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Blood, 1 September 2006, Vol. 108, No. 5, pp. 1698-1707. Prepublished online as a Blood First Edition Paper on May 9, 2006; DOI 10.1182/blood-2005-11-013672.
NEOPLASIA Multiple myeloma cell survival relies on high activity of protein kinase CK2From the Department of Clinical and Experimental Medicine, Hematology-Immunology Division, and the Department of Biological Chemistry, University of Padova School of Medicine, Padova, Italy; and the Venetian Institute of Molecular Medicine, Padova, Italy.
Casein kinase 2 (CK2) is a ubiquitous cellular serine-threonine kinase that regulates relevant biologic processes, many of which are dysregulated in malignant plasma cells. Here we investigated its role in multiple myeloma (MM). Analysis of MM cell lines and highly purified malignant plasma cells in patients with MM revealed higher protein and CK2 activity levels than in controls (normal in vitro-generated polyclonal plasma cells and B lymphocytes). The inhibition of CK2 with specific synthetic compounds or by means of RNA interference caused a cytotoxic effect on MM plasma cells that could not be overcome by IL-6 or IGF-I and that was associated with the activation of extrinsic and intrinsic caspase cascades. CK2 blockage lowered the sensitivity threshold of MM plasma cells to the cytotoxic effect of melphalan. CK2 inhibition also resulted in impaired IL-6-dependent STAT3 activation and in decreased basal and TNF-  -dependent I B degradation and NF-B-driven transcription. Our data show that CK2 was involved in the pathophysiology of MM, suggesting that it might play a crucial role in controlling survival and sensitivity to chemotherapeutics of malignant plasma cells.
Multiple myeloma (MM) is one of the most frequent hematologic malignancies arising from end-stage B lymphocytes.1,2 The growth of MM cells strictly depends on the surrounding bone marrow (BM) microenvironment1 through a number of paracrine and autocrine intercellular loops. For instance, several soluble mediators—such as IL-6, TNF- , IL-1 , IGF-I, TGF-, and VEGF— released by the BM stroma or by malignant plasma cells regulate plasma cell survival and proliferation, adhesiveness, migration, neoangiogenesis, and other features in the BM milieu.3 Several studies have identified genetic and biochemical abnormalities and play a relevant role in MM pathogenesis, but the mechanisms of MM cell growth are still largely unknown.4 In particular, a key point is the onset of resistance to drug-induced apoptosis underlying the poor outcome for patients with MM, whose molecular determinants are still elusive.5
CK2 (formerly known as casein-kinase II) is a constitutively active serine-threonine protein kinase with a tetrameric structure consisting of 2 catalytic (
Several stimuli, such as IL-6, TNF-
Moreover, IL-6 and IGF-I stimulate other important signaling pathways for MM cell growth, such as the JAK-STAT, the PI3K-AKT, and the MAPK cascades. Although CK2 has been evoked to regulate cell survival upon growth factors,26,27 its role downstream from these signaling pathways in MM and other B-cell malignancies is largely unknown. In addition, though CK2 activity has been shown to be elevated in solid tumors,9,10 no definitive studies are available regarding whether this kinase plays a role in the pathogenesis of B-cell malignancies and MM. In this work, we investigated CK2 levels and kinase activity in MM cells and the cellular and molecular consequences of hampering its function in these cells. In addition, we analyzed the effects of CK2 in regulating IL-6 and NF-
Patients and cell cultures Informed consent was obtained from patients in accordance with the Declaration of Helsinki. Sources of samples are listed in Table 1. Mononuclear cells from PB and BM were separated as in previous studies.28 CD138+ MM cells were purified by immunomagnetic sorting (Miltenyi Biotech, Bergish Gladbach, Germany) according to the manufacturer's protocol. MM cell lines OPM2, U266, and RPMI 8226 were purchased from the American Type Culture Collection (Rockville, MD); all cell lines were maintained in RPMI 1640 medium supplemented with 10% FCS, L-glutamine, penicillin, and streptomycin (Gibco Laboratories, Grand Island, NY) under controlled atmosphere at 37°C in the presence of 5% CO2. HEK293 cells were grown in DMEM with 10% FCS and other supplements. Plasma cells were generated in vitro, as previously described.29 B cells were enriched from PBMCs, as previously reported,30 and by positive selection with anti-CD19 MACS microbeads and were plated at 1.5 x 105/mL in the presence of 3.75 x 104/mL mitomycin C (Sigma-Aldrich, Milan, Italy)-treated CD40L-transfectant L-cells (a generous gift from Dr John Gordon, MRC Centre for Immune Regulation, Birmingham, United Kingdom) with various combinations of IL-2 (20 U/mL), IL-4 (50 ng/mL), IL-10 (50 ng/mL), and IL-12 (2 ng/mL) (PeproTech, Rocky Hill, NJ). On day 4, B cells were reseeded at 3 x 105/mL without CD40L transfectants and with IL-2 (20 U/mL), IL-10 (50 ng/mL), IL-12 (2 ng/mL), and IL-6 (5 ng/mL). On day 6, cells were double stained with FITC-conjugated anti-CD20 and PE-conjugated anti-CD38 (Becton Dickinson, San Jose, CA), and CD20-/CD38++ cells were sorted with a BD FACSAria (Becton Dickinson).
Chemicals and cytokines
TBB (4,5,6,7 tetrabrome benzotriazole) was synthesized as in previous studies,31 and IQA ([5-oxo-5,6-dihydroindole (1,2-a) quinazolin-7-yl]-acetic acid), formerly termed CGP029482,32 was kindly provided by Dr J. Schoepfer (Novartis, Basel, Switzerland). The TBB-derivative K27 (2-amino-4,5,6,7-tetrabromo-1H-benzimidazole), also termed 2A,33 was synthesized and kindly provided by Dr Z. Kazimierczuk (Warsaw, Poland). The remarkable specificity of TBB and IQA toward only CK2 out of a panel of more than 30 protein kinases (with the partial exception of DYRK1A) has been assessed elsewhere.32,34 K27/2a is closely related to DMAT/2C,33,35 whose specificity is similar to that of TBB. All these compounds are cell permeable.7,32,33 Melphalan, MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal), and the cytokines TNF- CK2 activity in cell lysates CK2 activity was determined as previously described.7,36 The CK2 activity was in a linear range according to the lysate protein amount used for the assay. Evaluation of growth and apoptosis MM cell growth was monitored using the MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide)-based assay, according to the manufacturer's protocol (Roche, Monza, Italy) and as described elsewhere.7 Apoptosis was assessed by annexin V/propidium iodide staining (BD PharMingen, San Francisco, CA) according to the manufacturer's instructions using a FACSCalibur cytofluorometer with the CellQuest analytic software (Becton Dickinson). Western blot and immunoprecipitation Whole cell extracts were prepared by lysis with 20 mM Tris, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.5% Triton X-100 supplemented with complete protease inhibitor cocktail (Complete Mini; Roche, Basel, Switzerland), 1 mM dithiothreitol (DTT; Amersham Biosciences, Little Chalfont, Buckinghamshire, United Kingdom), 1 mM phenyl-methyl-sulfonyl fluoride (PMSF; Sigma-Aldrich), 10 µM sodium fluoride (Sigma-Aldrich), 1 µM okadaic acid, and 1 mM sodium orthovanadate (Calbiochem, San Diego, CA). Extraction of cytosolic fractions was carried out as described elsewhere.37 Immunoprecipitation was performed according to standard protocols.
Proteins were subjected to SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with the following primary antibodies: CK2 RNA interference, plasmids, transfection, and luciferase assay
For RNA interference, 25 to 35 x 106 U266 MM cells in log phase of growth were transfected by electroporation with 600 pmol siGlo-Red or 600 pmol siGlo-Red plus control siRNA pool or CK2 Confocal microscopy MM cells seeded on polylysine-coated glass slides for 10 minutes were then fixed in 4% paraformaldehyde for 10 minutes at RT and permeabilized in PBS plus 0.1% Triton X-100 and sodium citrate (10 minutes at RT) before proceeding to blocking and staining. Specimens were mounted in Vectashield medium (Vector Laboratories, Peterborough, England) and analyzed using a dual-photon confocal microscope equipped with a 60x/1.4 numeric aperture (NA) or 100x/1.3 NA objective and fluorescence filters set for excitation at 488 nM and 595 nM (Bio-Rad, Milan, Italy), and images were acquired with Lasersharp 2000 software (Bio-Rad, Milan, Italy). Secondary conjugated antibodies Alexa Fluor 488 goat antirabbit (A-11008), Alexa Fluor 594 goat antirabbit (A-11012), and Alexa Fluor 594 donkey antigoat (A-11058) were purchased from Molecular Probes Europe (Leiden, The Netherlands). Statistical analysis Data were evaluated for their statistical significance with the 2-tail paired Student t test. Values were considered statistically significant at P values below .05.
CK2 activity and expression in MM cells We investigated CK2 activity and protein levels in MM cell lines (OPM2, U266, and RPMI 8226) and purified CD138+ plasma cells from MM patients (whose clinical features are summarized in Table 1). As controls, we used in vitro-generated normal polyclonal plasma cells/plasmablasts29 and purified normal CD19+ B lymphocytes from peripheral blood, spleen, or tonsil.
An in vitro kinase assay on whole cell extracts using a CK2-specific peptide40 as substrate revealed that CK2 activity was considerably higher in tumor cells than in normal plasma cells and lymphocytes (Figure 1); the activity was higher in cell lines (P < .02) and in cells from 6 of 7 patients (P < .05, with an increase of up to 5-fold). We then examined CK2 catalytic subunit levels. They paralleled the enzymatic activity and were higher in MM cell lines and in samples from MM patients (4 of 5 examined) than in normal plasma cells and lymphocytes (Figure 2A, top panel). Comparable results were obtained by Western blot experiments for the noncatalytic subunit of CK2, but the ' catalytic subunit was not significantly detected in any sample (not shown). We also investigated the phospho-radiolabeling of endogenous proteins on incubation of cell extracts with  33P-ATP and Mg++, and we observed significantly higher levels of protein phosphorylation in MM cells, with a pattern different from that found in whole cell extracts from normal plasma cells and lymphocytes. Remarkably, the intensity of the bands corresponding to some of these phosphorylated proteins was significantly reduced with the addition of the selective CK2 inhibitor TBB (data not shown). To confirm CK2 expression levels in MM cells as observed on Western blot, we performed immunofluorescence experiments on freshly isolated bone marrow cells from MM patients in whom more than 20% of plasma cell infiltrate was in the bone marrow. As seen in 2 representative patients (Figure 2B), we observed more intense CK2 staining in CD138+ plasma cells than in the remaining cell population, thus indicating that this kinase exhibits a preferential pattern of expression in malignant MM cells. Altogether, these results suggest that CK2 protein levels and enzymatic activity are higher in MM cells than in normal plasma cells, consistent with a role for CK2 in the biology of the malignant plasma cell. CK2 blockage causes apoptosis in MM cells To study the role of CK2 in MM cells, we used 3 synthetic CK2 inhibitors (2 of which are structurally unrelated) previously described as strong and specific. An MTT-based assay was used to perform cell proliferation/viability analysis.7,31-35 As shown in Figure 3A, MM cells cultured for 48 hours in the presence of increasing concentrations of the compounds K27, TBB, and IQA displayed a dose-dependent growth arrest. To prove that these inhibitors specifically affected CK2 kinase activity inside MM cells, we also performed kinase assays against a CK2-specific peptide substrate using K27 or TBB-treated MM cell lysates. As shown in Figure 3A, CK2 kinase activity was efficiently hampered in MM cells by K27 and TBB in a dose-dependent manner.
Moreover, the specificity of the CK2 inhibitors in causing growth inhibition was also determined by assessing their effects in cells transiently transfected with a CK2
Next, we tested whether the apoptotic process could be partly responsible for this effect. OPM2 and RPMI 8226 MM cells were treated with K27 (at 2.5-µM and 5-µM concentrations) for 24 hours and then were subjected to annexin V/propidium iodide (AV/PI) staining and FACS analysis. The percentage of AV/PI-negative (not apoptotic) cells after treatment progressively decreased in a dose-dependent manner for both cell lines (Figure 3B), thus indicating that apoptosis is triggered by CK2 blockage.
To confirm that CK2 protein sustains MM cell survival, we performed RNA-interference experiments. U266 MM cells were transiently transfected with irrelevant siRNA oligonucleotides or siRNA oligonucleotides directed against the Next, to check whether the inhibition of CK2 caused apoptosis of plasma cells in MM patients, we treated freshly isolated total PB or BM cells from MM patients with K27 (at 5-µM concentrations) for 8 hours. Remarkably, most of the CD138+ malignant plasma cell fractions underwent apoptosis, but the effect was less pronounced in the CD138- population (Figure 3D; top, results of 1 of 4 representative FACS experiments; bottom, results obtained by treating 4 different primary MM samples). Together, these results, showing MM cell apoptosis on pharmacologic and siRNA-induced down-regulation of CK2, support the solid evidence of a role for this kinase in MM cell survival. Blockage of CK2 in MM cells causes activation of the extrinsic and intrinsic apoptotic pathways and enhances the cytotoxic effect of melphalan We then sought to investigate the molecular consequences of CK2 blockage. OPM2 and RPMI8226 MM cells were treated with increasing doses of K27, and the activation of the caspase pathways was evaluated by Western blotting. As shown in Figure 4A, pro-caspase-8, pro-caspase-9, and pro-caspase-3 cleavage and the consequent cleavage of the downstream PARP enzyme were elicited in a dose-dependent manner by the addition of K27 to the cultures. Moreover, the release of cytochrome c and SMAC/DIABLO from mitochondria in the cytosol could be demonstrated in both cell lines (Figure 4B). Similar results were obtained using other MM cell lines and other CK2-specific inhibitors, notably TBB and IQA (data not shown). These findings indicate that molecular pathways associated with the induction of apoptosis are elicited in MM when CK2 is inhibited. Because we observed the induction of MM cell apoptosis by the ablation of CK2 function, we next tested whether CK2 blockage could also render MM cells more sensitive to the cytotoxic effect of a conventional chemotherapeutic agent used in MM therapy— namely, melphalan. To this end, we treated OPM2 cells with increasing doses of melphalan in the presence of vehicle (0.1% DMSO) or K27 at subtoxic concentrations (0.3 and 1.0 µM). At both concentrations of K27, the dose of melphalan that induced 50% growth inhibition was significantly lower than the corresponding dose in the absence of CK2 blockage (Figure 4C; * P < .01, ** P < .05). These data show that CK2 enhances MM-cell response to melphalan-induced cytotoxicity. IL-6 and IGF-I affect CK2 kinase activity and are unable to promote cell growth when CK2 is inhibited Next, we investigated whether pivotal growth signals for MM cells, namely IL-6 and IGF-I,42,43 could compensate for the loss of CK2 function, thus inducing cell growth even in the presence of CK2 inhibition. As shown in Figure 5A, IL-6 and IGF-I were unable to reverse the dose-dependent cytotoxic effect on MM cells caused by TBB or K27 on OPM2 MM cells. Comparable findings were obtained in similar experiments using IQA and other MM cell lines (data not shown). These results suggest that the pathways activated by these 2 growth factors in MM cells might rely on an intact CK2 function. Inhibition of CK2 kinase affects IL-6 signaling To gain more insight into the molecular mechanisms underlying the observed growth arrest and apoptosis on CK2 inhibition even in IL-6-stimulated MM cells, we analyzed the IL-6-triggered molecular events44 in OPM2 MM cell line and in purified CD138+ malignant plasma cells from MM patients. OPM2 cells were serum starved for 4 hours, incubated in serum free-medium with DMSO 0.1% or the CK2 kinase inhibitor K27 (5 µM) or TBB (20 µM) for another 2 hours, and exposed to IL-6 (10 ng/mL) for different time periods. Immunoblot analysis with antibodies against phospho-Tyr702-STAT3, phospho-Ser727-STAT3, and total STAT3 was then performed to check for activation of this IL-6-triggered signaling cascade (Figure 5B). On IL-6 stimulation of OPM2 cells (panels at the left), there was an increase in the levels of phosphorylated STAT3 at residues Tyr702 and Ser727; however, when OPM2 MM cells were preincubated with the CK2 inhibitor K27 or TBB, the IL-6-induced time-dependent STAT3 phosphorylation was decreased, with the strongest effect observed after 5 minutes and affecting Ser727 phosphorylation. In separate experiments (panels at the right), CD138+ MM cells from patients were left untreated or were treated with IL-6 for 5 minutes after incubation in the presence of 0.1% DMSO or with 5 µM or 20 µM TBB. As shown, we observed that even in freshly purified malignant plasma cells, IL-6-triggered STAT3 activation could be hampered by the blockage of CK2 (in these experiments, STAT phosphorylation on Tyr702 was analyzed). A possible inhibition of Jak tyrosine kinases and a general effect on total tyrosine phosphorylation is highly unlikely because the inhibitors used did not display any activity on a panel of tyrosine kinases tested34,35 and the pattern of total phosphotyrosine remained unchanged in treated MM cell lysates (data not shown). Although the exact significance of these findings warrants further investigation, they suggest that proper IL-6-triggered STAT3 activation is modulated by CK2 and involves this kinase in a crucial growth-promoting pathway in MM.
Regulation of I B degradation by CK2 in MM cells
We next asked whether CK2 could regulate the NF-
We then sought to analyze I B basal turnover once CK2 function was abrogated. We treated MM cells with vehicle or with TNF- or IQA or both for 12 hours and assessed total IB levels by Western blot. TNF- stimulation led to a considerable reduction in IB compared with untreated samples. On the contrary, the presence of IQA caused IB to accumulate. The addition of both TNF- and IQA resulted overall in accelerated IB degradation that was significantly less pronounced than the sample treated with TNF- alone (Figure 6C, left panels); similar results were obtained using K27 (not shown). Last, we checked the levels of IB in U266 MM cells transfected with control siRNA or CK2-specific siRNA. After 72 to 96 hours of transfection, we could observe that in MM cells in which CK2 was silenced, IB levels were more abundant. (Figure 6C, right panels). These results demonstrated that CK2 might be involved in stimulus-induced and basal IB phosphorylation and degradation in MM cells.
CK2 inhibition affects NF-
Next, because we observed that CK2-regulated I
CK2 associates with NF- B p50/p105 in MM cells
Given our findings, we wanted to determine whether CK2 could physically interact with NF-
In the present study we have shown that protein kinase CK2 is aberrantly active in MM cells and controls their survival. We have also demonstrated that CK2 might be involved in the control of the IL-6 and NF- B signaling pathways in these cells, and we have shown a previously unrecognized interaction of this kinase with the NF-B member p50/105.
The increased CK2 protein levels and activity we found in neoplastic MM cells (compared with normal in vitro-generated polyclonal plasma cells and B lymphocytes; Figures 1, 2) was likely not simply related to the proliferation rate because it was comparable in highly proliferating MM cell lines, in slowly proliferating purified CD138+ MM cells freshly isolated from the BM of patients, and in more aggressive extramedullary myelomas (Figure 1; Table 1), suggesting that the perturbation of CK2 function is a direct consequence of the transformed phenotype rather than an epiphenomenon of high cell proliferation, as indicated in other studies.12 Moreover, this concept was corroborated by the lower levels of CK2 expression and activity we detected in normal plasma cells, a significant fraction of which were highly proliferative.29 The biologic consequences of increased activity of CK2 in MM cells are predicted to be relevant because this kinase normally displays basal enzymatic activity directed toward a number of substrates involved in several important cellular processes. Therefore, physiologically, CK2 levels have to be tightly regulated to avoid perturbations of cell homeostasis and behavior. Given that we found that the increase in CK2 protein equally affected
We also found that MM cells contain an array of abnormally phosphorylated proteins, some of which are likely to be CK2 targets given their responsiveness to CK2 inhibitors. This finding indicated that plasma cell malignant transformation is accompanied by a deregulated signaling to which CK2 appears to contribute. Our experiments also showed that the pharmacologic disruption of CK2 signaling in malignant plasma cells with specific inhibitors caused apoptosis (Figures 3, 4A-B). The compounds used in some cases were structurally unrelated, and the concentrations required to induce the cellular effects were roughly proportional to their Ki for CK2 in vitro.31-33 Moreover, our findings that cells in which a CK2 mutant resistant to the inhibitors was transfected are resistant to cell death induced by the same inhibitors indicate that they act specifically on CK2.
Furthermore, we also provided strong molecular evidence of the pivotal role of CK2 kinase in MM cell survival by showing that knocking down CK2 It was known that CK2 is required for cell survival from in vivo deletion studies46,47 and other investigations, many of which were carried out with the same inhibitors used in this study.7,8,48-51 Our observation that both the intrinsic and the extrinsic apoptotic pathways are triggered on CK2 inhibition in MM cells might reflect a role for this kinase in checkpoints shared by the 2 apoptotic cascades in this cell type. This role might derive from the CK2-dependent control of the rate of caspase-triggered degradation of certain protein substrates as a more general mechanism of which a number of examples are already known.7,10,11,49,52 As previously demonstrated, it is also possible that CK2 prevents the activation of pro-caspase-8 in the absence of death receptor engagement.6 CK2 inhibition enhances TRAIL-induced apoptosis of rhabdomyosarcoma, colon cancer, and prostate cancer cells51,53,54 as well as MM cells (F.A.P., B.M., and G.S., unpublished data, 2006), and recently CK2 was found to control the activation of pro-caspase-8 by caspase-2.55 Furthermore, we have also revealed a role for CK2 in MM cell survival by showing that this kinase protects MM cells from the cytotoxic effect of melphalan, an alkylating DNA agent used as a chemotherapeutic drug in MM therapy (Figure 4C). In this regard, CK2 has recently been shown to play a role in the repair of DNA damage.56 In the present work, we have also shown that IL-6 and IGF-I, 2 MM cell survival factors, very likely involve CK2 activity in their signaling cascades because we found that these 2 important MM growth factors were unable to induce cell growth in the absence of functioning CK2 (Figure 5A). Moreover, with regard to the IL-6 pathway, we demonstrated that CK2 might be an important regulator of the downstream intracellular signaling events triggered by this cytokine, particularly STAT3 phosphorylation on Tyr702 and Ser727 (Figure 5B). Recently, this pathway has been demonstrated to be constitutively activated and to mediate MM cell survival.57 Based on our results, however, the exact mechanism through which CK2 controls the rate of IL-6-dependent STAT3 activation is still unclear. It is unlikely that CK2 inhibitors affected tyrosine phosphorylation because we could not find differences in the patterns of total phosphotyrosine expression in K27- or TBB-treated MM cells. Because Tyr702-STAT3 phosphorylation was found to be impaired, it is conceivable that CK2 could act on early events turned on in this pathway. These results, even though warranting further study, are remarkable because they unravel novel molecular interactions important for MM cell growth. The identification of CK2 targets downstream from IL-6 and other stimuli, such as insulin-like growth factor-I (IGF-I), vascular endothelial growth factor (VEGF),58 and fibroblast growth factor (FGF),59 would add insight into the regulation of these pathways in MM. Such identification is under way in our laboratory.
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Abstract
Introduction
), CD138+ purified plasma cells from 7 MM patients (
), in vitro-generated normal plasma cells (nPCs), and purified CD19+ B lymphocytes from healthy donors (
). Samples N1 to N3 were from peripheral blood; samples N4 and N5 were from tonsil; samples N6 and N7 were from spleen. Data are mean ± SD.
). OPM2 cells were cultured with the indicated stimuli in the absence or in the presence of TBB, K27, or both and then were subjected to MTT-based viability assay 48 hours later. Data are the mean ± SD. (B) Western blot analysis of the IL-6/STAT3 cascade in MM cells on CK2 inhibition. MM cells were serum starved for 4 hours (cell lines OPM2) or 2 hours (freshly isolated MM plasma cells from patients), incubated for 2 hours with 0.1% DMSO, (5 µM) K27, or 20 µM TBB and then stimulated for the indicated time points with 10 ng/mL IL-6. For MM cell lines, the levels of phospho-Tyr702 STAT3, phospho-Ser727 STAT3, and total STAT3, and for MM cells from patients the levels of phospho-Tyr702 STAT3 and total STAT3 were assessed, as indicated. 
