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Clinical Cancer Research 13, 6010, October 15, 2007. doi: 10.1158/1078-0432.CCR-07-0140
© 2007 American Association for Cancer Research
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Human Cancer Biology

Canonical Nuclear Factor {kappa}B Pathway Inhibition Blocks Myeloma Cell Growth and Induces Apoptosis in Strong Synergy with TRAIL

Mathilde Romagnoli1,2, Grégoire Desplanques1,2, Sophie Maïga1,4, Steven Legouill1,3, Michel Dreano5, Régis Bataille1,2,4 and Sophie Barillé-Nion1,2

Authors' Affiliations: 1 INSERM, UMR 601, 2 Université de Nantes, Faculté de Médecine, 3 Department of Clinical Hematology, CHR Nantes, Nantes, France; 4 Centre de Lutte contre le Cancer Rene Gauducheau, Saint Herblain, France; and 5 Merck Serono International SA, Geneva, Switzerland

Requests for reprints: Sophie Barillé-Nion, UMR 601, 9 quai Moncousu, Nantes F-44093, France. Phone: 33-24008-4766; Fax: 33-24008-4778; E-mail: sbarille{at}nantes.inserm.fr.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Intrinsic activation of nuclear factor {kappa}B (NF-{kappa}B) characterizes various hematologic malignancies. In this study, we specifically address the role of NF-{kappa}B blockade in mediated antimyeloma activity using the I{kappa}B kinase-2 pharmacologic inhibitor, AS602868.

Experimental Design: Human myeloma cell lines (n = 16) and primary myeloma cells (n = 10) were tested for their sensitivity to AS602868 in terms of proliferation and apoptosis. Both in vitro and in vivo experiments were conducted. Functional mechanisms regarding the apoptotic pathways triggered by AS602868 were studied. The potential proapoptotic synergy between AS602868 and tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) was also evaluated.

Results: Our results show that AS602868 efficiently targeted the canonical NF-{kappa}B pathway in myeloma cells and potently inhibited their growth in inducing apoptosis through Bax and caspase-3 activation. AS602868 also induced apoptosis in primary myeloma cells even in the presence of bone marrow mononuclear cells. Moreover, the I{kappa}B kinase-2 inhibitor targeted the paracrine effect on the bone marrow environment. Indeed, it decreased the intrinsic and myeloma-induced secretion of interleukin-6 from bone marrow stromal cells. In addition, AS602868 inhibited myeloma cell growth in the MM.1S xenograft myeloma model. Of particular interest, AS602868 strongly increased myeloma sensitivity to TRAIL in blocking TRAIL-induced NF-{kappa}B activation and in decreasing the expression of antiapoptotic proteins such as cFLIP and cIAP-1/2.

Conclusions: Taken together, our data point out the interest to inhibit the canonical NF-{kappa}B pathway in myeloma and clearly encourage clinical evaluation of novel therapies based on targeting NF-{kappa}B, especially in combination with TRAIL.

The nuclear factor {kappa}B (NF-{kappa}B) transcription factors are assembled through the dimerization of five members of the Rel protein family, of which p50-p65 is the most common. In resting cells, NF-{kappa}B dimers are retained in the cytoplasm in a latent, inactive form by I{kappa}B family inhibitors, which bind to the rel-homology domain and interfere with the nuclear localization sequence of NF-{kappa}B (1, 2). Various stimuli activate the NF-{kappa}B signaling pathways leading to translocation of NF-{kappa}B dimers from the cytoplasm to the nucleus. In the canonical pathway, proinflammatory cytokines such as the tumor necrosis factor {alpha} (TNF{alpha}), working through different receptors belonging to the TNF-receptor family, activate the I{kappa}B kinase (IKK) complex consisting of the IKK1 (or IKK{alpha}) and IKK2 (or IKKß) catalytic subunits and the IKK{gamma} regulatory subunit (or NEMO). This complex predominantly acting through IKK2 (IKKß) catalyzes the phosphorylation of I{kappa}Bs (at sites equivalent to Ser32 and Ser36 of I{kappa}B{alpha}), their polyubiquitination and subsequent degradation by the 26S proteasome. The released NF-{kappa}B dimers, typically heterodimers of p50 and p65 subunits, translocate to the nucleus where it binds to specific DNA sequences in the promotors of target genes and modulates their transcription. Recently, an alternative NF-{kappa}B pathway, strictly dependent on IKK1 was described. This pathway, based on processing of the NF-{kappa}B2/p100 precursor, is activated in response to either the engagement of LTß and BAFF receptors or CD40 and may play a central role in the development and maintenance of secondary lymphoid organs (2).

Constitutive activation of NF-{kappa}B was reported for various solid and hematologic tumors (3, 4). Actually, NF-{kappa}B promotes cell survival through the expression of genes coding for antiapoptotic proteins, i.e., c-FLIP, members of the Bcl-2 family, or the inhibitor of apoptosis proteins (IAP) and stimulates cell proliferation via the induction of growth factors, i.e., c-myc or cell cycle regulators (5, 6). Importantly, most actual anticancer drugs activate the NF-{kappa}B pathway, an event that interferes with their efficiency and participates in chemoresistance (7). Therefore, it has been proposed that inhibition of NF-{kappa}B could be an adjuvant therapy for cancer.

Among hematologic malignancies, constitutive activation of the NF-{kappa}B pathway was observed in acute myeloid leukemia (8), and in a subgroup of lymphomas with diffuse large and primary mediastinal B-cell lymphomas (9). NF-{kappa}B activation also contributes to malignant transformation of myelodysplastic syndromes (10). Interestingly, Endo et al. recently showed that inhibition of the canonical NF-{kappa}B pathway in B-CLL cells induced cell death in contrast to inhibition of the alternative pathway activated by the TNF superfamily member, BAFF (11). Regarding multiple myeloma (MM), several studies revealed the expression of constitutively active NF-{kappa}B in bone marrow aspirates from patients (12–14). However, the underlying mechanisms for expression of intrinsically activated NF-{kappa}B in MM are poorly understood. NF-{kappa}B could be activated in an autocrine or paracrine manner because several NF-{kappa}B–inducing cytokines including TNF{alpha}, lymphotoxin, BAFF/APRIL, interleukin-1, and RANKL are produced either by MM cells or by the bone marrow environment. Indeed, the principal site of tumor growth and propagation of MM is the bone marrow compartment, in which cells from the environment are thought to supply growth and survival factors and to provide contact-mediated drug resistance. Chauhan et al. previously reported that MM cell adhesion to bone marrow stromal cells (BMSC) induced NF-{kappa}B–dependent up-regulation of transcription of interleukin-6 (IL-6), a major growth and antiapoptotic factor in MM (15). They further showed that blocking NF-{kappa}B activation in MM cells overcame the growth and survival advantage conferred by tumor cell binding to BMSC (13). Despite the successful clinical development of immunomodulatory derivatives of thalidomide and proteasome inhibitors acting partially through NF-{kappa}B inhibition, such as bortezomib, the search for additional new agents and effective strategies against myeloma that is still an incurable cancer, remains a high priority. The TNF-related apoptosis-inducing ligand (TRAIL) emerges as a very attractive death ligand in targeted MM therapy because it is able to induce apoptosis in myeloma cells through caspase-8 activation (16, 17). However, TRAIL resistance has also been observed in myeloma cells but mechanisms underlying this resistance remain elusive (18, 19).

Therefore, for its potential use in the treatment of MM, we investigated the pharmacologic inhibitor of the canonical NF-{kappa}B pathway, AS602868, which has the ability to block IKK2. We describe here that AS602868 decreased in vitro myeloma cell growth related to cell cycle blockade and induction of apoptosis through the classical apoptotic pathway, targeted the paracrine effects of the bone marrow environment, inhibited the in vivo myeloma tumor progression, and sensitized cells to TRAIL-induced cell death in preventing TRAIL-induced NF-{kappa}B activation. Therefore, specific blockade of NF-{kappa}B signaling may represent a novel potent therapeutic approach in the treatment of MM, particularly in combination with TRAIL-targeted therapy.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Characteristics of AS602868. AS602868 is an anilinopyrimidine derivative and an ATP competitor that has been optimized through chemical modification after the in vitro screening of compounds for their inhibitory effect on IKK2EE, a constitutive form of IKK2. This compound was developed by Merck Serono International SA and is covered by patent application no. PCT WO 02/46171. AS602868 has an in vitro inhibitory concentration of 50% (IC50) of 60 nmol/L toward purified IKK2EE and has limited or no effect on IKK1 or on a panel of recombinant kinases. In a series of tests on different cell lines, AS602868 was shown to block the phosphorylation of I{kappa}B and subsequent NF-{kappa}B activation (20).

Myeloma cells and BMSC culture. The human myeloma cell lines (HMCL) XG1, XG2, XG5, XG6, XG7, NAN1, NAN3, NAN6, MDN, and SBN have been previously established in our laboratory and were cultured in the presence of 3 ng/mL of recombinant human IL-6 (rhIL6; Novartis; ref. 21). U266, LP1, L363, RPMI 8226, and NCI-H929 HMCLs are commercially available. MM.1S was a gift from Dr. S.T. Rosen (Northwestern University, Chicago, IL). Cell lines were maintained in RPMI 1640 supplemented with 5% FCS, 2 mmol/L of glutamine, and 5 x 10–5 mol/L of 2-ß-mercaptoethanol.

Primary myeloma cells were obtained from bone marrow aspirates or peripheral blood samples and BMSC from bone marrow samples from patients with MM following Ficoll-Hypaque density gradient centrifugation. For 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, myeloma cells were further purified using CD138 immunomagnetic beads (Miltenyi Biotec) as previously described (22). BMSC were derived from confluent adherent cell monolayer obtained from total bone marrow cells after two passages using trypsin/EDTA solution. Informed consent was provided according to the Helsinki Declaration of the World Medical Association. The University Hospital of Nantes Review Board approved these studies.

Tritiated [3H]thymidine incorporation assay. HMCLs (104 cells/well) were cultured in triplicate in 96-well plates in the presence or absence of AS602868 for 72 h. Cells were pulsed with 1 µCi of [3H]thymidine during the last 8 h of culture, harvested onto glass filters with an automatic cell harvester (Perkin-Elmer), and the uptake of [3H]thymidine was monitored using a 1450 Microbeta Jet beta counter (Perkin-Elmer). The IC50 for [3H]thymidine incorporation was calculated for each HMCL. These experiments were done thrice and a mean ± SD of inhibition was presented.

Cell viability assay. Viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. After 48 h of treatment with 3 x 104 primary purified myeloma cells with increasing (5, 10, and 20 µmol/L) AS602868 concentrations, cells were incubated with 50 µL of 2.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma-Aldrich) for 210 min. Absorption at the 570 nm wavelength was measured after total solubilization of formazan crystals by 100 µL of lysis solution. The inhibition of cell viability induced by the drug was tested in triplicate and expressed as a percentage compared with the untreated corresponding cells.

Apoptosis assay. The percentages of apoptosis were assessed by flow cytometry analysis of Apo2.7-PE staining (FACScalibur, Beckman Coulter) after 48 h of treatment. AS602868 treatment was done at a concentration of 10 µmol/L. In the experiments using the pancaspase inhibitor z-VAD-fmk (Promega), cells were incubated with the caspase inhibitor (50 µmol/L) for 1 h, then AS602868 was added for 24 h. In TRAIL experiments, HMCLs or primary myeloma cells were incubated for 48 h with 5 µmol/L of AS602868 and/or recombinant TRAIL (R&D Systems) at 100 ng/mL for XG6, LP1, U266, MM.1S, and L363, or at 10 ng/mL for more sensitive cell lines NCI-H929, RPMI 8226, XG7, NAN1, and NAN3. Total bone marrow cells from patients with MM were cultured with RPMI 1640 complemented with 5% FCS and 3 ng/mL of rhIL6, and after 48 h of incubation with or without TRAIL (100 ng/mL) in the presence or absence of 10 µmol/L of AS602868, cells were stained with anti–CD38-APC, CD138-PC5, CD45-FITC, and Apo2.7-PE monoclonal antibodies (Becton Dickinson) before flow cytometry analysis, as previously described (23).

Binding activity of nuclear p50 NF-{kappa}B subunit by ELISA. NF-{kappa}B activation was determined with a Trans-AM NF-{kappa}B p50 transcription factor assay kit (Panomics) according to the manufacturer's instructions. This ELISA used a 96-well plate coated with an oligonucleotide containing the NF-{kappa}B consensus binding site (5'-GGGACTTTCC-3'). Cells were treated for 18 h with 10 µmol/L of AS602868, then nuclear extracts were prepared with Panomics nuclear extraction kit and added to the ELISA plate. NF-{kappa}B binding to the target oligonucleotide was detected by incubation with primary antibodies specific for the activated form of the p50 subunit, visualized by anti-IgG horseradish peroxidase conjugate, and quantified at 450 nm. Each condition was run in triplicate.

Subcellular p65 NF-{kappa}B subunit localization by immunofluorescence. HMCLs were treated with or without AS602868 at 10 µmol/L for 3 h, then spotted on a glass slide, air-dried 15 min at room temperature, fixed with PBS + 4% formaldehyde, and permeabilized with PBS + 0.05% Tween 20 + 0.05% Triton 100. After saturation in PBS/3% bovine serum albumin for 30 min, slides were incubated for 1 h with anti-human p65 antibody (1:100; Santa Cruz Biotechnology Inc.), then cells were washed and incubated for 30 min with an FITC-conjugated anti-rabbit antibody (1:200; Becton Dickinson). Images were collected using a Leica TCS NT microscope with a 63 x 1.3 NA Fluotar objective (Leica).

Intercellular adhesion molecule-1 expression. HMCLs were cultured with recombinant TNF{alpha} (5 ng/mL; R&D Systems) in the presence or absence of AS602868 (10 µmol/L) for 48 h. Cells were then stained with mouse IgG isotype control or FITC-conjugated anti-human intercellular adhesion molecule-1 (ICAM-1; CD54) antibody. ICAM-1 expression was determined using FACScalibur (Becton Dickinson).

Bax activation. For the detection of activated Bax, 5 x 105 cells treated with 10 µmol/L of AS602868 for 48 h and controls, were washed in PBS and fixed using Intra Prep Permeabilization Reagent Kit (R&D Systems) following the recommendations of the manufacturer. Then, cells were incubated with anti-Bax (clone 6A7) antibody or IgG1 isotype control for 20 min. After washing in PBS, cells were incubated with FITC-conjugated anti-mouse antibody (R&D Systems) for 20 min, and resuspended in PBS + 1% formaldehyde. Flow cytometry analysis was done as above.

Immunoblot analysis. Cells (5 x 106) were resuspended in lysis buffer [10 mmol/L Tris-HCl (pH 7.6), 150 mmol/L NaCl, 5 mmol/L EDTA, 2 mmol/L phenylmethylsulfonyl fluoride, 1% Triton X-100, and 2 µg/mL aprotinin]. Antiphosphatases were added for phosphorylated protein detection. After 40 min on ice, lysates were cleared by centrifugation at 12,000 x g for 30 min at 4°C. Protein concentration was measured using bicinchoninic acid (BCA Protein Assay). Fifty micrograms of proteins were loaded for each lane. The proteins were separated by SDS-PAGE and then electrotransferred to polyvinylidene difluoride membranes. Western blot analysis was done by standard techniques with enhanced chemiluminescence detection (Pierce) using anti–caspase-3 (E-8), anti–c-myc (A-14), and anti–Mcl-1 (S-19) from Santa Cruz Biotechnology Inc.; anti–PARP-1 (Ab-2) from Calbiochem; anti–phosphorylated-I{kappa}B{alpha} (Ser32/36; 5A5) and anti-I{kappa}B{alpha} from Cell Signaling Technology; anti–c-FLIP (NF6) from Alexis Biochemicals; anti-XIAP, anti–cIAP-1, anti–cIAP-2, and anti–Bcl-xL from BD Biosciences; anti–Bcl-2 from Dako; and anti-actin from Chemicon International. Protein loading was controlled with anti-actin.

Determination of IL-6 production. BMSC (104) and MM.1S (5 x 105) cells were cultured separately or together (coculture) and were incubated for 48 h in the presence or absence of AS602868 (10 µmol/L). Then culture supernatants were harvested and tested by ELISA (Sanquin).

In vivo antitumor activity of AS602868. Six- to 8-week-old nonobese diabetic/severe combined immunodeficiency mice (Charles River Laboratories) were s.c. inoculated in the right flank with 106 MM.1S cells in 100 µL of RPMI 1640. Three to 4 weeks later, when palpable tumors developed, mice (n = 6) were treated with 100 mg/kg of AS602868 in 0.5% carboxymethylcellulose/0.25% Tween 20 (g/100 mL) in distilled water, every day (6 days a week) by oral gavage. Control mice (n = 6) received equal amounts of control vehicle administrated the same way at the same schedule. Tumor size was measured every 3 days in two dimensions using a caliper, and tumor volume (mm3) was calculated as 4 {pi}/3 x (tumor width/2)2 x (tumor length/2). Mice were humanely sacrificed when moribund or when subcutaneous tumors reached a diameter of 25 mm.

Statistical analysis. The Wilcoxon signed rank test was used to calculate the statistical significance between treated and control samples from patients and the Mann-Whitney test was used to compare treated and control mice groups in in vivo experiments. For all other experiments, the results are shown as mean ± SD.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
AS602868 inhibits the canonical NF-{kappa}B pathway in myeloma cells. To confirm that the pharmacologic IKK2 inhibitor AS602868 inhibited the canonical NF-{kappa}B pathway in myeloma cells, we first evaluated the NF-{kappa}B binding activity of the p50 NF-{kappa}B subunit in nuclear extracts from AS602868-treated cells and control cells using ELISA. As shown in Fig. 1A , 10 µmol/L of AS602868 decreased nuclear p50-binding activity by 44.7%, 46.7%, and 30% in NCI-H929, XG6, and MM1.S cells, respectively. As expected, the positive control TNF{alpha} enhanced p50-binding activity in MM1.S cells and AS602868 was able to prevent this effect. These results clearly indicate that AS602868 prevented the translocation of the p50 NF-{kappa}B subunit to the nucleus but promoted its cytosolic retention by interaction with nonphosphorylated I{kappa}B{alpha} protein, limiting NF-{kappa}B binding activity in myeloma cells. To further show the potent activity of AS602868 on the NF-{kappa}B pathway in myeloma cells, we evaluated its effect on TNF{alpha}-induced targets. We first focused on ICAM-1 (CD54) expression. ICAM-1 expression detected by flow cytometry was induced in HMCLs after a 48-h incubation with TNF{alpha}, as shown in Fig. 1B (top) for MM.1S cells. Treatment with AS602868 had no significant effect on ICAM-1 basal levels but it abolished TNF{alpha}-induced ICAM-1 expression. The same results were obtained in XG6 and NCI-H929 cells (data not shown). In addition, TNF{alpha} alone induced a weak apoptosis in MM.1S cells but its apoptotic potential was strongly revealed by AS602868 cotreatment: 70.1% of specific apoptotic cells in AS602868 + TNF{alpha}-treated cells versus 8.8% in TNF{alpha}-stimulated cells (Fig. 1B, bottom). These results indicate that AS602868 blocked the NF-{kappa}B pathway induced by TNF{alpha} in myeloma cells. Altogether, these data strongly argue for the potent activity of AS602868 on the canonical NF-{kappa}B pathway in myeloma cells.


Figure 1
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  		Fig. 1. AS602868 inhibits the NF-{kappa}B pathway in myeloma cells. A, p50 NF-{kappa}B binding activity was evaluated by ELISA in nuclear extracts of NCI-H929, XG6, and MM.1S cells treated with or without AS602868 at 10 µmol/L for 18 h. MM1.S cells were also pretreated for 30 min with or without AS602868 before stimulation with TNF{alpha} at 5 ng/mL for 30 min (columns, means; bars, SD; n = 3). B, MM.1S cells were evaluated for their ICAM-1 (CD54) expression (top) and apoptotic index with Apo2.7 staining (bottom) by flow cytometry analysis after the following culture conditions were established for 48 h: control, TNF{alpha} (5 ng/mL), AS602868 (10 µmol/L), and AS602868 + TNF{alpha} (10 µmol/L + 5 ng/mL). Results are from a representative experiment (n = 3).

 
AS602868 inhibits in vitro myeloma cell growth. We then asked whether the pharmacologic IKK2 inhibitor AS602868 affected the growth of HMCLs. Sixteen HMCLs were incubated with increasing AS602868 doses and were evaluated by [3H]thymidine incorporation. Then, the IC50 of each HMCL proliferation rate was calculated. As presented in Fig. 2A , AS602868 efficiently decreased HMCL growth with IC50's ranging from 1.2 ± 0.8 µmol/L (NAN3) to 9.2 ± 0.9 µmol/L (RPMI 8226; mean = 3.7 ± 1.75 µmol/L; n = 16). The AS602868-induced inhibition of proliferation was confirmed by cell cycle analysis using propidium iodure staining. Indeed, AS602868 treatment led to the decrease of cell numbers in S phase (data not shown). We further evaluated whether growth inhibition resulted from the induction of apoptosis. Drug-induced apoptosis was evaluated by Apo2.7 staining after treatment with 10 µmol/L of AS602868 for 48 h. We showed that the IKK2 inhibitor induced a specific apoptosis in HMCLs (mean = 30.4 ± 14.3%; n = 15) ranging from 8 ± 3.2% (MM.1S) to 53.6 ± 10.6% (NAN3; Fig. 2B). These results clearly show that myeloma cell growth inhibition induced by AS602868 resulted from both inhibition of cell cycle progression and apoptosis induction.


Figure 2
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  		Fig. 2. AS602868 inhibits proliferation and induces apoptosis in myeloma cells. A, IC50 was calculated for each HMCL as the concentration inhibiting 50% of [3H]thymidine incorporation after 72 h of incubation with increasing concentrations of AS602868 (columns, means; bars, SD; n = 3). B, HMCLs were incubated with AS602868 at 10 µmol/L for 48 h, then apoptosis was evaluated by Apo2.7 staining and analyzed by flow cytometry. Specific drug-induced apoptosis for each HMCL was defined as the percentage of Apo2.7-positive cells in treated versus nontreated conditions (columns, means; bars, SD; n = 3). C, the effect of AS602868 at 5, 10, and 20 µmol/L on CD138+-purified primary myeloma cell viabilities were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Triplicates of CD138+ purified primary myeloma cells from three patients (a, b, and c) were presented (columns, means; bars, SD).

 
Cell growth inhibition induced by the IKK2 inhibitor was further assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay on CD138+-purified primary myeloma cells from patients with MM. As shown in Fig. 2C, increasing concentrations of AS602868 inhibited myeloma cell viability in samples from three patients (two at diagnosis and one on relapse). Cell viability inhibition displayed a dose-response effect, arguing for a drug-specific effect. Moreover, apoptosis analysis of total bone marrow mononuclear cells from patients with MM indicated that AS602868 at 10 µmol/L induced specific apoptosis in a series of myeloma samples (n = 10), five patients at diagnosis and five patients at relapse (median = 18%; range, 6-53; P < 0.01), as assessed by flow cytometry analysis (Table 1 ). Interestingly, four out of five cases of MM relapse showed AS602868 sensitivity. It should be noted that these experiments were done in the presence of rhIL6 to limit spontaneous in vitro apoptosis of primary myeloma cells (median = 5%).


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  		Table 1. AS602868 induced apoptosis in primary myeloma cells

 
AS602868 induces apoptosis in myeloma cells. To gain insight into the apoptotic pathways activated by AS602868 in myeloma cells, we first sought to establish Bax activation. We found that in HMCLs, the IKK2 inhibitor induced the exposure of an occluded NH2-terminal sequence of Bax, reflecting the conformational change which occurs during its activation. The activation of Bax was detected in XG6 cells as depicted in one representative experiment in Fig. 3A , as well as in the other HMCLs tested. We further assessed caspase-3 cleavage by immunoblot analysis in AS602868-treated HMCLs. As shown in Fig. 3B, cleaved caspase-3 was detected in XG6 treated by AS602868 for 48 h. Caspase-3 cleavage could be detected as soon as 6 h after treatment with AS602868 (data not shown). In addition, the cleavage of the caspase-3 substrate PARP-1 was also detected in treated cells in contrast to control cells. Finally, the pancaspase inhibitor z-VAD-fmk efficiently abrogated both caspase-3 and PARP-1 cleavage as well as cell death induced by AS602868 (Fig. 3B). These results show that the IKK2 inhibitor, AS602868, activated a caspase-dependent cell death pathway in myeloma cells.


Figure 3
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  		Fig. 3. AS602868 induces apoptosis through Bax activation, targets the bone environment, and inhibits tumorigenesis. A, Bax activation was assessed in XG6 cells by flow cytometry analysis using the 6A7 clone monoclonal antibody specifically recognizing the activated form of Bax. Cells were treated for 48 h with or without AS602868 at 10 µmol/L. The percentage of Bax-positive cells is indicated. Thin lines, IgG1 isotypic control; thick lines, conformationally active Bax. B, caspase-3 and PARP-1 cleavages were determined by immunoblot analysis in XG6 cells treated by AS602868 (10 µmol/L) for 48 h, preincubated or not for 1 h with the pancaspase inhibitor z-VAD-fmk (50 µmol/L), in comparison to nontreated control cells. Actin was used as the quantitative control of loaded proteins. The percentage of specific cell death is indicated in both conditions compared with control condition. C, IL-6 production was measured by ELISA in supernatants of BMSC culture and BMSC + MM.1S coculture in the presence or absence of AS602868 at 10 µmol/L for 48 h. Two independent experiments were done in triplicate (columns, means; bars, SD). D, myeloma (MM.1S) bearing nonobese diabetic/severe combined immunodeficiency mice were treated for 24 d as indicated in the Materials and Methods. Two groups of mice received either the IKK2 inhibitor (n = 6) or the corresponding vehicle (n = 6). Tumor size was measured in two dimensions using a caliper, and tumor volume (mm3) was calculated. Tumor sizes were calculated in each group at each time point (points, means; bars, SD). *, P < 0.05; **, P < 0.01.

 
AS602868 decreases paracrine IL-6 production in the bone marrow environment. Because IL-6 is a major paracrine growth factor in MM, we next sought to establish the effect of AS602868 on IL-6 production by BMSC either alone or in coculture with MM.1S cells. We observed that AS602868 significantly decreased the constitutive production of IL-6 proteins by BMSC (decrease of 23.9% and 38.8% in the two representative experiments shown in Fig. 3C). Interestingly, the IKK2 inhibitor also strongly inhibited IL-6 production in BMSC/MM.1S coculture experiments (decrease of 51.8% and 90.5% in the two representative experiments shown in Fig. 3C).

AS602868 inhibits in vivo myeloma cell growth. Finally, the effect of the IKK2 inhibitor was evaluated in vivo on an established MM xenograft model (MM.1S) in nonobese diabetic/severe combined immunodeficiency mice. Both groups of mice (control and AS602868-treated groups) were monitored over a period of 24 days and mean tumor size values were reported in Fig. 3D. AS602868 administrated by oral gavage was well tolerated in mice at a dose of 100 mg/kg. Importantly, AS602868 significantly decreased myeloma tumor size from days 18 to 24 of treatment (P < 0.05 at day 18 and P < 0.01 at days 21 and 24).

AS602868 synergizes with TRAIL to induce apoptosis in myeloma cells. Although TRAIL is considered as an attractive target in MM therapy, MM cells are inequally sensitive to TRAIL-induced apoptosis. Then, we tested whether the IKK2 inhibitor, AS602868, might sensitize them to TRAIL's apoptotic effect. HMCLs were incubated with recombinant TRAIL (100 or 10 ng/mL, depending on the TRAIL sensitivity of each HMCL) for 48 h in the presence or absence of AS602868 at the suboptimal dose of 5 µmol/L. Apoptotic cells were then assessed by Apo2.7 immunostaining and flow cytometry analysis. As shown in Fig. 4A , in all HMCLs tested (n = 10), AS602868 strongly increased HMCL response to TRAIL-induced apoptosis (mean = 19.3 ± 16.6% of apoptotic cells in TRAIL-treated HMCLs versus 47.4 ± 17.2% in TRAIL + AS602868-treated HMCLs, whereas AS602868 induced 9 ± 4.2% of apoptotic cells in this panel).


Figure 4
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  		Fig. 4. AS602868 synergizes with TRAIL to induce cell death in myeloma cells. A, HMCLs were incubated with recombinant TRAIL (100 ng/mL for XG6, LP1, U266, MM.1S, and L363 or 10 ng/mL for NCI-H929, RPMI 8226, XG7, NAN1, and NAN3) in combination or not with AS602868 (5 µmol/L) for 48 h. After flow cytometry analysis, the percentage of Apo2.7-positive cells was calculated for each treated HMCL compared with nontreated control cells (columns, means; bars, SD; n = 3). B and C, total bone marrow mononuclear cells from four patients with MM were incubated with or without AS602868 (10 µmol/L) in the presence or absence of TRAIL (100 ng/mL) for 48 h. Then the proportion of apoptotic myeloma cells was measured by specific coexpression of CD38- and CD138-targeting myeloma cells and Apo2.7 as analyzed by flow cytometry. Four different samples from patients (patients 1-4) with MM were presented in the histogram (B) and one patient (patient 2) in the dot plot (C). AS, AS602868.

 
Interestingly, as presented for four patients in Fig. 4B, AS602868 also synergized with TRAIL to specifically induce apoptosis in primary myeloma cells incubated with total bone marrow cells. Dot plots obtained by flow cytometry analysis for patient 2 illustrated specific apoptosis triggered by drugs in Fig. 4C.

Moreover, caspase-3 cleavage detectable in AS602868-treated XG6 cells but not in TRAIL-treated cells, was strongly enhanced in TRAIL + AS602868–treated cells (Fig. 5B ). These results show that TRAIL and the IKK2 inhibitor strongly synergize to induce myeloma cell death.


Figure 5
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  		Fig. 5. AS602868 promotes TRAIL-induced apoptosis through inhibition of TRAIL-induced NF-{kappa}B activation and decrease of NF-{kappa}B regulated protein expression. A, subcellular localization of p65 NF-{kappa}B subunits was evaluated by immunofluorostaining and monitored by confocal microscopy. XG6 cells were treated with or without AS602868 at 10 µmol/L for 18 h before the addition or not of TRAIL at 100 ng/mL for 1 h. Results from a representative experiment (n = 3) are shown. B, level of NF-{kappa}B activation (P-I{kappa}B{alpha}) and expression of the proliferative protein (c-myc) and proapoptotic molecules (caspase-3, cFLIPL, XIAP, cIAP-1/2, Mcl-1, Bcl-xL, and Bcl-2) were determined by immunoblot analysis in XG6 cells treated with or without AS602868 (5 µmol/L), TRAIL (100 ng/mL), or the combination of AS602868 + TRAIL for 48 h. Results from a representative experiment (n = 3) are shown. *, cleaved form of caspase-3, XIAP, and Mcl-1.

 
To better understand the mechanisms involved in the apoptotic synergy between TRAIL and AS602868, we evaluated the ability of TRAIL to induce NF-{kappa}B activation in MM cells. In TRAIL- and/or AS602868-treated cells, we also assessed p65 localization, the phosphorylation levels of I{kappa}B{alpha} protein (P-I{kappa}B{alpha}), and the expression levels of various NF-{kappa}B–regulated proteins which play a role in cell proliferation (c-myc) or in tumor survival, and chemoresistance or radioresistance (c-FLIPL, XIAP, cIAP-1, cIAP-2, Mcl-1, Bcl-xL, and Bcl-2). Confocal microscopy analysis first indicated intrinsic NF-{kappa}B activation because p65 was found in the nucleus as well as in the cytoplasm of control cells. Second, this experiment suggested that TRAIL promoted p65 nuclear localization and that AS602868 abrogated both basal and TRAIL-induced p65 translocation (Fig. 5A). Moreover, immunoblot analysis showed that TRAIL treatment strongly enhanced P-I{kappa}B{alpha} protein levels in MM cells, as shown in XG6 cells, indicating that TRAIL was indeed able to activate NF-{kappa}B pathway in MM cells (Fig. 5B). Importantly, AS602868, which alone decreased P-I{kappa}B{alpha} level, completely abrogated the induction of TRAIL-dependent P-I{kappa}B{alpha} levels. Interestingly, AS602868 also significantly decreased the NF-{kappa}B–regulated proteins c-myc, c-FLIPL, and cIAP-1/2 in these cells, as well as slightly decreasing Mcl-1 and XIAP levels, at least by cleavage of the full-length protein (asterisks on Fig. 5B). TRAIL and AS602868 combination strongly decreased the expression of c-myc and of the antiapoptotic proteins c-FLIPL, XIAP, cIAP-1/2, Mcl-1, and Bcl-xL but not Bcl-2. Similar results were obtained in other HMCLs. These results argue for the potent synergy of the TRAIL/AS602868 combination for the extinction of antiapoptotic and proliferative proteins.


   Discussion
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Constitutive activation of NF-{kappa}B has been described in many hematologic malignancies leading to the proposition that targeting the NF-{kappa}B pathway may provide benefits in therapeutic strategies, particularly in oncohematology. In the present study, we show that the IKK2 inhibitor AS602868 decreases myeloma cell growth both in vitro and in vivo, targets the paracrine effects of the bone marrow environment, and sensitizes myeloma cells to TRAIL proapoptotic effects. These results strongly argue for further studies to suppress NF-{kappa}B activity as a MM therapeutic strategy.

We first show the ability of AS602868 to interfere with NF-{kappa}B activation in myeloma cells. Indeed, this inhibitor decreased either NF-{kappa}B–binding activity or nuclear translocation of both NF-{kappa}B subunits p50 and p65, respectively. It also decreased constitutive phospho-I{kappa}B{alpha} levels. These data corroborate those of Frelin et al. (8) who showed the decrease of NF-{kappa}B activation in acute myeloid leukemia cells treated with AS602868, and by electrophoretic mobility shift assay in parallel with the inhibition of IKK2 phosphorylation. In addition, AS602868 completely blocked TNF{alpha}-induced expression of ICAM-1 on myeloma cells, a well-known target of NF-{kappa}B (24). Moreover, the apoptotic effect of TNF{alpha} on myeloma cells was revealed by AS602868 treatment as previously observed in Jurkat cells (20). These results underscore the effect of TNF{alpha} in both caspase-8–dependent proapoptotic and NF-{kappa}B–dependent survival pathways in myeloma cells and that the NF-{kappa}B inhibitor tilts the balance towards apoptosis. Taken together, these results strongly argue for potent blockade of the canonical NF-{kappa}B pathway by AS602868 in myeloma cells.

Interestingly, AS602868 seemed to have an important inhibiting activity on myeloma cell growth, in a range of concentrations close to those determined in acute myeloid leukemia cells (0.6-14 µmol/L; ref. 8). Importantly, AS602868 also inhibited the cell growth of primary myeloma cells even in the presence of exogenous IL-6 or in patients in relapse. Thus, the canonical NF-{kappa}B pathway clearly takes part in myeloma cell growth as recently described in B-CLL (11). In addition, in our study, AS602868 specifically induced apoptosis in primary myeloma cells among total bone marrow mononuclear cells, suggesting that this molecule may selectively target myeloma cells. Nevertheless, further experiments would be necessary to precisely define the sensibility of CD34+ hematopoietic stem cells. Actually, in an abstract presented at the 2006 American Society of Hematology meeting, Jourdan et al. noticed that the IKK2 inhibitor induced growth inhibition in myeloma cells but also altered the survival of CD34+ cells among bone marrow cells (25). However, this potential cytotoxic effect of AS602868 was not reported by Frelin et al. in acute myeloid leukemias (8).

Decreased cell growth observed in myeloma cells was associated with the induction of apoptosis. To define the molecular apoptotic mechanisms triggered by AS602868, we studied Bax and caspase-3 activation and we showed that both mechanisms were induced. In addition, the pancaspase inhibitor z-VAD-fmk protected myeloma cells from AS602868-induced cell death as well as caspase-3 and PARP-1 cleavage. Moreover, AS602868 efficiently decreased the expression of endogenous inhibitors of caspases cIAP-1/2 and promoted cleavage in inactive fragments of both XIAP and Mcl-1, the most potent endogenous caspase inhibitor and the major regulator of mitochondrial proapoptotic activity in MM cells, respectively (26). It also inhibited the expression of c-FLIPL, which is an important NF-{kappa}B–regulated antiapoptogenic protein involved in TRAIL resistance through the suppression of either recruitment of procaspase-8 by FADD or autocatalytic activation of caspase-8. Altogether, these data indicate that inhibition of NF-{kappa}B in myeloma cells activated the mitochondrial cell death pathway leading to caspase cascade activation and caspase-dependent cell death and may promote an extrinsic caspase-8–dependent apoptotic pathway. In acute myeloid leukemia blasts, AS602868 also induced a strong apoptotic response related to mitochondrial potential failure and activation of caspases (8). Finally, the proliferative c-myc protein level was also down-regulated and might favor cell growth inhibition by AS602868. In addition, we observe that AS602868 targeted the bone marrow environment in decreasing paracrine IL-6 production by BMSC which supports myeloma cell growth. Importantly, we show that AS602868, by itself, inhibits tumor size in a xenograft model of MM.1S myeloma cells when orally administrated, without apparent toxicity.

TRAIL can selectively induce apoptosis in cancer cells and emerges as an attractive targeted therapy in MM. However, heterogenous TRAIL sensitivity has been previously observed in HMCLs (16, 18, 19, 27), but the mechanisms underlying TRAIL resistance have not been resolved. In addition to inducing apoptosis by caspase-8 recruitment through FADD, TRAIL binding to its receptors might also lead to NF-{kappa}B activation through TRADD (28, 29). We then wondered whether the combination of TRAIL and AS602868 might induce synergy in death response in MM cells. We indeed show that interrupting the NF-{kappa}B pathway with AS602868 strongly sensitized myeloma cells to lethal action of TRAIL in HMCLs and in primary myeloma cells, and even overcame TRAIL resistance. These effects were also observed when using the agonist monoclonal antibody HGS-ETR1 or HGS-ETR2, targeting the TRAIL receptors DR4 and DR5, respectively, in combination with AS602868 (data not shown). Interestingly, we point out for the first time that TRAIL potently activated the NF-{kappa}B pathway in MM cells. Indeed, NF-{kappa}B activation was detectable as early as 1 hour after TRAIL treatment (data not shown). Importantly, NF-{kappa}B inhibitor completely prevented cells from TRAIL-induced NF-{kappa}B activation. Therefore, inhibition of TRAIL-induced NF-{kappa}B activation by AS602868 associates with high increase of TRAIL proapoptotic capacity. Strongly decreased expression of the antiapoptotic proteins c-FLIPL, XIAP, cIAP-1/2, Mcl-1, and Bcl-xL was detected in MM cells when treated by the drug combination. XIAP and Mcl-1 decrease resulted from the cleavage of the full-length corresponding proteins, as previously observed for Mcl-1 (19). Of note, it has been recently shown that silencing c-FLIPL sensitized myeloma cells to TRAIL (30). Moreover c-FLIPL levels also regulate TRAIL sensitivity in mantle cell lymphoma B cells and the decrease of c-FLIPL by IKK inhibition sensitized these cells to TRAIL (31). Such an effect may occur in myeloma cells treated with AS602868, which decreased c-FLIPL expression, promoting caspase-8–dependent apoptotic pathways induced by TRAIL. In contrast to the proteasome inhibitor bortezomib that up-regulates DR5 expression (32), expression of the TRAIL receptors DR4 and DR5 was not modulated by the NF-{kappa}B inhibitor (data not shown). Altogether, these results point out that TRAIL resistance occurs at least through the activation of the canonical NF-{kappa}B pathway in myeloma cells, and suggests that AS602868 enhanced TRAIL's apoptotic effect in myeloma cells at least through c-FLIPL down-regulation. In addition, some reports showed interest in inhibiting NF-{kappa}B in order to increase drug sensitivity in myeloma cells. Indeed, Dai et al. previously reported that the pharmacologic NF-{kappa}B inhibitor Bay-11-7082 increased the sensitivity of myeloma cells to the checkpoint inhibitor UCN-01 (33). Bharti et al. also showed that curcumin (diferuloylmethane) inactivated NF-{kappa}B and potentiated the cytotoxic effects of chemotherapeutic agents (34), and Mitsiades et al. reported that the NF-{kappa}B inhibitory peptide SN50 enhanced doxorubicine's effect on myeloma cells (35). These effects might be related to the NF-{kappa}B activation frequently induced by drugs in treated cells.

Altogether, our data underscore the central role for NF-{kappa}B in MM. Indeed, NF-{kappa}B participated in (a) myeloma cell survival, (b) tumorigenesis, (c) resistance to TRAIL-induced apoptosis, and (d) in protumoral activation of the bone marrow environment. These findings suggest that a strategy combining anti-MM drugs with NF-{kappa}B inhibitors warrants attention in the treatment of MM and may constitute an additional approach to the oncologist's armamentarium.


   Acknowledgments
 
We thank Viviane Dubruille for providing bone marrow samples from patients with MM, Céline Seveno for her help in confocal analysis, and GEFLUC for financial support.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 1/18/07; revised 6/ 7/07; accepted 7/20/07.


   References
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 Materials and Methods
 Results
 Discussion
 References
 

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