We have shown that thalidomide (Thal) and its immunomodulatory derivatives (IMiDs), proteasome inhibitor PS-341, and As2O3 act directly on multiple myeloma (MM) cells and in the bone marrow (BM) milieu to overcome drug resistance. Although Thal/IMiDs, PS-341, and As2O3 inhibit nuclear factor (NF)-κB activation, they also have multiple and varied other actions. In this study, we therefore specifically address the role of NF-κB blockade in mediating anti-MM activity. To characterize the effect of specific NF-κB blockade on MM cell growth and survival in vitro, we used an IκB kinase (IKK) inhibitor (PS-1145). Our studies demonstrate that PS-1145 and PS-341 block TNFα-induced NF-κB activation in a dose- and time-dependent fashion in MM cells through inhibition of IκBα phosphorylation and degradation of IκBα, respectively. Dexamethasone (Dex), which up-regulates IκBα protein, enhances blockade of NF-κB activation by PS-1145. Moreover, PS-1145 blocks the protective effect of IL-6 against Dex-induced apotosis. TNFα-induced intracellular adhesion molecule (ICAM)-1 expression on both RPMI8226 and MM.1S cells is also inhibited by PS-1145. Moreover, PS-1145 inhibits both IL-6 secretion from BMSCs triggered by MM cell adhesion and proliferation of MM cells adherent to BMSCs. However, in contrast to PS-341, PS-1145 only partially (20–50%) inhibits MM cell proliferation, suggesting that NF-κB blockade cannot account for all of the anti-MM activity of PS-341. Importantly, however, TNFα induces MM cell toxicity in the presence of PS-1145. These studies demonstrate that specific targeting of NF-κB can overcome the growth and survival advantage conferred both by tumor cell binding to BMSCs and cytokine secretion in the BM milieu. Furthermore, they provide the framework for clinical evaluation of novel MM therapies based upon targeting NF-κB. We have shown that thalidomide (Thal) and its immunomodulatory derivatives (IMiDs), proteasome inhibitor PS-341, and As2O3 act directly on multiple myeloma (MM) cells and in the bone marrow (BM) milieu to overcome drug resistance. Although Thal/IMiDs, PS-341, and As2O3 inhibit nuclear factor (NF)-κB activation, they also have multiple and varied other actions. In this study, we therefore specifically address the role of NF-κB blockade in mediating anti-MM activity. To characterize the effect of specific NF-κB blockade on MM cell growth and survival in vitro, we used an IκB kinase (IKK) inhibitor (PS-1145). Our studies demonstrate that PS-1145 and PS-341 block TNFα-induced NF-κB activation in a dose- and time-dependent fashion in MM cells through inhibition of IκBα phosphorylation and degradation of IκBα, respectively. Dexamethasone (Dex), which up-regulates IκBα protein, enhances blockade of NF-κB activation by PS-1145. Moreover, PS-1145 blocks the protective effect of IL-6 against Dex-induced apotosis. TNFα-induced intracellular adhesion molecule (ICAM)-1 expression on both RPMI8226 and MM.1S cells is also inhibited by PS-1145. Moreover, PS-1145 inhibits both IL-6 secretion from BMSCs triggered by MM cell adhesion and proliferation of MM cells adherent to BMSCs. However, in contrast to PS-341, PS-1145 only partially (20–50%) inhibits MM cell proliferation, suggesting that NF-κB blockade cannot account for all of the anti-MM activity of PS-341. Importantly, however, TNFα induces MM cell toxicity in the presence of PS-1145. These studies demonstrate that specific targeting of NF-κB can overcome the growth and survival advantage conferred both by tumor cell binding to BMSCs and cytokine secretion in the BM milieu. Furthermore, they provide the framework for clinical evaluation of novel MM therapies based upon targeting NF-κB. NF-κB is a member of the Rel family of proteins and is typically a heterodimer composed of p50 and p65 subunits (1.Baldwin Jr., A.S. Ann. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5578) Google Scholar). NF-κB is constitutively present in the cytosol and inactivated by its association with IκB family inhibitors (2.Beg A.A. Baldwin A.S.J. Genes Dev. 1993; 7: 2064-2070Crossref PubMed Scopus (738) Google Scholar). IκBα is, therefore, a key molecular target regulating NF-κB activation. Various stimuli, including TNFα, 1The abbreviations used are: TNFαtumor necrosis factor αMMmultiple myelomaBMSCbone marrow stromal cellThalthalidomideIMiDsimmunomodulatory derivatives of thalidomideIL-6interleukin-6IKKIκB kinaseDexdexamethasoneICAM-1intracellular adhesion molecule-1VCAM-1vascular cell adhesion molecule-1MAPKmitogen-activated protein kinaseMEKMAPK kinaseERKextracellular signal regulated kinasesSTATsignal transducers and activators of transcriptionMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromideEMSAelectrophoretic mobility shift analysisAbantibodyELISAenzyme-linked immunosorbent assayPKAprotein kinase AJNKc-Jun NH2-terminal kinaseTdRthymidinePMSFphenylmethylsulfonyl fluoridePI3Kphosphatidylinositol 3-kinase lipopolysaccharide, phorbol esters, and viruses, trigger IκB protein phosphorylation on two serine residues located in its NH2terminus by the multisubunit IκB kinase (IKK) complex. Once phosphorylated, IκB is targeted for ubiquitination and degradation by the 26 S proteasome (3.Zandi E. Karin M. Mol. Cell. Biol. 1999; 19: 4547-4551Crossref PubMed Scopus (307) Google Scholar), allowing translocation of NF-κB into the nucleus where it binds to specific DNA sequences in the promoters of target genes and stimulates their transcription. The protein products of these genes include cytokines, chemokines, cell adhesion molecules, and proteins mediating cellular growth control. Importantly, many of these proinflammatory proteins also act, either in an autocrine or paracrine fashion, to further stimulate NF-κB activation. tumor necrosis factor α multiple myeloma bone marrow stromal cell thalidomide immunomodulatory derivatives of thalidomide interleukin-6 IκB kinase dexamethasone intracellular adhesion molecule-1 vascular cell adhesion molecule-1 mitogen-activated protein kinase MAPK kinase extracellular signal regulated kinases signal transducers and activators of transcription 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide electrophoretic mobility shift analysis antibody enzyme-linked immunosorbent assay protein kinase A c-Jun NH2-terminal kinase thymidine phenylmethylsulfonyl fluoride phosphatidylinositol 3-kinase Many studies have reported growth and anti-apoptotic roles of NF-κB in normal and malignant cells. For example, NF-κB promotes cell growth by up-regulating cyclin D transcription, with associated hyperphosphorylation of Rb, G1- to S-phase transition, and inhibition of apoptosis (4.Baldwin A.S. J. Clin. Invest. 2001; 107: 241-246Crossref PubMed Scopus (1199) Google Scholar). NF-κB also regulates transcription of the catalytic subunit of telomerase in mice (5.Yin L. Hubbard A.K. Giardina C. J. Biol. Chem. 2000; 275: 36671-36675Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). NF-κB is constitutively activated in Hodgkin's tumor cells, whereas inhibition of NF-κB blocks their growth (6.Bargou R.C. Emmerich F. Krappmann D. Bommert K. Mapara M.Y. Arnold W. Royer H.D. Grinstein E. Greiner A. Scheidereit C. Dorken B. J. Clin. Invest. 1997; 100: 2961-2969Crossref PubMed Scopus (699) Google Scholar). Moreover, inhibition of NF-κB via expression of the super-repressor of IκBα induces apoptosis, even in the presence of an oncogenic allele of H-Ras (7.Mayo M.W. Wang C.Y. Cogswell P.C. Rogers-Graham K.S. Lowe S.W. Der C.J. Baldwin Jr., A.S. Science. 1997; 278: 1812-1815Crossref PubMed Scopus (507) Google Scholar). Although the precise role of NF-κB activation in pathogenesis of multiple myeloma (MM) has not been fully characterized, we have previously shown that MM cell adhesion to bone marrow stromal cells (BMSCs) induces NF-κB-dependent up-regulation of transcription of IL-6, a growth and anti-apoptotic factor in MM (8.Chauhan D. Uchiyama H. Akbarali Y. Urashima M. Yamamoto K.I. Libermann T.A. Anderson K.C. Blood. 1996; 87: 1104-1112Crossref PubMed Google Scholar). In addition, TNFα secreted by MM cells: 1) activates NF-κB in BMSCs, thereby directly up-regulating IL-6 transcription and secretion in BMSCs; and 2) activates NF-κB in MM cells, thereby up-regulating intracellular adhesion molecule-1 (ICAM-1) (CD54) and vascular cell adhesion molecule-1 (VCAM-1) (CD106) expression on both MM cells and BMSCs and increasing MM cell to BMSC binding (9.Hideshima T. Chauhan D. Schlossman R. Richardson P. Anderson K.C. Oncogene. 2001; 20: 4519-4527Crossref PubMed Scopus (363) Google Scholar). Because IL-6 is the major growth and survival factor in MM cells (10.Borset M. Waage A. Brekke O.L. Helseth E. Eur. J. Haematol. 1994; 53: 31-37Crossref PubMed Scopus (65) Google Scholar, 11.Chauhan D. Kharbanda S. Ogata A. Urashima M. Teoh G. Robertson M. Kufe D.W. Anderson K.C. Blood. 1997; 89: 227-234Crossref PubMed Google Scholar, 12.Lichtenstein A. Tu Y. Fady C. Vescio R. Berenson J. Cell. Immunol. 1995; 162: 248-255Crossref PubMed Scopus (264) Google Scholar) and adherence of MM cells to fibronectin confers resistance to drug-induced apoptosis (13.Damiano J.S. Cress A.E. Hazlehurst L.A. Shtil A.A. Dalton W.S. Blood. 1999; 93: 1658-1667Crossref PubMed Google Scholar, 14.Hazlehurst L.A. Damiano J.S. Buyuksal I. Pledger W.J. Dalton W.S. Oncogene. 2000; 19: 4319-4327Crossref PubMed Scopus (309) Google Scholar), specific blockade of NF-κB signaling may represent a novel therapeutic strategy in MM. We and others have shown that novel agents with both preclinical and early clinical anti-MM activity, including thalidomide (Thal) (15.Keifer J.A. Guttridge D.C. Ashburner B.P. Baldwin A.S.J. J. Biol. Chem. 2001; 276: 22382-22387Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar) and its immunomodulatory derivatives (IMiDs), 2Mitsiades, N., Mitsiades, C. S., Poulaki, V., Hideshima, T., Chauhan, D., Treon, S. P., and Anderson, K. C. (2002) Blood, in press. proteasome inhibitor PS-341 (17.Palombella V. Conner E. Fuseler J. Destree A. Davis J. Laroux F. Wolf R. Huang J. Brand S. Elliot P. Lazarus D. McCormack T. Parent L. Stein R. Adams J. Grisham M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15671-15676Crossref PubMed Scopus (269) Google Scholar, 18.Hideshima T. Richardson P. Chauhan D. Palombella V.J. Elliot P.J. Adams J. Anderson K.C. Cancer Res. 2001; 61: 3071-3076PubMed Google Scholar), and arsenic trioxide As2O3 (19.Kapahi P. Takahashi T. Natoli G. Adams S.R. Chen Y. Tsien R.Y. Karin M. J. Biol. Chem. 2000; 275: 36062-36066Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar), all inhibit NF-κB activation and overcome conventional drug resistance in preclinical and early clinical trials, supporting this view. However, these novel drugs also have multiple other biologic actions, and the benefit of specifically targeting NF-κB in novel MM therapeutics is not yet defined. In the present study, we demonstrate the specific biologic sequelae of NF-κB activation and blockade on MM cell growth and survival in the BM microenvironment. The novel specific IKK inhibitor PS-1145 inhibits phosphorylation of IκBα in MM cells and modestly directly inhibits their growth. Importantly, however, PS-1145 abrogates NF-κB activation related up-regulation of adhesion molecules on MM cells, MM cell to BMSC adherence, as well as the MM cell growth and survival advantage conferred both by adherence and cytokine secretion in the BM milieu. Furthermore, PS-1145 blocks the protective effect of IL-6 against apoptosis induced by both conventional (dexamethasone, Dex) and novel (immunomodulatory derivative of Thal 3, IMiD3) therapies. These studies therefore confirm a central role for NF-κB in regulating growth and survival of MM cells in the BM milieu and further suggest the potential utility of novel therapeutics targeting NF-κB in MM. The Dex-sensitive human MM cell line MM.1S was kindly provided by Dr. Steven Rosen (Northwestern University, Chicago, IL). RPMI8226 and U266 human MM cells were obtained from American Type Culture Collection (Rockville, MD). All MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum (Sigma Chemical Co., St. Louis, MO), 2 μml-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin (Gibeo, Grand Island, NY). Patient MM cells were purified from patient BM aspirates using RosetteSep separation system (StemCell Technologies, Vancouver, Canada). The purity of MM cells was confirmed by flow cytometry using PE-conjugated anti-CD138 Ab (BD PharMingen, San Diego, CA). BM specimens were obtained from patients with MM. Mononuclear cells separated by Ficoll-Hypaque density sedimentation were used to established long term BM cultures, as previously described (20.Uchiyama H. Barut B.A. Mohrbacher A.F. Chauhan D. Anderson K.C. Blood. 1993; 82: 3712-3720Crossref PubMed Google Scholar). When an adherent cell monolayer had developed, cells were harvested in Hanks' buffered saline solution containing 0.25% trypsin and 0.02% EDTA, washed, and collected by centrifugation. A proteasome inhibitor PS-341 and an IKK inhibitor PS-1145 (Millennium Pharmaceuticals, Cambridge, MA) were dissolved in Me2SO and stored at −20 °C until use. K i value of PS-1145 against the IKK complex was determined by measuring K m, ATP against varying fixed concentration of the inhibitor. Briefly, partially purified IKK complex obtained from unstimulated HeLa S3 cells were pre-activated using the catalytic domain of MEKK1 expressed in sf9. Kinase activity was assessed using a biotinylated IκBα peptide (250 μm, RHDSGLDSMKD,K m, peptide = 30 μm,K m, ATP = 10 μm) and phospho-[Ser32]-phosphoantibodies in an ELISA format with appropriate standard curve for quantification. For PS-1145K i measurement, the activated IKK complex was first preincubated in varying fixed concentration of the inhibitor (0.1–1 μm) at 25 °C for 1 h. Then apparentK m measurement for MgATP was performed at each discrete inhibitor concentration. Specificity of PS-1145 inhibition against IKK complex was demonstrated by testing PS-1145 against 14 kinases, including NF-κB-inducing kinase, PKA (protein kinase A), protein kinase C, casein kinase II, Src, Lck, calmodulin-dependent kinase 2, interleukin-1 receptor-associated kinase, p38α (known as stress-activated protein kinase 2α), MEK (MAPK kinases) 1/2, MAPK-activated protein kinase 2, ERK2 (extracellular signal regulated kinases), JNK2 (c-Jun NH2-terminal kinase 2), and epidermal growth factor receptor tyrosine kinase. Percentage inhibition of these kinases was negligible at PS-1145 (100 μm) (Table I). Target selectivity of PS-1145 was confirmed in the receptor binding and formation assays by NovaScreen (NovaScreen Bioscience, Hanover, MD) and Cerep (Cerep Inc., Redmond, WA). PS-341 and PS-1145 were diluted in culture medium immediately before use; PS-341 and PS-1145 control media contained <0.1% Me2SO. MAPK kinase (MEK) inhibitor PD98059 was purchased from Cell Signaling (Beverly, MA).Table IInhibitory activity of PS-1145 on various kinasesKinaseIC50KinaseIC50μmμmIKK<0.1 (88 nm)IRAK>100NIK>100pp38α>100Protein kinase A>100MEK1>100Protein kinase C>100MAPKAP kinase 2>100Casein kinase II>100ERK2>100c-Src>100JNK2>100Lck>100EGF tyrosine kinase>100Calmodulin kinase>100 Open table in a new tab Proliferation was measured as previously described (21.Hideshima T. Chauhan D. Shima Y. Raje N. Davies F.E. Tai Y.-T. Treon S. Lin B. Schlossman R.L. Ridhardson P. Muller G. Stirling D.I. Anderson K.C. Blood. 2000; 96: 2943-2950Crossref PubMed Google Scholar). MM cells (3 × 104cells/well) were incubated in 96-well culture plates (Costar, Cambridge, MA) in the presence of media, PS-341, PS-1145 and/or Dex or recombinant human IL-6 (Genetics Institute, Cambridge, MA) for 48 h at 37 °C. DNA synthesis was measured by [3H]thymidine ([3H]TdR, PerkinElmer Life Sciences, Boston, MA) uptake. Cells were pulsed with [3H]TdR (0.5 μCi/well) during the last 8 h of 48-h cultures. All experiments were performed in triplicate. The inhibitory effect of PS-341 and PS-1145 on MM growth was assessed by measuring 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) dye absorbance of the cells. Cells from 48-h cultures were pulsed with 10 μl of 5 mg/ml MTT to each well for the last 4 h of 48-h cultures, followed by 100 μl of isopropanol containing 0.04n HCl. Absorbance was measured at 570 nm using a spectrophotometer (Molecular Devices Corp., Sunnyvale, CA). MM cells were cultured with PS-341 or PS-1145, harvested, washed, and lysed using lysis buffer: 50 mm Tris-HCl (pH 7.4), 150 mm NaCl, 1% Nonidet P-40, 5 mm EDTA, 5 mm NaF, 2 mmNa3VO4, 1 mm PMSF, 5 μg/ml leupeptin, and 5 μg/ml aprotinin. For detection of phospho-IκBα, IκBα, phospho-MAPK, phospho-STAT3, ERK2, or α-tubulin, cell lysates were subjected to SDS-PAGE, transferred to polyvinylidene difluoride membrane (Bio-Rad Laboratories, Hercules, CA), and immunoblotted with anti-phospho-IκBα, -IκBα, -phospho-MAPK, or -phospho-STAT3 Abs (Cell Signaling, Beverly, MA), and anti-α-tubulin (Sigma) Abs. Electrophoretic mobility shift analyses (EMSA) were carried out as in our previous studies (9.Hideshima T. Chauhan D. Schlossman R. Richardson P. Anderson K.C. Oncogene. 2001; 20: 4519-4527Crossref PubMed Scopus (363) Google Scholar, 18.Hideshima T. Richardson P. Chauhan D. Palombella V.J. Elliot P.J. Adams J. Anderson K.C. Cancer Res. 2001; 61: 3071-3076PubMed Google Scholar). Briefly, MM.1S cells were preincubated with PS-341 (5 μm for 1 h) and PS-1145 (10 μm for 90 min) before stimulation with TNFα (5 ng/ml) for 10 or 20 min. Cells were then pelleted, resuspended in 400 μl of hypotonic lysis buffer (20 mm HEPES, pH 7.9, 10 mm KCl, 1 mm EDTA, 0.2% Triton X-100, 1 mmNa3VO4, 5 mm NaF, 1 mmPMSF, 5 μg/ml leupeptin, 5 μg/ml aprotinin), and kept on ice for 20 min. After centrifugation (14,000 × g for 5 min) at 4 °C, the nuclear pellet was extracted with 100 μl of hypertonic lysis buffer (20 mm HEPES, pH 7.9, 400 mm NaCl, 1 mm EDTA, 1 mm Na3VO4, 5 mm NaF, 1 mm PMSF, 5 μg/ml leupeptin, 5 μg/ml aprotinin) on ice for 20 min. After centrifugation (14,000 × g for 5 min) at 4 °C, the supernatant was collected as nuclear extract. Double-stranded NF-κB consensus oligonucleotide probe (5′-GGGGACTTTCCC-3′, Santa Cruz Biotechnology.) was end-labeled with [γ-32P]ATP (50 μCi at 222 TBq/mm, PerkinElmer Life Sciences, Boston, MA). Binding reactions containing 1 ng of oligonucleotide and 5 μg of nuclear protein were conducted at room temperature for 20 min in a total volume of 10 μl of binding buffer (10 mm Tris-HCl, pH 7.5, 50 mm NaCl, 1 mm MgCl2, 0.5 mm EDTA, 0.5 mm dithiothreitol, 4% glycerol (v/v), and 0.5 μg of poly(dI-dC) (Amersham Biosciences, Inc., Peapack, NJ). The samples were loaded onto a 4% polyacrylamide gel, transferred to Whatman paper (Whatman International, Maidstone, UK), and visualized by autoradiography. For cell cycle analysis, MM cells cultured for 24–48 h in PS-1145 (10 μm), or control media were harvested, washed with phosphate-buffered saline, fixed with 70% ethanol, and treated with 10 μg/ml of RNase (Roche Diagnostics Corp., Indianapolis, IN). Cells were then stained with propidium iodine (Sigma) (5 μg/ml). For detection of ICAM-1 expression, MM cells cultured with TNFα (5 ng/ml) in the presence or absence of PS-1145 (10 μm) were harvested, washed with PBS, and stained with mouse IgG isotype control or fluorescein isothiocyanate-conjugated mouse anti-human CD54 Ab. Both cell cycle profile and ICAM-1 expression were determined using an Epics (Coulter Immunology, Hialeah, FL) flow cytometer, as in prior studies (9.Hideshima T. Chauhan D. Schlossman R. Richardson P. Anderson K.C. Oncogene. 2001; 20: 4519-4527Crossref PubMed Scopus (363) Google Scholar). To evaluate growth stimulation and signaling in MM cells adherent to BMSCs, 3 × 104 MM.1S cells were cultured in BMSC coated 96-well plates for 48 h, in the presence or absence of PS-1145. DNA synthesis was measured as described above. The Duoset ELISA (R&D System) was used to measure IL-6 in supernatants of 48-h cultures of BMSCs with or without MM.1S cells, in the presence or absence of PS-1145. Statistical significance of differences observed in drug-treated versus control cultures was determined using the Student's t test. The minimal level of significance was p < 0.05. The effect of PS-1145, a novel specific IKK inhibitor (Fig. 1,A and B) on IκBα phosphorylation and NF-κB activation in MM cells was first examined. Inhibition of IκBα phosphorylation by PS-1145 was assayed in MM.1S and patient MM cells triggered by TNFα. Serine phosphorylation and degradation of IκBα were significantly induced by TNFα at 5 and 10 min in cells cultured in Me2SO control media, whereas phosphorylation and degradation of IκBα were completely blocked by PS-1145 pre-treatment of MM.1S cells (Fig. 1 C). To study the dose-dependent effect of PS-1145, MM.1S cells were pre-treated with 1.25–40 μm PS-1145 for 90 min, and then stimulated by TNFα (5 ng/ml). Phosphorylation of IκBα was completely inhibited by ≥5 μm PS-1145 (Fig. 1 D). As in MM.1S cells, PS-1145 also inhibited phosphorylation and degradation of IκBα triggered by TNFα in patient MM cells (Fig. 1 E). These results demonstrate a time- and a dose-dependent inhibitory effect of PS-1145 on phosphorylation and degradation of IκBα. Because NF-κB activation requires phosphorylation, ubiquitination, and degradation of IκBα, we next examined whether PS-1145 could inhibit NF-κB activation, assessed by EMSA. MM.1S cells pre-treated with either Me2SO control media or PS-1145 (10 μm for 90 min) were stimulated by TNFα (5 ng/ml for 0–20 min). NF-κB activation was completely inhibited by PS-1145 pre-treatment (Fig. 1 F). PS-341 served as a positive control for inhibition of NF-κB activation, as previously reported (9.Hideshima T. Chauhan D. Schlossman R. Richardson P. Anderson K.C. Oncogene. 2001; 20: 4519-4527Crossref PubMed Scopus (363) Google Scholar,18.Hideshima T. Richardson P. Chauhan D. Palombella V.J. Elliot P.J. Adams J. Anderson K.C. Cancer Res. 2001; 61: 3071-3076PubMed Google Scholar). To study the direct effect of PS-1145 on MM cells, we measured [3H]TdR uptake by MM.1S, U266, and RPMI8226 cell lines cultured for 48 h in the presence of PS-1145 (1.5–50 μm). 20–50% inhibition in proliferation was observed at doses > 12.5 μm PS-1145 (Fig. 2 A). In contrast, PS-341 completely inhibited [3H]TdR uptake in all cell lines tested at IC50 of 0.02–0.005 (Fig. 2 B). These results indicate that complete blockade of NF-κB activation cannot achieve >50% inhibition of DNA synthesis in MM cell lines and that complete inhibition by PS-341 is mediated through inhibition of another signaling pathway, such as p42/44 MAPK (22.Ogata A. Chauhan D. Teoh G. Treon S.P. Urashima M. Schlossman R.L. Anderson K.C. J. Immunol. 1997; 159: 2212-2221PubMed Google Scholar). Cell cycle profile, assessed by propidium iodine staining, was also examined in these MM cell lines. Interestingly, PS-1145 induced G1 growth arrest, but not apoptosis, in U266 cells (Fig. 2 C). Similar results were observed in RPMI8226 cells (data not shown). These data show that NF-κB blockade induces G1 growth arrest in MM cells. We have previously shown that IL-6 stimulates p42/44 MAPK, JAK2/STAT3, and PI3K/Akt signaling pathways in MM.1S cells (23.Hideshima T. Nakamura N. Chauhan D. Anderson K.C. Oncogene. 2001; 20: 5991-6000Crossref PubMed Scopus (426) Google Scholar) and that PS-341 inhibits p42/44 MAPK, but not JAK2/STAT3 or PI3K/Akt, cascades triggered by IL-6 (18.Hideshima T. Richardson P. Chauhan D. Palombella V.J. Elliot P.J. Adams J. Anderson K.C. Cancer Res. 2001; 61: 3071-3076PubMed Google Scholar). IL-6 induces phosphorylation of both p42/44 MAPK and STAT3 in MM.1S cells, and the MEK1 inhibitor PD98059 selectively inhibits p42/44 MAPK phosphorylation (Fig. 2 D). As in our prior report (18.Hideshima T. Richardson P. Chauhan D. Palombella V.J. Elliot P.J. Adams J. Anderson K.C. Cancer Res. 2001; 61: 3071-3076PubMed Google Scholar), PS-341 also inhibits p42/44 MAPK phosphorylation; however, PS-1145 does not inhibit either p42/44 MAPK or STAT3 phosphorylation. Phosphorylation of Akt triggered by IL-6 was also unaffected by PS-1145 pre-treatment (data not shown). These results indicate that PS-1145 specifically blocks NF-κB, without affecting other known signaling pathways in MM.1S cells triggered by IL-6. We next determined whether PS-1145 enhances the inhibitory effect of Dex on NF-κB activation in MM.1S cells. Because Dex has been shown to up-regulate IκBα expression in monocytic cell lines and T cells (24.Scheinman R.I. Cogswell P.C. Lofquist A.K. Baldwin Jr., A.S. Science. 1995; 270: 283-286Crossref PubMed Scopus (1597) Google Scholar, 25.Auphan N. DiDonato J.A. Rosette C. Helmberg A. Karin M. Science. 1995; 270: 286-290Crossref PubMed Scopus (2163) Google Scholar), we first examined whether Dex could induce IκBα protein in MM cells. MM.1S cells were cultured in the presence of Dex (1 μm for 0–36 h), and expression of IκBα protein was assessed by Western blotting. Dex significantly induces IκBα expression, and IL-6 partially blocks Dex-induced up-regulation of IκBα (Fig. 3 A). NF-κB activation was next directly assayed in the presence of Dex, with or without IL-6. TNFα-induced activation of NF-κB in MM.1S cells is abrogated by pre-treatment with Dex (Fig. 3 B). Moreover, PS-1145 also inhibits NF-κB activation triggered by TNFα in MM.1S cells in a dose-dependent fashion, and pre-treatment with Dex enhances the inhibitory effect of PS-1145 (Fig. 3 C). These results suggest that Dex inhibits NF-κB activation via up-regulation of IκBα, with related G1 growth arrest and apoptosis. The effect of Dex, PS-1145, PS-341, and/or IL-6 on TNFα-induced phosphorylation of IκBα in MM.1S cells was next examined. Dex, PS-341, and/or IL-6 do not inhibit TNFα-induced phosphorylation of IκBα; moreover, PS-341 enhances phosphorylation of IκBα, due to inhibition of proteasome activity and accumulation of IκBα (Fig. 3 D). In contrast, PS-1145, in the presence or absence of IL-6, inhibits phosphorylation of IκBα. Dex inhibits TNFα-induced degradation of IκBα, whereas IL-6 blocks this effect. Both PS-341 and PS-1145 inhibit degradation of IκBα, in the presence or absence of IL-6. To define the functional sequelae of PS-1145-related NF-κB blockade in MM.1S cells, we first examined its inhibitory effect on growth of MM cells, in the presence of Dex and/or IL-6. Dex inhibits IL-6-induced proliferation of MM.1S cells, but PS-1145 does not enhance its effect (Fig. 4 A). Importantly, both constitutive and IL-6-induced DNA synthesis in MM.1S cells is abrogated in the presence of PS-1145 in a dose-dependent fashion (Fig. 4 B). Furthermore, the protective effect of IL-6 on Dex-induced growth inhibition is also abrogated by PS-1145 (Fig. 4 C). These data suggest that promotion of cell growth and survival by IL-6 is mediated, at least in part, via NF-κB signaling. Because we have previously reported that the immunomodulatory derivative of Thal 3 (IMiD3) inhibits MM.1S cell growth and that IL-6 abrogates this IMiD effect (21.Hideshima T. Chauhan D. Shima Y. Raje N. Davies F.E. Tai Y.-T. Treon S. Lin B. Schlossman R.L. Ridhardson P. Muller G. Stirling D.I. Anderson K.C. Blood. 2000; 96: 2943-2950Crossref PubMed Google Scholar), we next examined the effect of PS-1145 on DNA synthesis of MM.1S cells treated with IMiD3, in the presence or absence of IL-6. IMiD3 significantly (p < 0.001) inhibits DNA synthesis in MM.1S cells in a dose-dependent fashion (0.25–1 μm, Fig. 4 D). IL-6 (20 ng/ml) overcomes the effect of IMiD3; importantly, however, PS-1145 neutralizes the inhibitory effect of IL-6 against IMiD3. These studies suggest that NF-κB blockade can overcome resistance to IMiDs. We have previously shown that TNFα induces ICAM-1 and VCAM-1 expression on both BMSCs and MM cells and that proteasome inhibitor PS-341 blocks induction of these molecules (9.Hideshima T. Chauhan D. Schlossman R. Richardson P. Anderson K.C. Oncogene. 2001; 20: 4519-4527Crossref PubMed Scopus (363) Google Scholar). Because PS-341 is not a specific NF-κB inhibitor, we cannot conclude from these earlier studies that TNFα-induced up-regulation of ICAM-1 and VCAM-1 is mediated through NF-κB. However, PS-1145 also inhibits TNFα-induced up-regulation of ICAM-1 expression on MM.1S and RPMI8226 cells (Fig. 5 A). This result strongly suggests that up-regulation of ICAM-1 by TNFα is mediated via activation of NF-κB. We have previously shown that TNFα induces modest proliferation, but not apoptosis, in MM.1S cells (9.Hideshima T. Chauhan D. Schlossman R. Richardson P. Anderson K.C. Oncogene. 2001; 20: 4519-4527Crossref PubMed Scopus (363) Google Scholar). In this study, we hypothesized that TNFα would induce apoptosis in MM.1S cells when NF-κB activation is blocked by PS-1145. To test this hypothesis, MM.1S cells were cultured with TNFα, in the presence or absence of PS-1145. TNFα treatment does not affect viability of MM.1S cells, assessed by MTT assay; however, in the presence of PS-1145, viability of the cells is significantly (p < 0.001) decreased (Fig. 5 B). For example, TNFα (1 ng/ml) induces 60% growth inhibition of MM.1S cells in the presence of 10 μmPS-1145. This effect on MM.1S viability is also confirmed by trypan blue exclusion (data not shown). These studies suggest that NF-κB mediates protection of MM.1S cells against TNFα-induced apoptosis. To study the role of NF-κB activation in regulating IL-6 transcription and secretion, as well as related paracrine MM cell growth in the BM milieu, MM.1S cells were cultured with or without BMSCs, in the presence or absence of PS-1145. PS-1145 blocks constitutive secretion of IL-6 in BMSCs in a dose-dependent fashion (Fig. 6 A). Importantly, adhesion
