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J. Biol. Chem., Vol. 277, Issue 19, 16639-16647, May 10, 2002

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NF-B as a Therapeutic Target in Multiple Myeloma^*

Teru Hideshima, Dharminder Chauhan, Paul Richardson, Constantine Mitsiades, Nicholas Mitsiades, Toshiaki Hayashi, Nikhil Munshi, Lenny Dang^§, Alfredo Castro^§, Vito Palombella^§, Julian Adams^§, and Kenneth C. Anderson^¶

From the  Jerome Lipper Multiple Myeloma Center, Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts 02115 ^§ Millennium Pharmaceuticals, Inc., Cambridge, Massachusetts 02139

Received for publication, January 11, 2002, and in revised form, February 25, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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)-B activation, they also have multiple and varied other actions. In this study, we therefore specifically address the role of NF-B blockade in mediating anti-MM activity. To characterize the effect of specific NF-B blockade on MM cell growth and survival in vitro, we used an IB kinase (IKK) inhibitor (PS-1145). Our studies demonstrate that PS-1145 and PS-341 block TNF-induced NF-B activation in a dose- and time-dependent fashion in MM cells through inhibition of IB phosphorylation and degradation of IB, respectively. Dexamethasone (Dex), which up-regulates IB protein, enhances blockade of NF-B activation by PS-1145. Moreover, PS-1145 blocks the protective effect of IL-6 against Dex-induced apotosis. TNF-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-B blockade cannot account for all of the anti-MM activity of PS-341. Importantly, however, TNF induces MM cell toxicity in the presence of PS-1145. These studies demonstrate that specific targeting of NF-B 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-B.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

NF-B is a member of the Rel family of proteins and is typically a heterodimer composed of p50 and p65 subunits (1). NF-B is constitutively present in the cytosol and inactivated by its association with IB family inhibitors (2). IB is, therefore, a key molecular target regulating NF-B activation. Various stimuli, including TNF,¹ lipopolysaccharide, phorbol esters, and viruses, trigger IB protein phosphorylation on two serine residues located in its NH₂ terminus by the multisubunit IB kinase (IKK) complex. Once phosphorylated, IB is targeted for ubiquitination and degradation by the 26 S proteasome (3), allowing translocation of NF-B 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-B activation.

Many studies have reported growth and anti-apoptotic roles of NF-B in normal and malignant cells. For example, NF-B promotes cell growth by up-regulating cyclin D transcription, with associated hyperphosphorylation of Rb, G₁- to S-phase transition, and inhibition of apoptosis (4). NF-B also regulates transcription of the catalytic subunit of telomerase in mice (5). NF-B is constitutively activated in Hodgkin's tumor cells, whereas inhibition of NF-B blocks their growth (6). Moreover, inhibition of NF-B via expression of the super-repressor of IB induces apoptosis, even in the presence of an oncogenic allele of H-Ras (7). Although the precise role of NF-B 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-B-dependent up-regulation of transcription of IL-6, a growth and anti-apoptotic factor in MM (8). In addition, TNF secreted by MM cells: 1) activates NF-B in BMSCs, thereby directly up-regulating IL-6 transcription and secretion in BMSCs; and 2) activates NF-B 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). Because IL-6 is the major growth and survival factor in MM cells (10-12) and adherence of MM cells to fibronectin confers resistance to drug-induced apoptosis (13, 14), specific blockade of NF-B 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) and its immunomodulatory derivatives (IMiDs),² proteasome inhibitor PS-341 (17, 18), and arsenic trioxide As₂O₃ (19), all inhibit NF-B 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-B in novel MM therapeutics is not yet defined.

In the present study, we demonstrate the specific biologic sequelae of NF-B activation and blockade on MM cell growth and survival in the BM microenvironment. The novel specific IKK inhibitor PS-1145 inhibits phosphorylation of IB in MM cells and modestly directly inhibits their growth. Importantly, however, PS-1145 abrogates NF-B 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-B in regulating growth and survival of MM cells in the BM milieu and further suggest the potential utility of novel therapeutics targeting NF-B in MM.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

MM-derived Cell Lines and Patient MM Cells-- 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 µM L-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).

BMSC Cultures-- 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). 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.

Inhibitors-- 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 IB 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-1145 K_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 apparent K_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-B-inducing kinase, PKA (protein kinase A), protein kinase C, casein kinase II, Src, Lck, calmodulin-dependent kinase 2, interleukin-1 receptor-associated kinase, p38 (known as stress-activated protein kinase 2), 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).

DNA Synthesis-- Proliferation was measured as previously described (21). 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.

Growth Inhibition Assay-- 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 0.04 N HCl. Absorbance was measured at 570 nm using a spectrophotometer (Molecular Devices Corp., Sunnyvale, CA).

Immunoblotting-- 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 mM Na₃VO₄, 1 mM PMSF, 5 µg/ml leupeptin, and 5 µg/ml aprotinin. For detection of phospho-IB, IB, phospho-MAPK, phospho-STAT3, ERK2, or -tubulin, cell lysates were subjected to SDS-PAGE, transferred to polyvinylidene difluoride membrane (Bio-Rad Laboratories, Hercules, CA), and immunoblotted with anti-phospho-IB, -IB, -phospho-MAPK, or -phospho-STAT3 Abs (Cell Signaling, Beverly, MA), and anti--tubulin (Sigma) Abs.

Electrophoretic Mobility Shift Analysis-- Electrophoretic mobility shift analyses (EMSA) were carried out as in our previous studies (9, 18). 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 TNF (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 mM Na₃VO₄, 5 mM NaF, 1 mM PMSF, 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-B consensus oligonucleotide probe (5'-GGGGACTTTCCC-3', Santa Cruz Biotechnology.) was end-labeled with [-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.

Flow Cytometric Analysis-- 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 TNF (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).

Effect of PS-1145 on Paracrine MM Cell Growth in the BM-- 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 Analysis-- 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.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

PS-1145 Inhibits IB Phosphorylation and NF-B Activation in MM.1S Cells and Patient MM Cells-- The effect of PS-1145, a novel specific IKK inhibitor (Fig. 1, A and B) on IB phosphorylation and NF-B activation in MM cells was first examined. Inhibition of IB phosphorylation by PS-1145 was assayed in MM.1S and patient MM cells triggered by TNF. Serine phosphorylation and degradation of IB were significantly induced by TNF at 5 and 10 min in cells cultured in Me₂SO control media, whereas phosphorylation and degradation of IB were completely blocked by PS-1145 pre-treatment of MM.1S cells (Fig. 1C). 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 TNF (5 ng/ml). Phosphorylation of IB was completely inhibited by 5 µM PS-1145 (Fig. 1D). As in MM.1S cells, PS-1145 also inhibited phosphorylation and degradation of IB triggered by TNF in patient MM cells (Fig. 1E). These results demonstrate a time- and a dose-dependent inhibitory effect of PS-1145 on phosphorylation and degradation of IB.

View larger version (35K): [in this window] [in a new window]   Fig. 1.   PS-1145 blocks phosphorylation and degradation of IB triggered by TNF. A, structure of PS-1145; B, relative IKK inhibitory activity of PS-1145; C, MM.1S cells were pre-treated with PS-1145 (10 µM for 90 min) and then stimulated by TNF (5 ng/ml for 0-20 min); D, MM.1S cells were pre-treated with 0.125-40 µM PS-1145 for 90 min before stimulation by TNF (5 ng/ml for 5 min); E, patient MM cells were pre-treated with PS-1145 (10 µM for 90 min) and then stimulated by TNF (5 ng/ml for 0-10 min). The cells were lysed, electrophoresed, and then immunoblotted with anti-phospho IB and anti-IB Abs; F, MM.1S cell were pre-treated with PS-1145 (10 µM for 90 min) and then stimulated by TNF (5 ng/ml for 0-20 min). Nuclear extracts of the cells were subjected to EMSA. PS-341 served as a positive control for inhibition of NF-B activation, as previously described (9, 18).

Because NF-B activation requires phosphorylation, ubiquitination, and degradation of IB, we next examined whether PS-1145 could inhibit NF-B 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 TNF (5 ng/ml for 0-20 min). NF-B activation was completely inhibited by PS-1145 pre-treatment (Fig. 1F). PS-341 served as a positive control for inhibition of NF-B activation, as previously reported (9, 18).

PS-1145 Inhibits Proliferation of MM Cell Lines-- 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. 2A). In contrast, PS-341 completely inhibited [³H]TdR uptake in all cell lines tested at IC₅₀ of 0.02-0.005 (Fig. 2B). These results indicate that complete blockade of NF-B 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). 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. 2C). Similar results were observed in RPMI8226 cells (data not shown). These data show that NF-B blockade induces G₁ growth arrest in MM cells.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Effect of PS-1145 on DNA synthesis, cell cycle profile, and signaling cascades triggered by IL-6. MM.1S (), U266 (), and RPMI8226 () cells were cultured in the presence of (A) PS-1145 (1.5-50 µM) or (B) PS-341 (0.00001-10 µM) for 48 h. DNA synthesis was assessed by [3H]TdR uptake. C, U266 cells were cultured in the presence of PS-1145 (10 µM for 24, 36, and 48 h), harvested, stained with propidium iodine, and analyzed for cell cycle profile by flow cytometry. Percentages represent cells in G0/G1. D, MM.1S cells were pre-treated with PS-1145 (10 µM for 90 min) or PS-341 (5 µM for 60 min) and cultured with or without IL-6 (50 ng/ml for 5 min). The cells were harvested, lysed, electrophoresed, and immunoblotted with anti-phospho-STAT3, phospho-p42/44 MAPK, and p42/44 MAPK Abs.

We have previously shown that IL-6 stimulates p42/44 MAPK, JAK2/STAT3, and PI3K/Akt signaling pathways in MM.1S cells (23) and that PS-341 inhibits p42/44 MAPK, but not JAK2/STAT3 or PI3K/Akt, cascades triggered by IL-6 (18). 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. 2D). As in our prior report (18), 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-B, without affecting other known signaling pathways in MM.1S cells triggered by IL-6.

Dex Up-regulates IB Protein and Enhances the Inhibitory Effect of PS-1145 on NF-B Activation in MM.1S Cells-- We next determined whether PS-1145 enhances the inhibitory effect of Dex on NF-B activation in MM.1S cells. Because Dex has been shown to up-regulate IB expression in monocytic cell lines and T cells (24, 25), we first examined whether Dex could induce IB protein in MM cells. MM.1S cells were cultured in the presence of Dex (1 µM for 0-36 h), and expression of IB protein was assessed by Western blotting. Dex significantly induces IB expression, and IL-6 partially blocks Dex-induced up-regulation of IB (Fig. 3A). NF-B activation was next directly assayed in the presence of Dex, with or without IL-6. TNF-induced activation of NF-B in MM.1S cells is abrogated by pre-treatment with Dex (Fig. 3B). Moreover, PS-1145 also inhibits NF-B activation triggered by TNF in MM.1S cells in a dose-dependent fashion, and pre-treatment with Dex enhances the inhibitory effect of PS-1145 (Fig. 3C). These results suggest that Dex inhibits NF-B activation via up-regulation of IB, with related G₁ growth arrest and apoptosis.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Dex up-regulates IB protein and enhances the inhibitory effect of PS-1145 on NF-B activation. A, MM.1S cells were cultured in the presence of Dex (1 µM for 0-36 h), in the presence or absence of IL-6. The cells were harvested, lysed, electrophoresed, and immunoblotted with anti-phospho-IB and anti-tubulin Abs. B, MM.1S cells were cultured with either Me2SO control or Dex (1 µM for 18 h) before stimulation with TNF (5 ng/ml) or IL-6 (50 ng/ml). Nuclear extracts were subjected to EMSA to assess NF-B activation. C, MM.1S cells were pre-treated with either Me2SO control or Dex (1 µM for 18 h), cultured with PS-1145 (2.5-10 µM for 90 min), and then stimulated by TNF (5 ng/ml for 5 min). The cells were harvested, and nuclear extracts were subjected to EMSA to assess NF-B activation. D, MM.1S cells were cultured in the presence of Dex (1 µM for 18 h), IL-6 (50 ng/ml for 18 h), PS-341 (5 µM for 60 min), or PS-1145 (10 µM for 90 min) and then stimulated by TNF (5 ng/ml for 5 min). The cells were harvested, lysed, electrophoresed, and then immunoblotted with anti-phospho-IB and anti-IB Abs.

The effect of Dex, PS-1145, PS-341, and/or IL-6 on TNF-induced phosphorylation of IB in MM.1S cells was next examined. Dex, PS-341, and/or IL-6 do not inhibit TNF-induced phosphorylation of IB; moreover, PS-341 enhances phosphorylation of IB, due to inhibition of proteasome activity and accumulation of IB (Fig. 3D). In contrast, PS-1145, in the presence or absence of IL-6, inhibits phosphorylation of IB. Dex inhibits TNF-induced degradation of IB, whereas IL-6 blocks this effect. Both PS-341 and PS-1145 inhibit degradation of IB, in the presence or absence of IL-6.

PS-1145 Overcomes the Protective Effect of IL-6 against Dex and IMiD3 in MM.1S Cells-- To define the functional sequelae of PS-1145-related NF-B 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. 4A). 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. 4B). Furthermore, the protective effect of IL-6 on Dex-induced growth inhibition is also abrogated by PS-1145 (Fig. 4C). These data suggest that promotion of cell growth and survival by IL-6 is mediated, at least in part, via NF-B signaling.

View larger version (36K): [in this window] [in a new window]   Fig. 4.   PS-1145 inhibits growth and blocks the protective effect of IL-6 against Dex- and IMiD3-induced growth inhibition. A, MM.1S cells were cultured with Me2SO control (), or with 1.5 µM (), 3 µM (), 6 µM (), 12.5 µM (), 25 µM (), and 50 µM () PS-1145, in the absence or presence of 0.01 and 0.1 µM Dex for 48 h. B, MM.1S cells were cultured with Me2SO control () or with 1.5 µM (), 3 µM (), 6 µM (), or 12.5 µM () PS-1145, in the absence or presence of 0.5, 5, and 50 ng/ml IL-6 for 48 h. C, MM.1S cells were cultured with Me2SO control () or 0.4 µM (), 2 µM () or 10 µM () PS-1145 in the presence of Dex (0.1 µM) and/or IL-6 (50 ng/ml) for 48 h. D, MM.1S cells were cultured for 48 h with Me2SO control () or with 0.25 µM (), 0.5 µM (), or 1 µM () IMiD3, in the presence or absence of PS-1145 (10 µM) and with or without IL-6 (50 ng/ml). DNA synthesis was assessed by [3H]TdR uptake.

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), 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. 4D). 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-B blockade can overcome resistance to IMiDs.

PS-1145 Inhibits TNF-induced ICAM-1 Expression and Enhances TNF-induced Apoptosis in MM.1S Cells-- We have previously shown that TNF 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). Because PS-341 is not a specific NF-B inhibitor, we cannot conclude from these earlier studies that TNF-induced up-regulation of ICAM-1 and VCAM-1 is mediated through NF-B. However, PS-1145 also inhibits TNF-induced up-regulation of ICAM-1 expression on MM.1S and RPMI8226 cells (Fig. 5A). This result strongly suggests that up-regulation of ICAM-1 by TNF is mediated via activation of NF-B.

View larger version (37K): [in this window] [in a new window]   Fig. 5.   PS-1145 inhibits TNF-induced ICAM-1 expression and triggers TNF-induced apoptosis in MM cells. A, MM.1S and RPMI8226 cells were cultured for 24 h with Me2SO control (heavy solid line), TNF (5 ng/ml) alone (light solid line), or TNF + PS-1145 (). The cells were harvested, stained with isotype control (dotted line) or fluorescein isothiocyanate-conjugated anti-ICAM-1 Ab, and analyzed using flow cytometry. B, TNF induces apoptosis in MM.1S cells with PS-1145-related NF-B blockade. MM.1S cells were cultured for 48 h with 0.2 and 1 ng/ml TNF, in the absence () or presence of 2.5 µM (), 5 µM (), and 10 µM () PS-1145. Cell viability was assessed by MTT assay.

We have previously shown that TNF induces modest proliferation, but not apoptosis, in MM.1S cells (9). In this study, we hypothesized that TNF would induce apoptosis in MM.1S cells when NF-B activation is blocked by PS-1145. To test this hypothesis, MM.1S cells were cultured with TNF, in the presence or absence of PS-1145. TNF 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. 5B). For example, TNF (1 ng/ml) induces 60% growth inhibition of MM.1S cells in the presence of 10 µM PS-1145. This effect on MM.1S viability is also confirmed by trypan blue exclusion (data not shown). These studies suggest that NF-B mediates protection of MM.1S cells against TNF-induced apoptosis.

Effect of PS-1145 on Paracrine MM Cell Growth and IL-6 Secretion-- To study the role of NF-B 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. 6A). Importantly, adhesion of MM.1S cells to BMSCs triggers increased IL-6 secretion (1.6-fold, p < 0.01), and PS-1145 also blocks this response in a dose-dependent fashion (p < 0.01). Adherence of MM.1S cells to BMSCs also triggers increased MM.1S cell growth (1.9-fold, p < 0.01), and PS-1145 similarly inhibits this augmentation in a dose-dependent fashion (p < 0.01, Fig. 6B). These data confirm our earlier studies that induction of IL-6 secretion from BMSCs triggered by MM cell adhesion is mediated through NF-B (8), and importantly, show that PS-1145 can abrogate this effect.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   PS-1145 inhibits paracrine MM cell growth and IL-6 secretion in BMSCs triggered by MM cell adherence. MM.1S cells, BMSCs, and BMSCs with MM.1S cells were cultured for 48 h in the presence of Me2SO control () or with 0.4 µM (), 2 µM (), or 10 µM () PS-1145. A, IL-6 level was measured in culture supernatants by ELISA; B, DNA synthesis was assessed by [3H]TdR uptake.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Five NF-B family members have been identified and cloned, including RelA (p65), RelB, c-Rel, NF-B1 (p50/p105), and NF-B2 (p52/p100). All have highly conserved Rel homology domains mediating DNA binding and interaction with IB. The NF-B family regulates growth, cell differentiation, and apoptosis in cell lines and tissues, including embryonic limb, lymphocytes, lung and liver, skin, and bone (26-29). It has also been shown that NF-B activation inhibits apoptosis of Kaposi's sarcoma-associated herpes virus infected primary effusion lymphoma cells (30) and induces Bcl-xL expression in Hodgkin/Reed-Sternberg cells (31) and CD4+ T lymphocytes (32). In this study, we characterize the biologic sequelae of NF-B activation in MM pathogenesis. Specific NF-B blockade using the IKK inhibitor PS-1145 confirms the role of NF-B in growth, survival, and drug-resistance in MM cells within the BM milieu and suggests the therapeutic benefit of targeting NF-B in MM.

We first demonstrate the inhibitory effect of PS-1145 on serine phosphorylation of IB in MM.1S cells and patient MM cells in a dose-dependent fashion. Phosphorylation of IB triggered by TNF is completely abrogated by pre-treatment with 5 µM PS-1145, and inhibition of DNA binding of NF-B by PS-1145 is confirmed by EMSA. Even with NF-B blockade, however, PS-1145 decreases cell proliferation of MM cells by only 50%, assessed by [³H]TdR uptake. In contrast, as we have previously shown (18), proteasome inhibitor PS-341 completely blocks cell proliferation at an IC₅₀ of 0.002-0.005 µM in these same MM cell lines. Moreover, PS-1145 induces G₁ growth arrest, but not apoptosis or necrosis, as does PS-341 in MM cells. Our result is consistent with previous reports demonstrating that NF-B activity is increased during G₀ to G₁ cell cycle transition in mouse fibroblasts (33) and that inhibition of NF-B causes impairment of cell cycle progression in human glioma cells (34). The identification of an NF-B binding site in the cyclin D1 promoter may account, at least in part, for cell cycle regulation by NF-B (31) (35). Our results further indicate that a complete growth inhibitory effect cannot be induced in MM cells by NF-B blockade and that the complete block in MM cell growth by PS-341 is not solely due to NF-B blockade but also to targeting other signaling pathways. Indeed, PS-341 inhibits IL-6-triggered p42/44 MAPK activation, which mediates proliferation in MM cell lines as well as patient MM cells (22).

We have shown that Dex-induced apoptosis in MM cells is mediated via related adhesion focal tyrosine kinase activation; second mitochondria-derived activator of caspases, but not cytochrome c release from mitochondria; and caspase-9 activation (23, 36, 37). Conversely, IL-6 protects against Dex-induced apoptosis via PI3K/Akt signaling (23) and activation of SH2 domain containing protein tyrosine phosphatase SHP2, thereby blocking related adhesion focal tyrosine kinase activation (37). Although Dex up-regulates IB in monocytic cell lines and T cells (24, 25), it inhibits NF-B activation without affecting IB expression in epithelial cells (38). We therefore examined whether Dex induces IB protein and thereby inhibits NF-B activation in MM cells. As expected, Dex markedly increases IB protein expression in MM cells in a time-dependent fashion and inhibits NF-B activation triggered by TNF in MM.1S cells. Inhibition of NF-B activation by Dex is also augmented by pre-treatment with PS-1145; conversely, Dex-induced up-regulation of IB protein expression is inhibited by IL-6. Therefore Dex inhibits cell proliferation and IL-6 overcomes this effect, at least in part, by altering expression of IB protein. These results suggest that NF-B plays a critical role in DNA synthesis and protection against Dex-induced apoptosis in MM.1S cells and that PS-1145 may be useful to overcome clinical Dex resistance.

Recently, we (21, 39, 40) and others (41, 42) have reported that Thal and IMiDs overcome drug resistance in MM cell lines and MM patient cells in vitro and achieve clinical responses even in refractory relapsed MM (21, 41, 42). A recent report (15) shows that Thal at high concentration inhibits NF-B activity via indirect suppression of IKK activity. In this study, we further examine the effect of PS-1145 in Thal/IMiD3-treated MM.1S cells and demonstrate that PS-1145 enhances inhibition of MM cell growth by IMiDs. Importantly, our prior study shows that IL-6 protects against Thal/IMiDs (21), and the current experiments demonstrate that PS-1145 blocks this protective effect of IL-6 against IMiD-induced apoptosis in MM.1S cells. These results indicate that the protective effect of IL-6 against Thal/IMiDs-induced apoptosis is also mediated via NF-B activation. Importantly, they suggest the combined use of Thal/IMiDs with PS-1145 as a potential clinical strategy to overcome drug resistance.

Induction of NF-B with related up-regulation of adhesion molecules, including CD54 (ICAM-1) and CD106 (VCAM-1), has been demonstrated in TNF-stimulated MM cell lines and BMSCs, human umbilical vein endothelial cells, and fibroblast-like synoviocytes (9, 17, 43). Moreover, MM cell adhesion to fibronectin mediates an anti-apoptotic effect against chemotherapeutic agents (14). In our prior study, proteasome inhibitor PS-341 blocked TNF-induced up-regulation of adhesion molecules and related increased MM cell to BMSC binding by inhibiting NF-B activation (9, 18). In this study, IKK inhibitor PS-1145 also inhibits TNF-induced ICAM-1 expression in MM.1S and RPMI8226 cells, suggesting that NF-B directly targets ICAM-1 expression in MM. We further demonstrate that MM cell adherence to BMSCs induces up-regulation of IL-6 transcription and secretion in BMSCs, as well as proliferation of adherent MM cells, as we (18) and others (44) have previously reported. Importantly, PS-1145 abrogates this induction of IL-6 secretion in BMSCs and proliferation of adherent MM cells in a dose-dependent fashion, consistent with our previous report that adhesion-induced IL-6 transcription and secretion in BMSCs is conferred, at least in part, by NF-B activation (8). This PS-1145-mediated inhibition of adhesion molecule expression, abrogation of protection against apoptosis confessed by MM cell to binding to BMSCs adhesion, and blockade of cytokine secretion in the BM milieu, provides further rationale for targeting NF-B in novel MM therapies.

We have previously shown that TNF mediates moderate cell proliferation associated with p42/44 MAPK activation in MM.1S cells (9). TNF has also been reported to induce NF-B activation and apoptosis via caspase cleavage (45). When NF-B signaling was blocked by PS-1145 in this study, TNF significantly inhibited MM.1S cell growth in a dose-dependent fashion. This result strongly suggests that NF-B mediates protection against TNF-induced apoptosis in MM cells and is consistent with a recent report that activation of IKK mediates protection against TNF-induced apoptosis in T cells (16). It further suggests that TNF, which is secreted by MM cells (9), may promote tumor cell apoptosis in patients treated with PS-1145.

In summary, our data demonstrate that NF-B activation promotes growth, survival, and drug resistance of MM cells in the BM microenvironment and provide the framework for clinical trials of novel agents, such as PS-1145, specifically targeting NF-B in MM.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant PO-1 78378 and a Doris Duke Distinguished Clinical Research Scientist Award (to K. C. A.), and by Multiple Myeloma Research Foundation Senior Awards (to T. H. and D. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dana-Farber Cancer Institute, 44 Binney St., Boston, MA 02115. Tel.: 617-632-2144; Fax: 617-632-2140; E-mail: kenneth_anderson@dfci.harvard.edu.

Published, JBC Papers in Press, February 28, 2002, DOI 10.1074/jbc.M200360200

² Mitsiades, N., Mitsiades, C. S., Poulaki, V., Hideshima, T., Chauhan, D., Treon, S. P., and Anderson, K. C. (2002) Blood, in press.

	ABBREVIATIONS

The abbreviations used are: TNF, tumor necrosis factor ; MM, multiple myeloma; BMSC, bone marrow stromal cell; Thal, thalidomide; IMiDs, immunomodulatory derivatives of thalidomide; IL-6, interleukin-6; IKK, IB kinase; Dex, dexamethasone; ICAM-1, intracellular adhesion molecule-1; VCAM-1, vascular cell adhesion molecule-1; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; ERK, extracellular signal regulated kinases; STAT, signal transducers and activators of transcription; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; EMSA, electrophoretic mobility shift analysis; Ab, antibody; ELISA, enzyme-linked immunosorbent assay; PKA, protein kinase A; JNK, c-Jun NH₂-terminal kinase; TdR, thymidine; PMSF, phenylmethylsulfonyl fluoride; PI3K, phosphatidylinositol 3-kinase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES