Analysis of the anti-proliferative and the pro-apoptotic efficacy of Syk inhibition in multiple myeloma
- Ruth-Miriam Koerber†1,
- Stefanie Andrea Erika Held†1,
- Annkristin Heine1,
- Philipp Kotthoff1,
- Solveig Nora Daecke1,
- Anita Bringmann1 and
- Peter Brossart1Email author
© Koerber et al. 2015
Received: 1 July 2015
Accepted: 20 July 2015
Published: 5 August 2015
Multiple myeloma (MM) is a clonal B cell malignancy characterized by proliferation of malignant plasma cells in the bone marrow. Despite high-dose melphalan therapy with autologous stem cell transplantation (ASCT) and the introduction of immunomodulatory drugs like bortezomib or lenalidomide, that have been associated with improved survival, MM is still incurable and new treatment options are needed. In B cell malignancies such as chronic lymphocytic leukaemia (CLL) or diffuse large B cell lymphoma (DLBCL), Syk (spleen tyrosine kinase) inhibitors have shown promising in vitro and first clinical results. In our study, we analyzed the potential of Syk as a target in MM.
The MM cell lines AMO-1, U266 and RPMI8226 and primary MM cells were treated with the Syk inhibitors BAY61-3606, R406 or Piceatannol and proliferation, migration and apoptosis induction were analyzed. Effects on involved intracellular signaling cascades were determined by Western blotting. Furthermore, we analyzed synergistic and additive effects of Syk inhibitors in combination with established anti-myeloma drugs and experimental inhibitors (e.g. PI-3-Kinase inhibitor NVP-BEZ235).
Incubation of MM cell lines as well as primary MM cells with Syk inhibitors resulted in a reduced proliferation and stromal cell-derived factor-1 alpha (SDF-1 alpha) induced migration that was accompanied by a concentration dependent inhibition of the MAP-Kinase, characterized by reduced phosphorylation of ERK an p38 molecules, and NF-kappaB signalling pathways. Furthermore, Syk inhibition induced apoptosis in MM cells in a dose-dependent manner, characterized by reduced expression of pro-caspase 3, increased PARP-1 cleavage and enhanced release of cytochrome c. In addition combined treatment of MM cells with Syk inhibitors and NVP-BEZ235 (dual PI3-kinase/mTOR inhibitor) or MAPK inhibitors (PD98059, SP600125, U0126, SB203580) resulted in increased apoptotic activity of the drugs.
Our results show that Syk inhibition might represent a promising new treatment option in MM with an increased efficacy when combined with MAP kinase inhibitors. Furthermore, our study strongly underlines the potency of Syk inhibitors as a potential therapeutic treatment option for MM patients.
Multiple myeloma (MM) is a B cell clonal malignancy characterized by proliferation and accumulation of malignant plasma cells within the bone marrow (BM) . Several recently introduced therapies such as bortezomib, thalidomide or lenalidomide as well as high dose chemotherapy and autologous stem cell transplantation (ASCT) have improved the prognosis and overall survival of patients with MM. However, MM is still an incurable disease [2, 3].
Drug resistance represents one of the major difficulties to overcome in patients with malignant disease which often results in failure to eliminate minimal residual tumor cells [4, 5]. Studies performed in an attempt to determine the factors mediating drug resistance and survival of malignant cells led to the characterization of novel pathways and identification of potential targets for anticancer therapies [6, 7]. In malignant B cells and plasma cells, the involvement of signalling pathways via Janus kinases 1/2 (JAK1/2) and the signal transducers and activators of transcriptions (STAT) molecules as well as the events mediated by the phosphatidylinositol-3 kinases (PI3-K) and mitogen activated kinases (MAPK) were found to be critical for cell growth, differentiation and survival [8–10].
In addition, emerging evidence suggests that spleen tyrosine kinase (Syk), an intracellular tyrosine kinase, plays a central role upon activation of cells by B or T cell receptors (BCR or TCR) as well as by cytokines or adhesion molecules [11–13]. Syk activation is mediated when sarcoma kinases (Src kinases) phosphorylate conserved sequences within receptors containing immune tyrosine activation motifs (ITAMs) [14, 15]. Once activated, Syk propagates the phosphorylation of further downstream molecules including the PI3-K and MAP-kinase pathways [16, 17].
Syk and the zeta-chain-associated protein kinase 70 (ZAP-70) are the only members of the Syk family kinases. Syk was first identified by isolation of tyrosine kinase activity found in the thymus and spleen leading to the cloning of the 72 kDa protein [18, 19]. Mice with genetically disrupted Syk are deficient in B cell maturation and expansion and die prenatally due to severe bleedings. This indicates that Syk might play a role in angiogenesis and maintaining of vascular integrity [20, 21]. Subsequently, Syk has been found to be involved and play a broad role in hematopoietic cell signalling such as FCeRI receptor in mast cells, FCgRIIA in macrophages and platelets as well as the antigen receptors of B and T cells [22, 23]. In addition, it was demonstrated that Syk is also expressed in fibroblasts, hepatocytes, neuronal cells and vascular endothelium . Syk was shown to function as an oncogene and to be a negative regulator of tumorigenesis in some malignant diseases such as breast cancer, B and T cell lymphoma and myelodysplastic syndromes [25, 26].
Recently, several reports on preclinical studies demonstrated that Syk inhibition might represent a rational therapeutic target in B cell malignancies such as chronic lymphocytic leukaemia (CLL), diffuse large B-non-Hodgkin lymphoma (DLBCL), follicular and mantle cell lymphoma by interfering with the signalling promoted by the activation via the BCR, cytokines and stromal factors [27–29]. In DLBCL and CLL, first clinical experiences with the clinically available Syk inhibitor R406 provided encouraging and promising results in heavily pre-treated patients [30–32].
In our study we analyzed the effects of the three currently available Syk inhibitors, Piceatannol, Bay-61-3606 and R406 on the viability and functional properties of myeloma cells in order to provide a mechanistic understanding and a rationale for a possible use of these compounds as therapeutic option.
Syk inhibitors reduce the proliferation and migration of MM cells
Additionally, we performed MTT (tetrazolium salt 3-4,5-dimethylthiazol-2yl]-2,5-diphenyl-tetrazolium bromide) cytoxicity assays to ensure that the effects on migration and proliferation induced by Syk inhibitors are not due to cytotoxicity as a mechanism of action. The MTT assays showed no decline in cell viability when incubated with Syk inhibitors. This was proved in three cell lines at three different concentrations. Based on these results, the effects of the various Syk inhibitors on migration and proliferation are not due to cytotoxic effects (see Additional file 1).
Inhibition of Syk induces apoptosis in MM cells
Likewise, we wanted to determine the efficacy of the compounds to induce apoptosis in purified MM cells. As shown in Fig. 4c, incubation of primary MM cells from patients with plasma cell leukemia with R406, Piceatannol or Bay61-3606 significantly reduced cell viability.
Because bone marrow (BM) microenvironment promotes MM survival and drug resistance, we next determined the apoptosis induction in MM cells in this context. The majority of MM patients expresses the chemokine receptor CXCR4 on neoplastic MM cells which is crucial for myeloma cell migration and homing to the BM microenvironment. Additionally, it supports tumor cell survival and mediates chemotherapy resistance. For contacts of cell–cell and cell to extracellular matrix, a group of cellular adhesion molecules such as CD11a or CD49d are essential, likewise their ligands, i.e. CD102 (ICAM 2). Surface antigens were examined by flow cytometric analysis. Despite reduced migration of cells when treated with Syk inhibitors, we could not detect any effect on the expression of CXCR4, CD49d or CD102 on the cell surface of MM cells (data not shown).
Combined treatment of MM cells with Syk inhibitors
NVP-BEZ235, an orally available dual PI3 kinase/mTor inhibitor, was recently shown to suppress survival and growth of MM cells in preclinical studies. We found that combined treatment of MM cells with Piceatannol and NVP-BEZ235 significantly increased the cytotoxic effect as compared to each drug alone (Fig. 7a). This was predominantly observed in AMO-1 cells. Significant results could be observed in both cell lines when correlated to Piceatannol, in U266 cells significances correlated to NVP-BEZ235 were not significant, but in AMO-1 they were significantly higher (see Additional file 1).
As mentioned above, MAP-Kinases play a critical role in differentiation, cell growth and survival in MM. Combination of Syk inhibitors with MAP-Kinase inhibitors such as U0126 (a potent, selective inhibitor of MAP2K), PD98059 (an inhibitor of MAP2K/MEK), SP600125 (a selective inhibitor of JNK) and SB203580 (a selective inhibitor of p38 MAPK) resulted in synergistic cytotoxic effects in combination with U0126 (Fig. 7b) and PD98059 (Fig. 7d). Additive effects were observed when combined with SP600125 (a selective inhibitor of JNK, Fig. 7c) or SB203580 (Fig. 7e).
In our study we demonstrate that a proportion of MM cells express Syk and pSyk and Syk inhibitors R406, Piceatannol and Bay61-3606 effectively block proliferation, migration and survival of MM cells. This indicates that Syk inhibition might represent a potential new therapeutic option in the treatment of MM. However, the compounds we used in our experiments are not entirely specific for Syk and do display some lower activity against JAK, Lck, Lyn, STAT3 and STAT 5 as well as against some potential additional, yet to be determined, tyrosine kinases [34, 36, 37].
R406 and BAY61-3606 have previously been reported to inhibit proliferation and to induce apoptosis in non-Hodgkin lymphoma (B-NHL) and CLL cells [27, 38]. R406 is currently used in clinical trials and has demonstrated promising activity in patients with B-NHL, ITP and rheumatoid arthritis [39, 40]. The so far reported side effects of the compound are generally mild and consist primarily of gastrointestinal side effects and neutropenia, which was dose related and rapidly reversible upon dose reduction or discontinuation of treatment . Interestingly, in mice treated with R406, additional immunomodulatory effects including lymphopenia, reduced thymus and spleen weight and decreased cellularity of the BM were observed, which resolved within 2 weeks after stopping the drug application .
MM cells express the chemokine receptor CXCR4 and its ligand SDF-1α controls their α4β1-mediated adhesion to the vascular adhesion molecule 1 (VCAM-1), fibronectin and endothelial cells as well as their transendothelial migration and homing in the BM [43, 44]. MM cells are highly dependent on BM microenvironment, where signals provided by cytokines and adhesion molecules promote the survival, proliferation and expansion of MM cells [45, 46]. In our study we found that treatment of MM cells with Syk inhibitors blocked the SDF-1α mediated migration of MM cells and abrogated the survival signals provided by IL-6, TLR ligands or BM stroma. These blocking effects were accompanied by the inhibition of downstream pathways as demonstrated by reduced ERK1/2 and p38 phosphorylation and nuclear localized RelB and RelA, indicating that MAP-Kinase and NF-κB signalling pathways are involved in the observed inhibitory effects.
Syk inhibitors efficiently induced apoptosis in established MM cell lines and purified MM cells. This was characterized by an enhanced caspase-3 activity and PARP-1 cleavage, a known caspase-3 substrate. The induction of cell death was inhibited by the addition of the pan-caspase inhibitor zVAD-FMK, further confirming the critical role of caspases in this setting. In addition, we found that Syk inhibition resulted in enhanced release of cytochrome c indicating that the apoptotic cell death was mediated via the internal, mitochondrial pathway.
Our findings are in line with previous findings in CLL cells  where treatment of CLL cells with BAY61-3606 caused caspase-3 activation, cleavage of PARP-1 and loss of mitochondrial potential. However, in contrast to this report we could not detect any regulation of MCL-1 protein expression in our experiments indicating that the effects and mechanisms induced by the utilized compounds may vary depending on the used cell lines and models.
The introduction of recently developed agents such as the proteasome inhibitor bortezomib or lenalidomide has dramatically improved the prognosis and overall survival in MM patients [47, 48]. Some of the induced effects by these targeted therapies are mediated by interfering with the MAP-Kinase and NF-kB signalling pathways. Therefore, we hypothesized that Syk inhibition might represent a rationale combination partner. While there were no additive effects by bortezomib, the combined treatment of MM cells with MAP-Kinase inhibitors resulted in an increased cytotoxic effect. In addition, we observed that simultaneous exposure to NVP-BEZ235 , an orally available dual inhibitor of PI3 kinase/mTor signalling significantly enhanced the efficacy of Syk inhibitors.
Syk inhibitors already showed promising results in B cell malignancies such as CLL and DLBCL. Our data show successful findings of Syk inhibition in MM. Syk inhibition in MM resulted in decreased proliferation and migration of MM cells. Additionally, Syk inhibition induces apoptosis and is effective in combination with established anti myeloma drugs and experimental new kinase inhibitors, such as a PI3-Kinase inhibitor. In summary, our study provides a mechanistic insight and a rationale for Syk inhibition as a novel therapeutic option for the treatment of MM.
The cells lines AMO-1, U266, RPMI8226 and MM1-S were a kind gift from Helmut Salih from the University Hospital Tuebingen. The cells were cultured in RP10 medium (RPMI 1640 containing GlutaMAX, supplemented with 10% heat-inactivated fetal calf serum and 100 units/ml penicillin/streptomycin, all from Gibco, Karlsruhe, Germany) in a humidified atmosphere (37°C, 5% CO2). Cells were seeded into 75 cm2 flasks at 104/10 ml/flask (BD Heidelberg, Deutschland).
After informed consent, blood samples were collected from patients with multiple myeloma hospitalized at the University Hospital Bonn. PBMCs were isolated by Ficoll/Paque (Biochrom, Berlin, Germany) density gradient centrifugation.
Cells were preincubated with zVAD (Bachem Distribution Services GmbH, Weil am Rhein, Germany) for 1 h. Piceatannol, applied at concentrations of 10, 25 and 50 µM (Sigma-Aldrich Chemie GmbH, Munich, Germany), R406, applied at concentrations of 1 and 5 µM, and BAY61-3606, applied at concentrations of 1, 2.5 and 5 µM, (Sigma-Aldrich Chemie GmbH, Munich, Germany) were added for 24 h. After 24 h, cells were prepared for further experiments.
To analyse possible synergistic effects we tested in addition thalidomide(MP Biochemicals, Solon, Ohio, USA), lenaldomide (Selleck Biochemicals, Houston, TX, USA) TRAIL (R&D Systems, Minneapolis, MN, USA), NVP-BEZ235 (Novartis Deutschland GmbH, Nürnberg, Germany), Bortezomib (Millenium Pharmaceuticals Limited, Cambridge, MA, USA) and five MAPK Inhibitors (PD98059, SB203580, SP600125, U0126, SB201290 all purchased from Tocris Bioscience, MO, USA).
Preparation of whole cell lysates and nuclear extracts
For the generation of whole cell lysates, cells were lysed in RIPA buffer containing 1% Igepal CA-630, 0.5% Na-Deoxycholat, 0.1% SDS, 2 mM EDTA, 2 µg/ml Aprotinin, 1 mM PMSF. Protein concentration was measured using a bicinchoninic acid (BCA) assay (Pierce, Perbio Science, Bonn, Germany).
Nuclear extracts were prepared as described. In brief, 106 cells were incubated in 400 µl Buffer A containing 10 mM HEPES pH 7.9, 10 mM KCL, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT and 0.5 mM PMSF. Cellular membranes were destroyed by addition of 10% Igepal CA-630 and vigorous vortexing. Nuclei were pelleted by centrifugation and resuspended in buffer C containing 20 mM HEPES pH 7.9, 0.4 M sodium chloride, 1 mM EDTA, 1 mM EGTA, 1 mM DTT and 0.5 mM PMSF. Nuclear proteins were recovered by centrifugation for 5 min at 20,000g.
PAGE and Western blotting
20 µg of whole cell lysates or nuclear extracts of 1 × 106 cells were separated by SDS-PAGE and subsequently transferred onto nitrocellulose membranes (Whatman, Dassel, Germany). The blots were probed with antibodies against bcl-x, cytochrome c, caspase-3, XIAP (all purchased from BD Biosciences, Heidelberg, Germany), ERK 1/2, mcl-1, survivin, Rel-A, Rel-B (all purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, USA), p38, pp38 (all purchased from Cell Signaling Technology®, MA, USA). GAPDH (Santa Cruz Biotechnology, Inc., Santa Cruz, USA) was used as a loading control. Signals were detected using the ECL detection system (GE Healthcare, Munich, Germany).
To estimate the expression of the protein tyrosine kinase syk in our multiple myeloma cell lines we performed immunoprecipitation. Cell lysates were assembled as explained above (see preparation of whole cell lysates). For immunoprecipitation Dynabeads® Protein A or Protein G from Invitrogen (Life Technologies GmbH, Darmstadt, Germany) were used. The primary antibodies (3 µl/sample) were pooled to 50 µl Dynabeads® Protein A and G. To isolate the generated complexes we washed (each with 200 µl AB Binding und Wasch Puffer) the solution and the suspension was passed by two magnets where the magnetic beads were reversibly trapped in the magnetic field and formed a compact dense plug.
To avoid co-elution of the antibody we cross linked the primary antibody to Dynabeads®. DSS was eluted in DMSO and a working solution consisting of 20× Coupling Buffer (2.5 µl), 2.5 mM DSS and ultrapure water (38.5 µl) was established. The Dynabeads®–antibody complex was resuspended in 50 µl working solution and incubated for 30–60 min in this solution.
Subsequent the Beads were resuspended with the whole cell lysates (100–500 µg protein). The Dynabeads®-antibody–antigen-complex was then incubated for 1 h, followed by a couple of steps of washing (3 × 200 µl Wash Buffer, 1× 100 µl Wash Buffer), magnetic field exposition and resuspending in 20 µl Elution buffer. Afterwards the pure precipitate was eluted from the beads of the supernatant and then analyzed by western blotting (explanation see PAGE and Western Blotting). The blots were probed with an antibody against p-syk (purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, USA).
Measurement of apoptosis
Apoptosis was measured by the method of Nicoletti . 1 × 105 cells were incubated in a hypotonic buffer containing 1% sodium citrate, 0.1% Triton X-100, 50 µg of propidium iodide per ml and subsequently measured by flow cytometry on a Cytomics FC 500 (Beckmann Coulter, Krefeld, Germany) using CXPAnalysis software. The hypodiploid cells to the left of the 2N peak were considered as apoptotic.
Fluorimetric assay of caspase-3 activity
Caspase activity was determined from cytosolic extracts, by the cleavage of the fluorogenic caspase substrate N-acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin (DEVD-AMC, Bachem, Heidelberg, Germany). In brief, for fluorimetric assay of caspase activity, cytosolic cell extracts were prepared by lysing cells in a buffer containing 0.5% NP-40, 20 mM HEPES pH 7.4, 84 mM KCl, 10 mM MgCl2, 0.2 mM EDTA, 0.2 mM EGTA, 1 mM DTT, 5 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin and 1 mM PMSF. Cell lysates were incubated with 50 mM DEVD-AMC in 200 µl buffer containing 37.5 mM HEPES pH 7.3, 75 mM NaCl, 7.5% sucrose, 0,075% CHAPS and 10 mM DTT. The release of aminomethylcoumarin was determined by fluorometry using an excitation length of 360 nm and an emission wave-length of 460 nm.
Phenotyping of MM cells
Cells were treated as mentioned above. After incubation staining for fluorescence-activating cell sorting (FACS) followed. 5 × 105 cells/tube were incubated with 10 µl anti-CD49d PE (purchased from BD Biosciences Europe, Erembodegem, Belgium), CD102 PE (purchased from BD Biosciences Europe, Erembodegem, Belgium) or CXCR4 PE (purchased from Beckman Coulter GmbH, Krefeld, Germany) and 10 µl anti-mouse IgG1, κ PE for 30 min at 18–22°C in the dark. When examined the next day, cells were fixed in 250 µl FACS-Buffer and 250 µl 2% formaldehyde and then analyzed by flow cytometry.
Multiple myeloma cells were seeded into 96-well microtiter plates (Greiner Bio-One GmbH, Frickenhausen, Germany) at 106, 105 or 104 cells/well and incubated for 24 h at 37°C, 5% CO2. The assay was performed in fourfold replicates. Cells were pulsed with 3H-thymidine (Hartmann Analytic, Braunschweig, Germany) and incubated for another 16 h at 37°C, 5% CO2. Cells were harvested then using a Filter Mate Harvester (Perkin Elmer, Waltham, USA) and uptake of 3H-thymidine was measured by a Microbeta TriLux (Perkin Elmer).
Cells were seeded into transwell chambers (8 µm, BD) in 24-well plates (BD) and migration was induced through SDF-1α (Biovision, Mountain View, CA, USA).
After 3 h migrated multiple myeloma cells were analyzed by counting gated cells for a time interval of 60 s on a Cytomics FC 500 (Beckmann Coulter).
TACS®-MTT cell proliferation assay
Measurement of cell viability and proliferation were performed with TACS®-MTT assay to quantify the cell population‘s response to Syk inhibitors. The yellow tetrazolium salt 3-4,5-dimethylthiazol-2yl]-2,5-diphenyl-tetrazolium bromide (MTT) is reduced by metabolic active cells and can then be quantitated by spectroscopic means. Measurement of proliferation and cell viability can be determined over the metabolic events that lead to apoptosis or necrosis.
Cells were cultured and treated with Syk Inhibitors as mentioned above and then plated in the wells. After adding the MTT agent, cells were incubated in the incubator until a purple dye was visible. In the next step Detergent Reagent was added and then the flat-bottomed 96-well plates were read after 2 h with the microplate reader at 570 nm. The average value was determined from triplicate reading.
Piceatannol (Sigma-Aldrich Chemie GmbH, Munich, Germany), a plant secondary product (3,4,3,5-tetrahydroxy-trans-stilbene), was identified as a tyrosine kinase inhibitor in thymocytes that also displays some activity against Lyn and STATs. The used concentrations were adjusted to reported working concentrations in literature. BAY61-3606 (Sigma-Aldrich Chemie GmbH, Munich, Germany), (orally available 2-(7-(3,4-dimethoxyphenyl)-imidazol(1,2-c)pyrimidin-5-ylamino)nicotinamide dihydrochloride), is a specific Syk inhibitor that acts as an ATP competitor blocking its catalytic activity and was shown to inhibit antigen-induced airway inflammation in rodents . The used concentrations were adjusted to reported working concentrations in literature.
R406 is an adenosine triphosphate competitive, small molecule that was developed as a specific Syk inhibitor by Rigel Pharmaceuticals (San Francisco, CA, USA) with lower activity against FMS-like tyrosine kinase 3 (Flt3), JAK 1/2 and lymphocyte-specific protein tyrosine kinase (Lck) and is currently used in clinical trials for rheumatoid arthritis, idiopathic thrombocytopenic purpura (ITP), B-CLL and DLBCL. The used concentrations of this compound in our experiments represent serum levels obtained in treated patients. In volunteers and patients, plasma levels after a single dose of R406 between 1 and 5 µg/ml were reported .
Variance analysis was performed using SPSS 17.0 of SPSS Inc. Evaluation of synergistic effects was performed using SAS 9.2 from SAS Institute Inc.
RMK carried out the proliferation assays, immunoassays, western blots, caspase assays and statistics and participated in the sequence alignment and drafted parts the manuscript. SND carried out the proliferation and migration assays, immunoassays, western blots. AH participated in the sequence alignment. SAEH and AB participated in the design of the study and wrote parts of the manuscript. PB conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
We thank Anika Beckers and Kati Riethausen for their excellent technical assistance. This work was supported by grants from Deutsche Forschungsgemeinschaft (DFG) (PB), Deutsche Krebshilfe (PB), BONFOR Forschungsförderung (AH). Parts of this work were presented on the annual meeting of the American Society of hematology (ASH) as a poster under a different title.
Compliance with ethical guidelines
Competing interests The authors declare that they have no competing interests.
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