pathway was examined by Western blot analysis.Galiximab inhibited phospho-AKT and phospho-IKKa/bbut not the unphosphorylated forms as shown byWesternblot analysis and densitometry (Fig. 2D).
Figure 1. Galiximab inhibits cell proliferation ofB-NHL cell lines and sensitizesB-NHLcells to apoptosis byCDDPandTRAIL. A, surface expression ofCD80onB-NHL cell lines. The surface expression of CD80 was analyzed by flow cytometry. A, representative histogram is shown for the various B-NHL celllines and an isotype control is shown. In addition, the mean fluorescence intensity (MFI) is represented in the table. B, sensitization to apoptosis. B-NHLcell 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 additional18 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-Vas described (right). C, galiximab-induced inhibition of Raji cell viability and cell recovery. The B-NHL cell line Raji was treated with various concentrationsof galiximab and incubated for different time periods (6–24 hours), and cell viability was determined by trypan blue dye exclusion and total cell recoverywas recorded. Raji cells that were not treated with galiximab represented 100% viability. The data represent the mean � SD from 3 independentexperiments �, P < 0.05. D, galiximab sensitizes resistant B-NHL cell lines to apoptosis by CDDP and TRAIL. Sensitization of B-NHL Raji cells bygaliximab to apoptosis byCDDP or TRAIL is synergistic. Raji cells were treatedwith various concentrations of galiximab (10–100 mg/mL) for 18 hours and thentreated 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, and10 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 isrepresented left of D. AAD, aminoactinomycin D.
Inhibition of the resistant factors YY1 and Snail bygaliximab; inhibition of the expression and activity ofboth YY1 and Snail by galiximab. We and others havereported that several antiapoptotic gene products regu-lated byNF-kB participate in the acquisition of tumor cellresistance to apoptotic stimuli (37–39). For instance, thetranscriptional repressor YY1 was shown to inhibit theresponse to TRAIL apoptosis via the TRAIL receptor DR5(40). Furthermore, the transcription repressor Snail wasalso reported to participate in the antiapoptotic activity oftumor cells (41). NF-kB regulates the transcription of both
Snail (31) andYY1 (32) and, in addition, Snail transcriptionis also regulated by YY1 (42). Therefore, we examinedwhether galiximab-induced inhibition of NF-kB alsoinhibited the expression and activity of both YY1 andSnail. Raji cells were treated with various concentrationsof galiximab (25, 50, and 100mg/mL) for 18hours andbothnuclear and total cell lysates were prepared. Western blotanalysis revealed that galiximab significantly inhibitedboth YY1 and Snail expressions in a concentration-depen-dent manner and quantified by densitometric analyses(Fig. 3A). The inhibition of the DNA-binding activities of
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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 cellswere 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 celllysates as described in Materials and Methods. A, Western blot analysis for NF-kB expression. Total cell lysates were tested for various gene products ofthe 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-actinbands.B, inhibition ofNF-kBDNA-binding activity by galiximab. Raji cellswere treatedwith galiximab (25mg/mL). Nuclear lysateswere tested forNF-kBDNA-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 4�C before the analysis by EMSA. C, galiximab-inducedinhibition 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 theNF-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 additional24 hours and apoptosis was determined. The data represent the mean � SD from 3 independent experiments. �, P < 0.01. D, inhibition of the AKTpathway by galiximab. Total cell lysates were tested for various gene products of the AKT pathway. b-Actin was used as a loading control. Densitometricanalysis is also shown.
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Mol Cancer Ther; 11(3) March 2012 Molecular Cancer Therapeutics576
YY1 and Snail by galiximab was determined by EMSA(Fig. 3B).
The direct role each of YY1- and Snail-induced inhibi-tion by galiximab in the sensitization of Raji tumor cellsto apoptosis by CDDP and TRAIL. Raji cells were trea-ted with YY1 siRNA or control siRNA and Western blotanalysis was conducted. Treatment with YY1 siRNAinhibited YY1, Snail, and p-p65. Treatment with SnailsiRNA inhibited Snail, p-p65, and p-AKT (Fig. 4A). ThesiRNA-transfected cells were then treated with CDDP orTRAIL and examined for apoptosis. Whereas treatmentwith control siRNA did not reverse resistance, treatmentwith YY1 siRNA significantly sensitized the cells to apo-ptosis by both CDDP and TRAIL (Fig. 4B, top). Treatmentof Raji cells with Snail siRNA, unlike control siRNA,significantly sensitized the tumor cells to apoptosis byboth CDDP and TRAIL (Fig. 4B, bottom).
Mechanism of YY1- and Snail-induced inhibition bygaliximab in the reversal of resistance: the role of Bcl-XL–induced inhibition by galiximab, YY1 and Snail in thesensitization to apoptosis by CCDP and TRAIL. Theantiapoptotic Bcl-2/Bcl-XL factors in tumor cell resistancearewell established (26, 27). Both Bcl-2 andBcl-XL expres-sions were inhibited by galiximab in a concentration-dependent manner (Fig. 4C). We examined whether thesensitization wasmediated by the inhibition of Bcl-2/Bcl-XLgeneproducts byYY1 andSnail in Raji cells. Treatmentwith YY1 siRNA, and not with control siRNA, resulted inthe specific inhibition of both Bcl-2 and Bcl-XL (Fig. 4D).However, treatment with Snail siRNA inhibited signifi-cantly Bcl-XL expression and modestly Bcl-2 (Fig. 4D).Thesefindings showed that bothSnail andYY1participate
in the regulation of Bcl-XL expression and their role insensitization was tested following treatment of Raji cellswith the pan-Bcl-2 family inhibitor 2MAM-A3. Such treat-ment reversed the resistance of Raji cells to apoptosis byboth CDDP and TRAIL (Fig. 4E).
Altogether, the above findings show that galiximabinhibits NF-kB activity and downstream both YY1 andSnail. Individually, the inhibitions of either one of thosefactors reverse the resistance. In addition, those factorshave in common the regulation of the antiapoptoticgene product Bcl-XL whose inhibition reverses theresistance.
DiscussionGaliximab (anti-CD80monoclonal antibody) is current-
ly being investigated as a novel therapeutic against B-NHL and is currently in phase III clinical trials (43). Arandomized, double-blind study of galiximab in combi-nation with rituximab was compared with rituximab incombination with placebo for the treatment of subjectswith relapsed or refractory, follicular NHL. The resultsshow that the addition of galiximab to rituximab reducedthe hazard for disease progression or death by 26% com-paredwith the rituximabþ placebo group. Galiximab hasshown some clinical benefits; however, themechanism bywhich galiximab mediates its antitumor effects is notclear. The present in vitro findings show that galiximabexerts an antiproliferative response on Burkitt’s B-NHLcell lines and also sensitizes the resistant tumor cells toapoptosis by both chemotherapeutic and immunothera-peutic drugs. Galiximab signals the cells via the CD80receptor and inhibits intracellular survival/antiapoptotic
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Figure 3. Galiximab inhibits the expression and the activity of the transcription factors YY1andSnail in Raji cells. A, inhibition of YY1 andSnail by galiximab.Rajicells were treated with various concentrations of galiximab (25, 50, and 100 mg/mL) for 18 hours and total cell lysates were prepared forWestern blot analysis.b-Actinwas used as a loading control. TheWestern blots analyseswere also analyzed by densitometry and is shownbelow theWestern 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 forDNA-binding activities for YY1 and Snail as described in Materials and Methods. The specificity of DNA-binding activity was determined by the use of acorresponding competitive cold probe and in the absence of nuclear extracts in the assay.
pathways such as theNF-kB andAKTpathways and theirtargets, namely, the antiapoptotic transcription factorsYY1 and Snail and each leading to inhibition of the anti-apoptotic gene product Bcl-XL. Individually, inhibitionby galiximab of NF-kB, YY1, Snail, or Bcl-XL in B-NHLcells results in the sensitization to both CDDP- andTRAIL-mediated apoptosis. These findings establish thepresence of a dysregulated NF-kB/Snail/YY1/Bcl-XLresistant circuit in B-NHL cells and its inhibition bygaliximab leads to the reversal of drug resistance.
Galiximab inhibited the expression of phospho-p65, amember of the NF-kB pathway, as well as NF-kB DNA-
binding activity. It is not clear that how galiximab inhibitsNF-kB activity. CD80 is a transmembrane immune costi-mulatory glycoprotein involved in the regulation of T-cellactivation (44). CD80 also serves as a receptor that trans-duces distinct signals to the cells expressing CD80 uponengagement byCD28 (45). It is possible that it translocatesthe CD80 receptor into lipid rafts and inhibits Src kinasesupstream of NF-kB as we and others have previouslyshown following treatment of B-NHL cellswith rituximab(26, 27). The involvement of galiximab-induced inhibitionof NF-kB in sensitization was corroborated by the use ofthe NF-kB–specific inhibitor DHMEQ which mimicked
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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 apoptosisby 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 siRNAinhibited 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 lysateswere 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. Rajicells were transfected with either YY1 siRNA, Snail siRNA, or control siRNA and lysates were examined for Bcl-XL and Bcl-2 expression by Westernblot 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 withthe 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 wasdetermined. �, P < 0.05.
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Mol Cancer Ther; 11(3) March 2012 Molecular Cancer Therapeutics578
galiximab in sensitizing Raji cells to apoptosis by bothCDDP and TRAIL.We report here amechanismbywhich galiximab-medi-
ated inhibition of NF-kB and AKT resulted in the reversalof resistance.We show the presence of a dysregulatedNF-kB/YY1/Snail/Bcl-XL resistant circuit and whose inhi-bition by galiximab reverses resistance. Each of the geneproducts individually participated in the reversal of resis-tance following their inhibition by galiximab. Galiximabinhibited the expression of the transcription factor YY1that is regulated, in part, byNF-kB (40).We show here thedirect role of YY1 inhibition by galiximab in the reversal ofresistance using YY1 siRNA in agreement with our pre-vious findings (40, 46). The involvement of YY1 in theregulation of the apoptotic pathway is shown here by itsability to regulate the expression of Bcl-XL and Bcl-2 asanalysis of the promoters revealed the presence of puta-tive YY1-binding sites.Galiximab inhibited Snail expression and Snail is
involved in the regulation of tumor cell response to CDDPand TRAIL as shown here in cells transfected with SnailsiRNA. Like YY1, treatment with Snail siRNA also inhib-ited Bcl-XL expression. The mechanism by which Snailinhibits Bcl-XL is not clear. Because Snail is primarily arepressor and there are no putative binding sites of Snailon the Bcl-XL promoter, we envisaged that Snail inhibitsthe regulation of Bcl-XL indirectly (47). Studies throughactivation of the mitogen-activated protein kinase(MAPK) and PI3K/AKT pathways showed that Snail-expressing cells show hyperactivation of MAPK andPI3K/AKT activities. Both pathways can modulate theupregulation of Bcl-XL expression (48).We showhere thatthe treatment with Snail siRNA inhibited phospho-AKT.Also, Snail negatively regulates Wnt, which encodes asecreted Wnt family of proteins that negatively regulateNF-kB activity and downstream Bcl-XL (49). The involve-ment of galiximab-induced inhibition of Snail and YY1and their regulation of Bcl-2 and Bcl-XL leading to sen-sitization was corroborated in experiments showing thatthe pan-Bcl-2 inhibitor 2MAM-A3 sensitized the tumorcells to CDDP and TRAIL apoptosis.CD80 is expressed in pediatric B-cell lymphoblastic
lymphoma, Burkitt’s lymphoma, and diffuse large B-celllymphoma (50). Therefore, our findings here with theBurkitt’s lymphoma cell line, Raji, may be consistent withnon-Burkitt’s CD80-expressing lymphomas.Our findings in this report are schematically dia-
grammed in Fig. 5 and can be summarized as follows:the constitutively activated NF-kB and AKT pathways inRaji cells lead to the expression of YY1 and Snail and to theoverexpression of antiapoptotic gene products such asBcl-2 and Bcl-XL. Treatment with galiximab inhibits NF-kB and AKT activities and downstream the expression ofYY1 and Snail, leading to inhibition of Bcl-2 and Bcl-XLand reversal of resistance. The present findings supportthe existence of a dysregulatedNF-kB/YY1/Snail/Bcl-2/Bcl-XL resistance circuit as each of the gene products inthis circuit is inhibited by galiximab and each directly
regulates tumor cell resistance. These findings offer apotential therapeutic approach using the combination ofgaliximab and either subtoxic chemoimmunotherapeuticdrugs or specific inhibitors of the resistant factors in thecircuit in the treatment of drug-resistant CD80-expressinghematologic malignancies.
Disclosure of Potential Conflicts of InterestNo potential conflicts of interests were disclosed.
AcknowledgmentsThe authors thank the assistance of Kerry Choy, Daphne Liang, and
Melissa Cao in preparation of themanuscript andDr. KazuoUmezawa fortheNF-kB inhibitor. They also thankProgramade Posgrado; Doctorado enCiencias Biom�edicas, Facultad de Medicina UNAM, and Dr. OtonielMartinez-Maza for valuable input and support.
Grant SupportThis study was supported, in part by, academic support CONACYT,
Mexico (165639 to M.A. Martinez-Paniagua), Jonsson ComprehensiveCancer Center (B. Bonavida and M.I. Vega), UCLA AIDS Institute (M.I.Vega), and Fogarty International Center Fellowship (D43 TW00013-14; M.I. Vega & S. Huerta-Yepez).
The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.
Received August 16, 2011; revised December 21, 2011; acceptedDecember 22, 2011; published OnlineFirst January 19, 2012.
Galiximab
CD80
AKT P
P
P
IKKα/β
NF-κB
Snail
Bcl-2/Bcl-XL
Resistance to CDDP- or TRAIL-
mediated apoptosis
Sensitization to CDDP- or TRAIL-
mediated apoptosis
YY1
Figure 5. Schematic diagram representing the mechanism by whichgaliximab sensitizes B-NHL cells to both chemo- and immunocytotoxicdrugs. Raji cells exhibit constitutively activated NF-kB and AKT activitiesand resulting downstream in the expression of Snail, YY1, and Bcl-2/Bcl-XL. These resistant gene products regulate the resistance to apoptoticstimuli. However, treatment with galiximab inhibits NF-kB and AKTactivities and downstream in the inhibition of Snail, YY1, and Bcl-2/Bcl-XL expressions. Each of these gene products, namely, Snail, YY1, or Bcl-2/Bcl-XL, inhibited by galiximab result in the reversal of resistance andsensitization to both CDDP and TRAIL apoptosis. Hence, the tumor cellsexhibit a constitutively dysregulated NF-kB/Snail/YY1/Bcl-2-Bcl-XLresistant circuit whose inhibition by galiximab results in the reversal ofresistance when used in combination with cytotoxic drugs.
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