Targeting early B-cell receptor signaling induces apoptosis in leukemic mantle cell lymphoma
© Boukhiar et al.; licensee BioMed Central Ltd. 2013
Received: 5 February 2013
Accepted: 5 February 2013
Published: 19 February 2013
We previously showed that B-cell receptor (BCR) signaling pathways are important for in vitro survival of mantle cell lymphoma (MCL) cells. To further identify early BCR-activated signaling pathways involved in MCL cell survival, we focused our study on BCR-proximal kinases such as LYN whose dysregulations could contribute to the aggressive course of MCL.
Primary MCL cells were isolated from 14 leukemic patients. Early BCR-induced genes were identified by qRT-PCR array. The basal and BCR-induced phosphorylation of LYN and JNK were evaluated by immunoblottting. Cell survival signals were evaluated by apoptosis using flow cytometry.
We showed that LYN was constitutively phosphorylated in MCL cell lines and in 9/10 leukemic MCL cases. Treatment with dasatinib or with a specific inhibitor of Src kinases such as PP2 suppressed constitutive LYN activation and increased in vitro spontaneous apoptosis of primary MCL cells. BCR engagement resulted in an increase of LYN phosphorylation leading to activation of c-JUN NH2-terminal kinase (JNK) and over-expression of the early growth response gene-1 (EGR-1). Inhibition of JNK with SP600125 induced apoptosis and reduced level of basal and BCR-induced expression of EGR-1. Furthermore, decreasing EGR1 expression by siRNA reduced BCR-induced cell survival. Treatment with PP2 or with dasatinib suppressed BCR-induced LYN and JNK phosphorylation as well as EGR-1 upregulation and is associated with a decrease of cell survival in all cases analysed.
This study highlights the importance of BCR signaling in MCL cell survival and points out to the efficiency of kinase inhibitors in suppressing proximal BCR signaling events and in inducing apoptosis.
KeywordsMantle cell lymphoma LYN BCR EGR-1 Dasatinib
Mantle cell lymphoma
Src family kinase
c-JUN NH2-terminal kinase
Early growth response gene-1
Immunoglobulin heavy chain variable-region
Mitogen-activated protein kinases
Extracellular signal-regulated kinase
Chronic lymphocytic leukemia
Diffuse large B cell lymphoma
Peripheral blood mononuclear cells
Transforming growth factor-β activated kinase-1
Immunoreceptor tyrosine-based activation motif
Spleen tyrosine kinase
Protein kinase C
Signal transducer and activator of transcription 3
Mantle cell lymphoma (MCL) constitutes about 6%–10% of non-Hodgkin lymphoma and despite recent advances in the treatment, the disease has not generally been cured with a poor progression-free survival for a large number of patients. New therapies that target specific signaling molecules are therefore of potential value. In recent years, some studies tried to reveal new suitable therapeutic targets [1–9] and have clarified the impact of several signaling pathways for increased proliferation and resistance to apoptosis of MCL cells. Constitutively active B-cell receptor (BCR)-mediated signaling has been implicated in the pathogenesis of a number of NHLs including diffuse large B cell lymphoma (DLBCL), follicular lymphoma, gastric mucosa-associated lymphoid tissue lymphoma and B-cell chronic lymphocytic leukaemia (CLL) [10–13]. Recently, we demonstrated in primary MCL cells a central role for active BCR signals in survival of MCL cells . The activated forms of the BCR-associated kinases LYN and spleen tyrosine kinase (SYK) were present in MCL tumor tissues therefore supporting an in vivo role of active BCR signaling in this pathology . Moreover, MCL is characterized by a highly restricted immunoglobulin gene repertoire with stereotyped VH CDR3s and precise Somatic Hyper Mutation targeting, thus strongly implying a role for antigen-driven selection of the clonogenic progenitors .
Upon antigen engagement, Igα–Igβ heterodimer (CD79a-CD79b) are phosphorylated on immunoreceptor tyrosine-based activation motif (ITAM) tyrosines by the BCR-associated kinase LYN, which belongs to the Src family kinases (SFK). SYK protein is then recruited through its SH2 domain to the phosphorylated Igα–Igβ heterodimer, resulting in the triggering of different signaling cascades . Among them, the PLCγ2/PKC pathway is crucial for activation of various mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinase (ERK) and c-JUN NH2-terminal kinase (JNK). Extensive work by several groups has established that MAP kinase pathways play critical roles in the pathogenesis of various hematologic malignancies, providing new potential molecular targets for future therapeutic approaches . Indeed, gene expression profiling of DLBCL revealed enhanced expression of JNK mRNA in at least 60 percent of cases . Moreover inhibition of JNK activation by the pharmacological inhibitor SP600125 induced growth arrest in myeloma cell lines . Of interest, JNK was showed to be constitutively activated in MCL and inhibition of phospho-JNK with SP600125 resulted in growth arrest in MCL cell lines (Jeko-1, HBL-2, UPN-1, Granta-519) .
A key downstream target of JNK activation is the early growth response gene-1 (EGR-1) transcription factor playing an important role in cell cycle regulation, cell proliferation and apoptosis [22, 23]. EGR-1 was first identified as a putative G0/G1 switch regulatory gene in lymphocyte cultures . Constitutive EGR-1 expression is involved in the self-renewal capacity of B-1 lymphocytes  and hematopoietic stem cells . EGR-1 is also constitutively expressed in immature BKS-2 B lymphoma and inhibition of EGR-1 using specific antisense oligonucleotides induced apoptosis . Alternatively, mature B2 cells undergo proliferation with an increase of EGR-1 expression upon BCR engagement . Moreover, EGR-1 is down-regulated upon JNK inhibition by SP600125, and its overexpression partially protects against JNK inhibitor-induced apoptosis in B lymphoma cell lines .
Given the importance of BCR signaling in tumor cell survival including MCL cells, we hypothesized that targeting BCR-associated kinases such as SFK represents a potentially useful strategy to treat MCL. LYN kinase is the major SFK expressed in B cells and its constitutive phosphorylation was previously reported in Jeko-1 cell line . However its role in MCL has not yet been explored to date. Therefore we analysed the activation status of LYN in primary MCL cells and evaluated the in vitro impact of its inhibition on MCL cells survival. We showed that LYN was constitutively phosphorylated in most MCL cases tested and that BCR engagement led to an increased LYN phosphorylation. Treatment with dasatinib, the oral broad inhibitor of tyrosine kinases, suppressed BCR-induced LYN and JNK phosphorylation in primary MCL cells. Similarly, treatment with dasatinib inhibited BCR-dependent EGR-1 upregulation and cell survival. Using PP2, a more specific inhibitor of BCR-associated SFK, we proved the efficiency of blocking BCR-emanating signals in suppressing MCL cell survival.
EGR-1 and c-MYC are rapidly induced upon BCR engagement in MCL
We have previously described that BCR engagement induces a survival signal in MCL through an IL6/IL10-dependent activation-loop of STAT3 . To further investigate which BCR-induced signaling pathways are critical, we screened purified B cells from primary leukemic MCL for the differential expression of 84 genes upon anti-IgM stimulation using RT2 Profiler PCR Arrays (SA Biosciences PAHS-027). Fifteen genes exhibited significant increased or decreased expression as compared to unstimulated cells (Additional file 1: Table S2; average fold-change from 4 individual experiments using UPN 5, 10, 13 and 14). Four genes were down-regulated (APAF1, CCNG2, ATM, BTG2), all corresponding to proapoptotic proteins. Conversely, eleven genes were overexpressed (CDK4, MCL1, NFkB1, IFNB1, SESN2, BCL2A, IL6, TNF, HK2, EGR-1, c-MYC), all of them being involved in cell cycle progression or inhibition of apoptosis. Within this group, three genes encoded for transcription factors; namely NF-kB, c-MYC and EGR-1 the two later being the two most upregulated genes upon anti-IgM stimulation (average of 23.6- and 17.6-fold increase, respectively).
Inhibition of JNK suppresses both BCR-induced EGR-1 upregulation and cell survival
Inhibition of LYN activity is associated with an increase of apoptosis in MCL cells
Inhibition of the BCR-induced LYN phosphorylation by PP2 or dasatinib is associated with a suppression of BCR-mediated cell survival
In the present study, we showed that primary MCL cells displayed a constitutive and BCR-induced activation of LYN and that treatment with dasatinib or with a more specific inhibitor of LYN suppressed both BCR-induced JNK phosphorylation and EGR-1 upregulation and is associated with a decrease of cell survival.
Recent studies have shown the importance of tonic BCR signaling in survival of DLBCL cells  and CLL cells  but few studies focused on the role of BCR signaling in MCL cell survival [14, 15]. We have previously shown in MCL cells that BCR engagement induced a cell survival signal through an IL6/IL10 autocrine dependent activation of STAT3 . To further identify early genes involved in BCR-induced survival, we looked at the differential gene expression upon BCR stimulation. We evidenced that BCR engagement led to a rapid but transient induction of mRNA and protein levels of EGR-1. EGR-1 is a zinc finger transcription factor whose expression has been described as directly dependent on antigen receptor signaling [31, 32]. EGR-1 is a downstream target of JNK and it regulates the expression of several genes like CD44, NF-kB1, thymidine kinase, cyclin D1 and platelet-derived growth factor that are important for cell survival and proliferation [23, 33–35]. We thus evaluated the role of EGR-1 in MCL cell survival and showed that inhibition of JNK by SP600125 induced a decrease of constitutive and BCR-induced EGR-1 expression, associated with an increase of apoptosis and a suppression of BCR-induced survival. We confirmed the JNK-dependent upregulation of EGR-1 by blocking the activity of TAK1, the upstream activator of JNK, which was recently described to play an essential role in MCL survival . Our results indicate that in MCL cells, EGR-1 is a downstream target of BCR signaling and its expression can be enhanced in response to antigen stimulation leading to cell survival. In addition to EGR-1, we observed that the BCR engagement also led to an increase of c-MYC in patients’ cells only. This differential response between cell lines and primary cells might reflect higher levels of c-MYC expression in cell lines as compared to patient’s cells . Cell lines might therefore become unresponsive to further stimulation through the BCR. The delayed kinetic induction of c-Myc as compared to EGR-1 in patient’s cells might argue for a latter induction of c-Myc. Whether this induction is related to expression of EGR-1 as proposed in CLL  and BKS2 cells activated by CpG ODN  remains to be determined. Nevertheless, our results suggest that EGR-1 and c-MYC upregulations could play an essential role in BCR-induced survival of MCL cells.
The importance of BCR signaling in MCL was recently investigated using a high-throughput phospho-proteomic technique which identified more than 300 tyrosine-phosphorylated proteins . The most abundant peptides were part of proteins constituting the BCR-associated signalosome. Among them, the kinases LYN and SYK were found to be constitutively strongly phosphorylated, thus reflecting an active BCR signaling even in absence of antigen stimulation. The importance of the BCR signaling in MCL was also suggested through the activation of SYK probably due to a constitutively activated signalosome made of LYN and CSK-binding protein/phosphoprotein associated with glycosphingolipid-enriched microdomains (Cbp/PAG) membrane adaptor [6, 40]. In the present study, we showed in a subset of primary MCL cells that LYN was in a constitutively active form as revealed by phosphorylation of the active Tyr397 LYN residue. LYN is believed to be a key component of cell membrane lipid rafts. Moreover, a subset of transmembrane proteins with aberrant expression was identified in MCL plasma membranes . In particular, Cbp/PAG that participates to the negative regulation of LYN in resting B cells through CSK recruitment was underexpressed in MCL primary cells compared to normal B cells. This low expression of Cbp/PAG could thus contribute to the constitutive activation of LYN in MCL cells.
Dasatinib, a dual BCR/ABL and SFK inhibitor, has demonstrated its efficacy in inhibiting cell proliferation of lymphoma B cells exhibiting a constitutive activation of Src kinase. Yang et al. demonstrated that inhibition of the Src-SYK-PLCg2 pathway by dasatinib induced G1 arrest in DLBCL . In the present study, we evidenced a constitutive and BCR-induced phosphorylation of LYN, thus justifying the rationale to evaluate the impact of dasatinib in MCL cell survival. We showed that dasatinib, which targeted the ATP binding pocket of LYN , inhibited phosphorylation of Tyr397 LYN probably by blocking its trans-autophosphorylation. .We also showed for the first time that dasatinib induced apoptosis of primary MCL cells and suppressed BCR-induced survival after antigen-triggering at nanomolar range. Of interest, the concentration levels of dasatinib (100nM) required to induce in vitro MCL cell apoptosis are in agreement with clinically achievable doses . A phase II study of dasatinib in relapsed or refractory CLL showed partial responses in 3 of 15 patients and among the remaining 12 patients, five patients had nodal responses. The investigators thus concluded that dasatinib as a single agent had activity in relapsed and refractory CLL . A phase I/II study of dasatinib is currently conducted by recruiting patients in relapsed or refractory non-hodgkin’s lymphoma (NHL) including mantle cell lymphoma (NCT00550615).
In conclusion, this study performed on primary MCL lymphocytes evidenced a dysregulation of early BCR signaling characterized by a constitutive LYN phosphorylation which can be enhanced in response to BCR engagement. Furthermore, targeting proximal BCR-associated kinases efficiently induced apoptosis of MCL cells. Thus, inhibition of LYN kinase and downstream JNK/EGR-1 pathway could be a new therapeutic strategy in MCL to overcome pro-survival signal emanating from the BCR.
MCL samples and cell lines
Peripheral blood mononuclear cells were obtained from 14 MCL leukemic patients by Ficoll-Hypaque density gradient (Stem Cell Technologies, Grenoble, France). Lymphocytosis was greater than 8.0×109/L and 10 out of 14 samples contained at least 80% of B lymphocytes (Additional file 5: Table S1). All B lymphocytes are monoclonal tumor B cells as evidenced through flow cytometry phenotyping of the surface immunoglobulin light chain (monotypic kappa or lambda). Seven cases (7/14) showed mutated IGHV and none of them displayed mutation in ITAM sequences of CD79B. The diagnosis of MCL was ascertained by immunophenotyping, cytogenetic and FISH analysis of t(11;14) and overexpression of cyclin D1 was detected by competitive RT-PCR according to the World Health Organization classification. All patients were homogeneously treated in a prospective trial of the GOELAMS group “Lymphome du manteau 2006 SA” . All patients were provided written informed consent, validated by the Ethics Committee from the GOELAMS group, in accordance with the Declaration of Helsinki. Patients usually received treatment very quickly after sampling, making it difficulties to repeat all experiments several times on the same sample. Jeko-1, and Granta-519 cell lines were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunshwieig, Germany) and the HBL-2 cell line was a generous gift from Dr B. Sola (Caen, France) .
Patients’ cells were either used freshly isolated or cryopreserved in liquid nitrogen in the presence of 10% dimethyl sulfoxide and 20% heat-inactivated FCS. MCL leukemic cells (3 × 106 cells/ml) were cultured in complete RPMI 1640 medium supplemented with 10% heat-inactivated foetal calf serum. Jeko-1, HBL-2 and Granta-519 cell lines were maintained in culture in the same media. For BCR stimulation, plates were pre-coated with rabbit anti-human IgM antibody (10 μg/mL; Jackson ImmunoResearch, Baltimore, MD) as previously described  or the anti-IgM antibody was added to the culture medium at the same concentration for short stimulation time.
Antibodies and reagents
Antibodies to EGR-1 (mAb 44D5), c-MYC (mAb 9B11), phospho-Src family (mAb 100 F9) also reactive with phospho-Tyr397 LYN (catalytic site) and phospho-JNK (Thr183/Tyr185) were from Cell Signaling (Beverley, MA). Monoclonal mouse antibody (H-6) and polyclonal rabbit antibody (44) to LYN were from Santa-Cruz (Santa Cruz, CA). Anti-phosphotyrosine monoclonal antibody (clone 4 G10) was from Millipore (Billerica, MA). Dasatinib (Clini Sciences, Montrouge, France) was used at 100nM, unless otherwise stated. JNK inhibitor SP600125 and PP2 (4-Amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine) was from Sigma (Saint Quentin Fallavier, France) and (5Z)-7-Oxozeaenol (TAK1 inhibitor) was from Tocris Bioscience (Bristol, UK).
RT2 profiler PCR arrays
Tumor B-lymphocytes from MCL patients were purified by the RosetteSep® Human B Cell Enrichment Cocktail (Stemcell technologies, Grenoble, France). Cells were cultured for 3 hours upon BCR stimulation or left untreated. Total RNA were extracted and analyzed with “p53 signaling pathway” array (SA Biosciences PAHS-027) according to the manufacturer’s instructions (SA Biosciences, Frederick, MD) with an Applied Biosystems 7500 Fast Real-Time PCR Systems. Each gene expression was normalized to the mean Ct values from the four housekeeping genes available in the PCR array (β2-microglobulin, β-actin ribosomal protein L13A, hypoxanthine phosphorybosyl transferase-1 and β-actin), then normalized to unstimulated control cells to determine the fold-change. Relative fold change of expression was calculated by the ΔΔCt method and the values are expressed as 2-ΔΔCt. All points were done in duplicate.
Cell apoptosis was evaluated using flow cytometry (FACSCantoTM II Becton Dickinson) on leukemic MCL PBMC after gating on CD19+ cells using Annexin V-FITC and propidium iodide staining (BD Biosciences, San Jose, CA). Percentage of apoptotic cells corresponded to% of annexin V-positive, including PI-negative and PI-positive cells. All measurements were done in duplicate and the mean is indicated.
Quantification of EGR-1 and c-MYC mRNA by qRT-PCR
EGR-1 forward primer (5′CGAGCAGCCCTACGAGCACCTGAC3′),
EGR-1 reverse primer (5′TGCGCAGCTCAGGGGTGGGCTCTG3′),
c-MYC forward primer (5′GCTGCTTAGACGCTGGATTTTT3′),
c-MYC reverse primer (5′ACCGAGTCGTAGTCGAGGTCAT3′),
cyclophilin A forward primer (5′GCACTGGAGAGAAAGGATTTGG3′) and
cyclophilin A reverse primer (5′AGTGCCATTATGGCGTGTGA3′).
Western blotting and immunoprecipitation
Total protein extracts from 3×106 MCL cells (PBMC) were separated on 10% polyacrylamide denaturing gel, transferred to a nitrocellulose membrane and incubated overnight with the appropriate antibody followed by a secondary horseradish peroxidase-conjugated antibody (Bio-Rad). Detection was performed using ECL (Amersham Biosciences, Buckinghamshire, UK) and autoradiography (Biomax, Kodak, Rochester, USA). For LYN immunoprecipitation, HBL-2 cells were lysed in 1% Nonidet P-40 (NP-40) lysis buffer for 30 minutes on ice. Insoluble material was removed by centrifugation at 27 000 g for 10 minutes at 4°C and soluble proteins were immunoprecipitated with a rabbit anti-LYN (H-6) antibody for 2 hours at 4°C. Immunocomplexes were solubilized in SDS sample buffer, analyzed on SDS-PAGE, transferred and subjected to immunoblotting as described above using either a mouse anti-phosphotyrosine antibody (clone 4 G10) or a mouse anti-LYN (44) antibody.
Three million cells were resuspended in 100 μL of Human B Cell Lymphoma Nucleofector® Kit (Amaxa) containing either 1 μM of EGR-1 siRNA (On-Target plus SMART pool Human EGR-1, Dharmacon) or 1 μM of control siRNA (Ctrl1: On-Target plus non-targeting pool; Ctrl2: On-target plus non-targeting siRNA#1; Dharmacon). Cells were transfected in a Nucleofector II device (Lonza) by using U-015 program, transferred to culture plates and western blot and apoptosis assays were performed as described above.
Differences between groups were determined using the Student’s t test. Statistical analyses were performed using GraphPad Prism software (San Diego, CA). P values below 0.05 were considered statistically significant.
This work was supported by the GOELAMS group (Groupe Ouest-Est des Leucémies Aiguës et Maladies du Sang.) and by the Institut National du Cancer (INCA) for the tumor biobank. M.A.B. was supported by the Société Française d’Hématologie (SFH).
FBM and DL are co-senior authors.
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