Inhibitors of BCR signalling interrupt the survival signal mediated by the micro-environment in mantle cell lymphoma Sophie Bernard 1,2,3 , Damien Danglade 3 *, Laura Gardano 1,2 *, Christelle Laguillier 4 , Gregory Lazarian 3 , Claudine Roger 3 , Catherine Thieblemont 5 , Jacek Marzec 6 , John Gribben 6 , Florence Cymbalista 1,2,3 , Nadine Varin-Blank 1,2 , Dominique Ledoux 1,2† and Fanny Baran-Marszak 1,2,3† 1 U978 Institut National de la Sante et de la Recherche Medicale, Bobigny, France 2 Labex Inflamex, Universite Paris 13, Sorbonne Paris Cite, Bobigny, France 3 Service d’Hematologie Biologique, H^ opital Avicenne, Assistance Publique–H^ opitaux de Paris, Bobigny, France 4 Service de Biochimie, H^ opital Jean Verdier, Assistance Publique–H^ opitaux de Paris, Bondy, France 5 Hematologie, H^ opital Saint-Louis, Assistance Publique–H^ opitaux de Paris, Paris, France 6 Barts Cancer Institute, Queen Mary, University of London, London, United Kingdom Several studies provide evidences for mantle cell lymphoma (MCL) cell survival relying on B-cell receptor (BCR)-mediated sig- nalling pathways, whereas the nature of this activation is unknown. Significant progress in MCL treatment is achieved through therapies targeting BCR-associated kinases, i.e., Ibrutinib and Fostamatinib, inhibitors of BTK and SYK, respectively. Our study addresses survival signals emanating from the BCR or the tumour environment and how inhibiting BCR signalling effectors might impact these survival signals. We found that BTK was constitutively activated and that SYK phosphorylation was highly increased and sustained upon BCR activation of primary MCL cells. Moreover, MCL cells from leukaemic patients secreted high amount of IL-1b, IL-6, IL-8 and CCL5. Activation of the BCR induced (i) cell survival, (ii) STAT3 activation and (iii) increased autocrine secretion of IL-1b, IL-6, IL-8, CCL5, IL-10, TNFa and VEGF. Specific inhibition of BTK by Ibrutinib or SYK by Fostamati- nib (R406) reversed these protective effects and decreased both basal and BCR-induced autocrine cytokine secretions associ- ated with STAT3 phosphorylation. Interestingly, targeting BTK and SYK prevented and inhibited BCR-induced MCL cell adhesion to human bone marrow stromal cells (HMSCs) in short- and long-term co-culture. We demonstrated that BCR- induced survival relies on autocrine secretion of IL-1b, TNFa and CCL5 that might facilitate adhesion of MCL cells to HMSC. Treatment with Ibrutinib or Fostamatinib blocked the chemotactic signal thus increasing apoptosis. Mantle cell lymphoma (MCL) is a rare, aggressive and incur- able malignant lymphoma with a median survival of 5 years. The majority of MCL patients exhibit advanced-stage criteria at the time of diagnosis with frequent extranodal manifesta- tions, i.e., high prevalence of circulating MCL cells, bone marrow and gastrointestinal tract involvement. 1 These clinical features suggest that MCL cells have a high propensity to dis- seminate and home at other tissues. Besides these aggressive forms of the disease, clinical studies have evidenced a sub- group of MCL patients with an indolent course and a pre- dominantly non-nodal leukaemic disease. Despite new chemotherapeutic combinations, MCL is characterised by a poor overall response owing to a rapid relapse after initial treatment or primary resistance to conventional drugs. 2 How- ever, phase II studies are currently evaluating the efficacy of new tyrosine kinase inhibitors targeting the B-cell receptor Key words: mantle cell lymphoma, B-cell receptor, Ibrutinib, Fostamatinib, STAT3, cytokines, microenvironment Abbreviations: BCR: B-cell receptor; BTK: Bruton’s tyrosine kinase; CLL: chronic lymphocytic leukaemia; DLBCL: diffuse large B-cell lymphoma; EGR-1: early growth response gene-1; ERK: extracellular signal-regulated kinase; HMSC: human marrow stromal cells; IGHV: immunoglobulin heavy chain variable-region; IL: interleukin; MCL: mantle cell lymphoma; PBMC: peripheral blood mononuclear cells; STAT3: signal transducer and activator of transcription 3; SYK: spleen tyrosine kinase; TNF: tumour necrosis factor; VEGF: vas- cular endothelial growth factor Additional Supporting Information may be found in the online version of this article. *D.D. and L.G. are co-second authors † D.L. and F.B.M. are co-senior authors Grant sponsors: INSERM, INCa (Institut National du Cancer), ARC (Fondation ARC pour la Recherche sur le Cancer), University Paris 13; Grant sponsor: Labex INFLAMEX; Grant number: ANR11 IDEX 00502 DOI: 10.1002/ijc.29326 History: Received 5 May 2014; Accepted 27 Oct 2014; Online 12 Nov 2014 Correspondence to: Fanny Baran-Marszak and Nadine Varin-Blank, UMR U978, UFR SMBH, 74 rue Marcel Cachin, 93000 Bobigny, France, Tel.: 133-0-1-48-95-56-46, Fax: 133-0-1-48-95-56-48, E-mail: [email protected] and E-mail: [email protected]Cancer Cell Biology Int. J. Cancer: 00, 00–00 (2014) V C 2014 UICC International Journal of Cancer IJC
Transcript
Inhibitors of BCR signalling interrupt the survival signalmediated by the micro-environment in mantle cell lymphoma
Sophie Bernard1,2,3, Damien Danglade3*, Laura Gardano1,2*, Christelle Laguillier4, Gregory Lazarian3, Claudine Roger3,
Catherine Thieblemont5, Jacek Marzec6, John Gribben6, Florence Cymbalista1,2,3, Nadine Varin-Blank1,2,
Dominique Ledoux1,2† and Fanny Baran-Marszak1,2,3†
1 U978 Institut National de la Sant�e et de la Recherche M�edicale, Bobigny, France2 Labex Inflamex, Universit�e Paris 13, Sorbonne Paris Cit�e, Bobigny, France3 Service d’H�ematologie Biologique, Hopital Avicenne, Assistance Publique–Hopitaux de Paris, Bobigny, France4 Service de Biochimie, Hopital Jean Verdier, Assistance Publique–Hopitaux de Paris, Bondy, France5 H�ematologie, Hopital Saint-Louis, Assistance Publique–Hopitaux de Paris, Paris, France6 Barts Cancer Institute, Queen Mary, University of London, London, United Kingdom
Several studies provide evidences for mantle cell lymphoma (MCL) cell survival relying on B-cell receptor (BCR)-mediated sig-
nalling pathways, whereas the nature of this activation is unknown. Significant progress in MCL treatment is achieved through
therapies targeting BCR-associated kinases, i.e., Ibrutinib and Fostamatinib, inhibitors of BTK and SYK, respectively. Our study
addresses survival signals emanating from the BCR or the tumour environment and how inhibiting BCR signalling effectors
might impact these survival signals. We found that BTK was constitutively activated and that SYK phosphorylation was highly
increased and sustained upon BCR activation of primary MCL cells. Moreover, MCL cells from leukaemic patients secreted high
amount of IL-1b, IL-6, IL-8 and CCL5. Activation of the BCR induced (i) cell survival, (ii) STAT3 activation and (iii) increased
autocrine secretion of IL-1b, IL-6, IL-8, CCL5, IL-10, TNFa and VEGF. Specific inhibition of BTK by Ibrutinib or SYK by Fostamati-
nib (R406) reversed these protective effects and decreased both basal and BCR-induced autocrine cytokine secretions associ-
ated with STAT3 phosphorylation. Interestingly, targeting BTK and SYK prevented and inhibited BCR-induced MCL cell
adhesion to human bone marrow stromal cells (HMSCs) in short- and long-term co-culture. We demonstrated that BCR-
induced survival relies on autocrine secretion of IL-1b, TNFa and CCL5 that might facilitate adhesion of MCL cells to HMSC.
Treatment with Ibrutinib or Fostamatinib blocked the chemotactic signal thus increasing apoptosis.
Mantle cell lymphoma (MCL) is a rare, aggressive and incur-able malignant lymphoma with a median survival of 5 years.The majority of MCL patients exhibit advanced-stage criteriaat the time of diagnosis with frequent extranodal manifesta-tions, i.e., high prevalence of circulating MCL cells, bonemarrow and gastrointestinal tract involvement.1 These clinicalfeatures suggest that MCL cells have a high propensity to dis-seminate and home at other tissues. Besides these aggressive
forms of the disease, clinical studies have evidenced a sub-group of MCL patients with an indolent course and a pre-dominantly non-nodal leukaemic disease. Despite newchemotherapeutic combinations, MCL is characterised by apoor overall response owing to a rapid relapse after initialtreatment or primary resistance to conventional drugs.2 How-ever, phase II studies are currently evaluating the efficacy ofnew tyrosine kinase inhibitors targeting the B-cell receptor
(BCR) effectors with encouraging results in relapsed andrefractory MCL cases.3–5
BCR is usually composed of a membrane immunoglobulin(IgM) non-covalently bound to a heterodimer of CD79A andCD79B that initiates the signalling cascade. Upon antigenbinding, CD79A and CD79B are phosphorylated on tyrosineresidues of the immunoreceptor tyrosine-based activationmotif (ITAM) by the BCR-associated kinase LYN, a memberof the Src family of kinases (SFK).6 SYK protein is thenrecruited through its SH2 domain to the phosphorylatedCD79A–CD79B heterodimer, resulting in the activation ofdifferent signalling cascades.7 Bruton’s tyrosine kinase (BTK)is also phosphorylated by LYN at Y551 followed by auto-phosphorylation at Y223 and then associates with BLNKadaptor protein and phospholipase C g2 (PLCg2). ThePLCg2/PKC axis is crucial for the activation of several signal-ling effectors such as extracellular signal-regulated kinase(ERK) and c-JUN NH2-terminal kinase (JNK), early growthresponse transcriptional factor (EGR1–2) and the NF-jB.Additional inhibitory regulation is also attributed to LYN.After contributing to the initial BCR activation, LYN initiatesa negative feedback loop.6 In particular, LYN allows therecruitment of the SH2 domain-containing phosphatase(SHP-1), which dephosphorylates several ITAM-containingmolecules, including CD79A and CD79B.8
Most of MCL cells show high surface IgD and IgMexpression. Several kinases downstream of the BCR, includingSYK, LYN and BTK, are phosphorylated in primary MCLcells, suggesting that BCR signalling is constitutively activatedin these cells.9 Furthermore, amplification and overexpressionof the spleen tyrosine kinase (SYK) and the PI3K catalyticsubunit are found in a subset of MCL.10–12 In several MCLcases the negative feedback loop is altered by hypermethyl-ation of SHP-1 promoter resulting in a sustained activationof BCR-mediated pathways.13 However, in the majority ofcases the mechanism of this chronic activation is stillunknown and a detailed functional study is needed to appre-ciate the integrity of the BCR-mediated pathway. MCL cellsexpress high levels of integrin VLA-4 (CD49d) and ofCXCR4, the SDF1 chemokine receptor.14 These two proteinstrigger the migration and the adhesion of MCL cells to bonemarrow stromal cells (BMSC) in vitro, which produce signalsto support MCL cell survival and drug resistance.14,15 Fur-thermore, BCR activation triggers integrins, such as VLA-4,and CXCR4-dependent adhesion and homing, which contrib-
ute to cell survival of another CD51 B-cell lymphoma,Chronic lymphocytic leukaemia.16,17 Thus, on one hand, theBCR and its downstream effectors appear as central modula-tors of B-cell homing, survival and drug resistance in aninside-out activation of VLA-4 and CXCR4.14,18–21 On theother hand, the chemokine SDF1/CXCR4 and the integrinVLA-4/VCAM1 axis can induce outside-in activation of BCReffectors including LYN, SYK, BTK and PI3K.22–25 Interest-ingly, both outside-in and inside-out activation are disruptedby the inhibition of BCR-linked tyrosine kinases, such asSYK or BTK inhibitors.19,24,26 These inhibitors representemerging therapeutic strategies for controlling lymphomacells proliferation.
A fundamental aspect of the BCR signalling pathway isthe autocrine secretion of prosurvival cytokines, IL-6 and IL-10. Previously, we reported that IL-6 and IL-10 are importantmodulators for MCL growth and survival through the activa-tion of the Jak/STAT3 pathway.27 Interestingly, a constitutiveactivation of STAT3 is observed preferentially in MCLs withmutated rather than unmutated IGHV (immunoglobulinheavy-chain variable region) genes, suggesting that these twosubtypes of MCL may use different cell survival mechanisms.In addition, cells of the bone marrow environment, e.g.,BMSC, are also able to secrete large amounts of IL-6, BAFFand VEGFa that contribute to the protection of B cells fromapoptosis.15,28,29
We hypothesised that both BCR engagement andmicroenvironment-induced cell survival in MCL might reflecthigh paracrine and autocrine BCR-induced secretion of pro-survival factors (IL-6, IL-8, IL-1b, TNFa, IL-10 and VEGFa).We then focused our interest on the effect of BCR triggeringand HMSCs secretion of growth factors towards the induc-tion of MCL cell survival. We also analysed the impact ofIbrutinib (BTK inhibitor) and Fostamatinib/R406 (SYKinhibitor) treatment on the secretion of prosurvival factors byMCL cells and on the crosstalk with their microenvironmentduring short- and long-term co-culture with HMSC.
Material and MethodsMCL samples and cell lines
Peripheral blood mononuclear cells were obtained from 16MCL leukaemic patients by Ficoll-Hypaque density gradient(Stem Cell Technologies, Grenoble, France). Lymphocytosiswas superior to 10.0 Giga/L (Supporting Information Table 1)and all samples contained more than 70% of CD191 CD51
What’s new?
While there is evidence that Mantle Cell Lymphoma (MCL) cells rely on B-cell receptor (BCR)-mediated signalling pathways for
their survival, the nature of such activation remains unknown. Significant progress in MCL treatment was nonetheless
achieved through therapies targeting BCR-associated kinases such as Ibrutinib and Fostamatinib. Here, the authors showed
that the inhibition of SYK by Fostamatinib or BTK by Ibrutinib altered BCR-induced secretion of IL1b, IL6, IL8, VEGFa, TNFa,
and CCL5. These effectors could mediate survival of MCL cells through their homing and adhesion to bone marrow stromal
cells. Moreover, Fostamatinib and Ibrutinib impaired NF-jB-dependent STAT3 activation, leading to apoptosis.
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malignant lymphocytes as assessed by flow cytometry. Thediagnosis of MCL was ascertained by immunophenotyping,cytogenetic and FISH analysis of t(11;14) and overexpressionof cyclin D1 was detected by competitive RT-PCR accordingto the World Health Organization classification. All experi-ments were performed with peripheral blood mononuclearcells (PBMCs) containing at least 80% of MCL cells. Allpatients provided written informed consent, validated by theEthics Committee from the GOELAMS group (Groupe Ouest-Est des Leuc�emies Aigu€es et Maladies du Sang), in accordancewith the Declaration of Helsinki. Patients usually receivedtreatment shortly after sampling which made challenging torepeat experiments on the same patient. Jeko-1 cell lines werepurchased from the German Collection of Microorganismsand Cell Cultures (DSMZ, Braunshwieig, Germany). The iden-tity and purity of the cell lines were verified by flow cytometryand IGHV rearrangement sequencing.
Cell culture and reagents
Patients’ cells were either used freshly isolated or cryopre-served in liquid nitrogen in the presence of 10% dimethylsulphoxide and 20% heat-inactivated FCS at the cellulothequeof Avicenne Hospital. Results obtained with fresh or frozencells were identical and reproducible. MCL leukaemic cells (33 106 cells/mL) and Jeko-1 were cultured for 24 hr or 15days in complete RPMI 1640 medium supplemented with10% heat-inactivated foetal calf serum (FCS), 100 U/mL peni-cillin, 10 mg/mL streptomycin (Gibco BRL, Life Technologies,Saint Aubin, France), 1 mM sodium pyruvate (Gibco BRL),MEM vitamins 1003 (Gibco BRL) and 5 mg/mL plasmocin(Cayla InvivoGen, Toulouse, France). For BCR stimulation,F(ab0)2 fragment donkey anti-human IgM antibody (10 mg/mL; Jackson ImmunoResearch, Baltimore, MD) was added.Human primary bone marrow stromal cells (HMSCs) wereobtained from donors not affected by MCL. Mononuclearcells were purified from bone marrows and cultured in com-plete RPMI supplemented with 20% FCS. After 2 days of cul-ture, non-adherent cells were washed off and adherent cellswere maintained in culture in the same conditions. HMSC ofpassages not beyond six were used in all experiments. HMSCwere seeded in four-well plates coated with collagen and cul-tured in complete RPMI with 10% FCS until they reachedconfluency. MCL cells (3 3 106 cells per well) were seeded infour-well plates coated or not with HMSC at confluence forshort-term or long-term co-culture. Ibrutinib (PCI-32765) andR406 were purchased at Selleck Chemicals (Euromedex, Souf-felweyersheim, France).
Apoptosis assay
Cell apoptosis was evaluated using flow cytometry (FACS-CantoTM II Becton Dickinson) on leukaemic MCL PBMC insuspension (P1 gate Supporting Information Fig. S4) usingannexin V–FITC and propidium iodide staining (BDBiosciences, San Jose, CA). Percentage of apoptotic cellscorresponded to percent of annexin V-positive, including PI-
negative and PI-positive cells. All measurements were donein duplicate. In co-culture, 10,000 events were recorded andpercentage of apoptosis of adherent lymphocytes was ana-lysed in a CD191 gate.
Quantification of IL-1b, CCL5, IL-8 and IL-6 mRNA
by qRT-PCR
RNAs were extracted with Trizol by a Qiacub extractor (Qia-gen, Coutaboeuf, France). The expression of IL-1b, CCL5,IL-8 and IL-6 mRNAs was analysed in unstimulated andanti-IgM-stimulated cells using TaqMan Gene ExpressionAssays probes (IL6 Hs 00985639_m1, IL8 Hs 00174103_m1,IL1b Hs 01555410_m1 and CCL5 Hs 00174575_m1) on a7500 Real-time PCR system (Life Technologies, Saint Aubin,France). Target gene expression was normalised to the meanCt values of the housekeeping gene HPRT1 (HPRT1 Hs99999909_m1) and then normalised to unstimulated controlcells to determine the fold change. Relative fold change ofexpression was calculated by the DDCt method and the val-ues are expressed as 22DDCt. All the points were performedin duplicate.
Western blotting
Whole protein extracts were obtained as follows: 3 3 106
cells were resuspended in lysis buffer (Tris-HCl, 50 mM pH6.8, sodium dodecyl sulphate 2%, glycerol 2% and b-mercaptoethanol 2%). Extracts were sonicated and 30 mg ofprotein was loaded onto a 10% polyacrylamide denaturinggel (Mini Protean TGX gel Bio-Rad) and transferred to anitrocellulose membrane (Protran, Whatman GmbH, Protei-gene, Saint-Marcel, France). The membrane was blocked in1% non-fat milk (R�egilait, Saint-Martin Belle Roche, France)and incubated overnight with the following antibodies: rabbitpolyclonal anti-STAT3 antibody, anti-phospho-STAT3(Y705) antibody, anti-SYK antibody, anti-phospho-SYK(Y525–526) antibody, anti-phospho-BTK (Y223) antibody,anti-phospho-ERK (Y42/44) antibody, anti-EGR1 antibody(Cell Signaling Technology, Ozyme, Saint Quentin Yvelines,France), followed by a secondary horseradish peroxidase-conjugated antibody (Bio-Rad). Detection was performedusing ECL (Amersham Biosciences, Fisher Scientific, Illkirch,France) in a UVP ChemiDoc-It imaging system and analysedwith a VisionWorksLS software (Sciencetec, Les Ulis, France).
Quantification of secreted factors in cell culture
supernatants by Luminex immunoassay
MCL cells (3 3 106) were grown in 1 mL of culture mediumfor 24 hr. Interleukin (IL)21b/IL-1F2, IL-6, IL-8/CXCL8, IL-10, RANTES/CCL5, TNF-a and VEGF levels were deter-mined with the Magnetic LuminexVR Performance AssayHuman Cytokine Premixed Kit A (R&D Systems Europe,Lille, France). Data were collected with the Bio-Plex Manager5.0. The sensitivities of these assays were as follows: 0.57 pg/mL for IL-1b, 1.11 pg/mL for IL-6, 1.97 pg/mL for IL-8/CXCL8, 0.30 pg/mL for IL-10, 1.91 pg/mL for RANTES/
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CCL5, 1.50 pg/mL for TNF-a and 1.84 pg/mL for VEGF.Coefficients of variation for intra-assay determination rangedfrom 3.7 to 12.2% as specified by the manufacturer.
Statistical analyses
Differences between groups were determined using theunpaired Student’s t-test or the Wilcoxon-signed rank test asappropriate. Statistical analyses were performed using Graph-Pad Prism software (San Diego, CA). The p values <0.05were considered statistically significant.
ResultsKinetics of BCR signalling in MCL
BCR activation is central to the pathophysiology of MCL andinhibition of this pathway is emerging as a promising thera-peutic strategy for the treatment of the disease.30 We soughtof further investigate BCR signalling pathway, analysing thephosphorylation status of three kinases involved in BCR-mediated signal, BTK, SYK and ERK on tyrosine residues rep-resentative of their kinase activity. In three MCL cell lines(Granta, HBL-2 and Jeko-1) we observed basal phosphorylatedform of the kinases BTK or ERK (Fig. 1a and SupportingInformation Fig. S1) with different intensities between thethree cell lines. However, the amount of phospho-SYK (Y525/526) was very weak, almost below the limit of detection, underthe conditions used in the Western blot. Similarly, the phos-phorylated forms of BTK or ERK were detected in primarycirculating MCL cells from leukaemic patients (UPN-1 andUPN-2) although with highly variable intensities (Figs. 1b and1c). In addition, phospho-SYK (Y525/526) signal remainedvery faint (Figs. 1b and 1c). A similar profile was observed innormal B lymphocytes (data not shown). Next, we analysedthe kinetics of BCR signalling by following the phosphoryla-tion dynamics of BTK, SYK and ERK kinases upon BCR stim-ulation with a soluble anti-IgM on Jeko-1, HBL-2, Granta celllines and primary circulating MCL lymphocytes from two leu-kaemic patients (Figs. 1a–1c and Supporting Information Fig.S1). A rapid increase of phosphorylation of BTK, SYK andERK was observed in the three cell lines and primary MCLcells already at 5 min and up to 15 min following BCR stimu-lation. Phosphorylation was maintained for about 1 hr andreturned to low levels after this time.
In conclusion, we observed some constitutive activation ofthe BCR-associated kinases BTK or ERK in MCL cells and arapid response to BCR stimulation with high and sustainedphosphorylation levels of BTK, SYK and ERK suggesting atonic BCR signalling profile.
BCR survival signal is associated with upregulation of
secreted factors
We previously published that activation of the BCR for 24 hrinhibits spontaneous apoptosis in MCL cells and associateswith an autocrine secretion of IL-6 and IL-10.27 We con-firmed this effect of BCR stimulation by analysing the rate ofapoptosis triggered by stimulation of MCL primary cells
from 11 leukaemic patients with soluble anti-IgM for 24 hr.Using flow cytometry we found that the mean percentage ofapoptotic cells in unstimulated sample (control) was 52%;this percentage decreased to 33% (mean inhibition 36.5%,Student’s t-test, p5 0.0001) upon BCR stimulation (anti-IgM) (Fig. 2a). Most likely, IL-6 and IL-10 are not the onlysecreted cytokines that can sustain cell survival in MCL cellsupon BCR stimulation. In order to ascertain this point, weanalysed the differential transcriptome profile of primaryMCL cells of one patient after 3 hr of BCR stimulation whencompared to unstimulated cells for several putative secretedproteins (Supporting Information Table 2). Among 15 candi-date genes, IL-1b, IL-6, IL-8 and CCL5 were selected for fur-ther investigation (fold change expression greater than 2) asearly BCR-induced genes. Interestingly, all these genes corre-sponded to NF-jB target genes (http://www.bu.edu/nf-kb/gene-resources/target-genes/). We confirmed the upregulationof IL-6, IL-8, CCL5 and IL-1b mRNAs expression starting at30 min after BCR stimulation in Jeko-1 cells (Fig. 2b). Wealso measured the concentration of seven secreted factors inthe culture supernatants of MCL primary cells from 12patients after 24 hr of BCR stimulation (anti-IgM) comparedto unstimulated control cells (Fig. 2c). Primary unstimulatedcells secreted high amounts of IL-1b, IL-6, CCL5 and IL-8(median concentrations 48, 357, 327 and 2,541 pg/mL,respectively) in contrast to the cell lines that secreted verylow levels of IL-1b and high levels of VEGF (SupportingInformation Fig. S2C). Furthermore, after BCR stimulation,secretion of IL-1b, IL-6, CCL5, IL-8, IL-10, TNFa andVEGFa increased significantly (median concentrations were722, 2,958, 584, 3,900, 103, 185 and 45 pg/mL, respectively).Interestingly, all these cytokines are implicated in inhibitionof apoptosis. As frequently indolent leukaemic MCL harbourmutated IGHV genes, these results were further analysedwith respect to the IGHV mutational status27 (Fig. 2d). Albeitnot statistically significant, we observed noticeable differencesin BCR-induced secretions of IL-1b, IL-6, IL-10 and TNFa
between the mutated (n5 5) and the unmutated (n5 5)IGHV groups. Overall, these results show that BCR stimula-tion induces survival signals associated with high secretion ofautocrine prosurvival factors in the tumour environment.
Inhibitors of BCR-dependent kinases Ibrutinib and
Fostamatinib/R406 affect both BCR-dependent cascade
and STAT3 activation
We previously showed that in MCL, STAT3 is constitutivelyphosphorylated and is further activated upon BCR stimula-tion through an autocrine, NF-jB-dependent loop driven bysecreted IL-6 and/or IL-10 that results in MCL prolifera-tion.27 Hence, we analysed the impact of BTK and SYKinhibitors on BCR signalling and cell survival. Jeko-1, HBL-2and Granta cells were treated with increasing concentrationsof Ibrutinib or R406. Only Jeko-1 cells showed some sensitiv-ity to Ibrutinib and R406 at a concentration beyond 5 mM,after 24 hr of treatment (Supporting Information Figs. S2A
and S2B). Thus, only Jeko-1 cells were used in further inves-tigations. As 24-hr BCR stimulation of Jeko-1 cell line didnot induce cell survival (data not shown), the effects of Ibru-tinib and R406 treatments were rather analysed upon short-term stimulation (30 min) only. First, cells were treated withincreasing concentrations (from 0.1 to 10 mM) of Ibrutinib
for 90 min before a short-term BCR stimulation (30 min)with soluble anti-IgM. BCR-mediated phosphorylation ofBTK and Syk was already reduced at Ibrutinib concentrationof 0.1 mM, whereas ERK phosphorylation was maintained ata concentration of Ibrutinib up to 5 mM. In agreement withthis finding, we also observed a decreased expression of the
Figure 1. Constitutive and BCR-induced activation of SYK, BTK and ERK kinases in MCL cell lines or primary MCL cells from a leukaemic
patient. (a) Total cell lysates from Jeko-1 cells were analysed by Western blotting. The membrane was probed with an anti-phospho-BTK
(Y223) antibody and reprobed with anti-BTK antibody, anti-phospho-SYK (Y525–526) and reprobed with anti-SYK antibody and anti-tubulin
antibody to verify an equal loading and anti-phospho-ERK (Y42–44) and reprobed with anti-ERK antibody. (b and c) Primary MCL cells from
two leukaemic patients UPN-1 and UPN-2 were cultured in absence (2) or in the presence of soluble anti-IgM antibody (10 mg/mL) for 5,
15, 30, 60 and 240 min. BCR-induced phosphorylation of BTK, SYK and ERK was analysed by Western blotting as in a.
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Figure 2.
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early transcription factor EGR1 following similar dynamics(Fig. 3a). As BCR signalling was drastically inhibited by apretreatment with 10 mM Ibrutinib for all kinases, we chosethe concentration of 5 mM Ibrutinib that had maximumeffect with a weak toxicity to treat primary MCL cells (Sup-porting Information Fig. S2A). Similarly, we treated Jeko-1cells with increasing concentrations (from 0.5 to 4 mM) ofR406 (Fig. 3b). BCR-mediated SYK phosphorylationremained unchanged at all concentrations; though, BCR-mediated BTK phosphorylation weakly decreased after a pre-treatment with 2 mM of R406. At the same time, ERK phos-phorylation was fully inhibited and EGR1 expression reducedto basal levels already with R406 treatment of 1 mM. Thus, 2mM R406 concentration was chosen to treat primary MCLcells. Primary MCL cells (UPN1 and UPN2) were treatedwith Ibrutinib 5 mM and R406 2 mM and BCR-mediatedSYK, BTK, ERK phosphorylation were analysed upon short-term stimulation (30 min, Fig. 3c). Treatment with R406inhibited to variable extent BCR-mediated phosphorylation ofSYK, BTK and ERK; treatment with Ibrutinib showed an effi-cient inhibition for BTK and ERK phosphorylation only (Fig.3c and Supporting Information Fig. S3A). Moreover, after24-hr treatment we observed a complete inhibition of theBCR-mediated STAT3 phosphorylation with R406 only (Fig.3d and Supporting Information Fig. S3B).
In conclusion, we evidenced in MCL a BCR-dependentactivation of several important effectors such as SYK, BTK,ERK and STAT3. Moreover, inhibition of the proximal BCRsignalosome results in a complete inhibition of downstreameffectors such as ERK or STAT3.
Blocking BCR signalling induced apoptosis through the
inhibition of the autocrine secretion of prosurvival factors
Treatment of primary MCL cells (n5 11) with Ibrutinib andR406 increased significantly apoptosis after 24 hr (mean apo-ptosis of 66 and 62%, respectively, when compared tountreated cells 52%), suggesting the existence of a basal BCRsurvival signal. Indeed, stimulation of the cells with anti-IgMreduced apoptosis. This survival effect was blocked by Ibruti-nib and R406 pretreatment (mean apoptosis 57 and 54% inIbrutinib and R406-treated cells, respectively, when compared
to anti-IgM-stimulated cell apoptosis 33%) (Fig. 4a). Asalready mentioned, BCR signalling induces the secretion ofprosurvival factors; thus, we investigated the impact of thetreatment with Ibrutinib on this secretion. Treatment withIbrutinib mainly decreased the basal secretion of CCL5, IL-8,IL-10 and VEGF in Jeko-1 cells and IL-1b, IL-6, IL-8 andTNFa in three MCL patients’ cells (Figs. 4b and 4c and Sup-porting Information Fig. S4), suggesting that the constitutiveactivation of BCR-associated kinases BTK and SYK leads tothe secretion of prosurvival factors. Further activation of theBCR induced the secretion of these factors both in Jeko-1and in primary cells with a major effect on the latter. Pre-treatment with Ibrutinib decreased to variable extent BCR-induced secretion in MCL patients’ cells. A major effect wasobserved on IL-10 secretion in all these primary MCL cellsdemonstrating a direct link between BCR signalling andsecretion of prosurvival factors (Fig. 4c and Supporting Infor-mation Fig. S4).
Bone marrow stromal environment protects MCL cells from
spontaneous apoptosis through paracrine secretion of
prosurvival factors
As the survival of MCL cells depends on the interaction withthe microenvironment, we used a co-culture model of MCLcells and HMSCs to analyse the impact of paracrine secretedfactors on apoptosis (Figs. 5a and 5b). We observed in 24-hrco-culture that MCL cells were found: (i) in suspension inthe culture medium or (ii) adherent to and under the HMSClayer (Figs. 5a and 5b). Already in the upper suspension, weobserved a marked reduction of spontaneous apoptosis ofprimary MCL cells when cultured in the presence of HMSC(mean apoptosis in the presence or absence of HMSC 41%when compared to 61%, respectively; p5 0.001) (Fig. 5c andSupporting Information Fig. S5). In addition, a substantialprotection against apoptosis was observed for adherent/undercells that had established direct contact with HMSC (meanapoptosis in the presence or absence of HMSC 22% whencompared to 61%, respectively; p5 0.011) (Fig. 5c). In orderto assess whether this protecting role of HMSC towards MCLcells might be attributed to the secretion of prosurvival fac-tors, we measured the concentration of seven selected
Figure 2. BCR-induced cell survival is associated with an induced secretion of prosurvival factors. (a) Spontaneous apoptosis of PBMC from
11 leukaemic MCL patients was measured using flow cytometry. PBMCs were cultured for 24 hr in the absence (control) or presence of
soluble anti-IgM antibody (10 mg/mL). Percentage of apoptotic cells corresponded to the number of annexin V-positive, including PI-
negative and PI-positive cells on the 10,000 events recorded. Results are shown as mean 6 quartile (box) 6 SEM (bars) (n 5 11). The statis-
tical significance of the differences between groups was calculated using paired Student’s t-test. All measurements were done in duplicate
and the mean value is reported as a line on the graph. (b) Jeko-1 cell line was cultured in the absence (0) or presence of anti-IgM antibody
for 30, 60 and 240 min. mRNA expression of IL-1b, CCL5, IL-6 and IL-8 was analysed by qRT-PCR and the fold change was calculated when
compared to unstimulated cells. (c) The amount of secreted IL-1b, IL-6, CCL5, IL-8, IL-10, TNFa and VEGFa in absence (control) or upon of
BCR stimulation (anti-IgM 10 mg/mL) for 24 hr was evaluated by Luminex immunoassay on the culture supernatant of 12 MCL patients’ pri-
mary cells. Results are shown as median 6 quartile (box) 6 SEM (bars) of the concentration in pg/mL of the indicated secreted factors. The
statistical significance of the results was analysed between unstimulated and anti-IgM-stimulated samples by the Wilcoxon signed-rank
test. The asterisks indicate ***p<0.001, **0.001<p<0.01 and *p<0.02, respectively. (d) Fold change of secreted factors concentrations
upon 24-hr BCR stimulation were analysed with respect to the mutational status of IGHV genes. Results are shown for non-mutated IGVH
(NM) (n 5 5) group and in the mutated IGVH (M) (n 5 5) group. Results are shown as mean 6 quartile (box) 6 SEM (bars). Statistical signifi-
cance was calculated using the paired Student’s t-test.
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Bernard et al. 7
Int. J. Cancer: 00, 00–00 (2014) VC 2014 UICC
secreted factors in the supernatant of HMSC. Indeed, wefound high amounts of IL-6, IL-8 and VEGF that are impor-tant cytokines for B lymphocytes survival (Fig. 5d). More-over, the co-culture increased the basal secretion of IL-1b,IL-6, IL-8, IL-10, TNFa and VEGF by primary MCL cells(UPN2) (Fig. 5e, Control). This effect of the co-culture wasconfirmed upon BCR triggering for few cytokines (Fig. 5e,
anti-IgM). Next, we compared the secretion levels of thecytokines in the co-culture treated or not with Ibrutinib orR406. We observed an increase of secretion of IL-6, IL-8 andVEGF when cells were co-cultured with either Ibrutinib orR406 (Fig. 5e, Ibrutinib and R406). Importantly, we did notobserve by microscopy nor flow cytometry the detachment ofHMSC under treatment with the two inhibitors (Supporting
Figure 3. Impact of Ibrutinib and R406 inhibitors on BCR kinase activation. (a) Jeko-1 cells were pretreated for 90 min with increasing con-
centrations of Ibrutinib (0.1–10 mM) (a) or R406 (0.5–4 mM) (b). BCR was further stimulated with anti-IgM antibody for 30 min. Total cell
lysates were analysed by Western blotting. Primary MCL cells of a leukaemic patient were pretreated or not with Ibrutinib (5 mM) and R406
(2 mM) for 90 min. BCR was further stimulated or not with anti-IgM for 30 min (c) or 24 hr (d). Total cell lysates were analysed by Western
blotting with the indicated antibodies as previously described.
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8 BCR inhibition in MCL
Int. J. Cancer: 00, 00–00 (2014) VC 2014 UICC
Information Fig. S5 and data not shown). This suggestedthat the viability of HMSC was not affected by the inhibi-tors. As cytokines production measured in the co-culturesresulted from both HMSC and MCL cells, we extrapolatedthe contribution of the stromal cells by subtracting the
secretion of MCL alone from that of the co-culture. Thissecretion did not seem to vary in the presence or not ofinhibitors, suggesting that Ibrutinib and R406 do notaffect cytokines secretion by the stromal layer (data notshown).
Altogether, these results suggest that soluble factors anddirect contacts between MCL cells and the bone marrowmicroenvironment contribute to protect MCL cells from apo-ptosis and enhance autocrine secretions.
Blocking BCR signalling both prevents and inhibits
adhesion to HMSC leading to MCL cells apoptosis
As the treatment with Ibrutinib leads to an egress from thelymph node of MCL cells into peripheral blood of patients,31
we hypothesised that BCR signalling can promote the adhe-sion of MCL cells to HMSC. Indeed, we observed that thenumber of CD191 MCL cells adherent to the HMSC layerincreased in co-cultures when BCR was stimulated for 24 hr(Fig. 6a). Next, we asked whether the inhibitors had animpact on BCR-mediated MCL cell adhesion on the HMSClayer. We pretreated MCL cells with Ibrutinib or R406 for 90min before stimulating the BCR for 24 hr. The treatmentwith Ibrutinib or R406 blocked BCR-dependent adhesion ofprimary MCL cells (the mean percentage of CD191 adherentcells was 52% in Ibrutinib and 43% in R406-treated cells,respectively, when compared to 65% for BCR-stimulated cellsin the absence of treatment; n5 3). In addition, inhibitorypretreatment of MCL cells in the absence of stimulation didnot significantly impact their adhesive properties (Fig. 6a). Interms of apoptosis, the protective effects of both anti-IgMand co-culture were reversed by Ibrutinib and R406 pretreat-ment. Interestingly, a similar effect was observed for MCLcells both in suspension and in contact with HMSC (Fig. 6b,grey bars) Altogether, these results show that co-culture withHMSC provide a strong protective signal for MCL cells thatcan be reversed by inhibition of BCR signalling.
Effect of long-term co-culture on MCL survival and
adhesion
When MCL primary cells were co-cultured with HMSC for15 days, spontaneous apoptosis decreased in both suspensionand adherent pool of MCL cells (mean apoptosis 37 and17%, respectively, when compared to 95% dead cells withoutco-culture), thus mimicking the physiological situation (Fig.6c). Interestingly, in long-term co-culture the treatment withR406 for 24 hr partially reversed the protective effect of stro-mal cells and inhibited the adhesion of MCL cells to HMSC(Figs. 6c and 6d). Ibrutinib treatment resulted in a strongereffect on both apoptosis for cells in suspension and adhesion.However, in such conditions, the number of remainingadherent cells did not allow quantification of apoptotic cells(Figs. 6c and 6d).
Altogether these results suggest a central role for BCR sig-nalling in the induction of factors that potentiate the survivalsignals and promote adhesion to stromal cells.
Figure 4. Treatment with ibrutinib and R406 induced apoptosis and
inhibited BCR-mediated survival through the modulation of secreted
prosurvival factors. (a) Apoptosis of PBMC from 11 leukaemic MCL
patients was measured by flow cytometry. PBMCs were cultured for
24 hr in the absence (control) or in the presence of soluble anti-
IgM antibody (10 mg/mL) and treated with Ibrutinib (5 mM) or R406
(2 mM). Apoptotic cells corresponded to percentage of annexin V-
positive, including PI-negative and PI-positive cells. Results are
shown as mean 6 SEM (bars) (n 5 11). Differences between groups
were determined using the paired Student’s t-test. The asterisks
indicate ***p<0.001, **0.001<p<0.01 and *p<0.02, respec-
tively. All measurements were done in duplicate and the mean
value is represented on the graph by a bar. (b) Jeko-1 cells or (c)
primary MCL cells of a leukaemic patient (UPN-2) were cultured for
24 hr in the absence (control) or in the presence of anti-IgM and
treated or not with Ibrutinib (5 mM). The amount of seven selected
secreted factors was measured in the culture supernatant by Lumi-
nex immunoassay. Fold change secretion was calculated as the
ratio between cytokine concentrations in the samples and in the
untreated unstimulated cells (control). [Color figure can be viewed
in the online issue, which is available at wileyonlinelibrary.com.]
DiscussionIn this manuscript, we show the impact of BCR signalling onthe regulation of MCL cells cross-talk with their microenvir-onment. We provide evidences for a role of secreted factorsin the BCR-mediated survival facilitating bone marrow inter-actions. As targeting BCR-associated tyrosine kinases hasemerged as a promising strategy for the treatment of B-lymphoid neoplasms, we first evaluated the integrity of theBCR signalling pathway in MCL cells by the analysis of thephosphorylation on specific tyrosine residues of three kinases
involved in BCR response, BTK, SYK and ERK.26 In Jeko-1cell line and MCL patients cells, we observed a constitutivephosphorylation of BTK (Y223) and ERK (Y42/44) but avery weak basal phosphorylation of SYK on Y525/526,known to increase upon BCR stimulation.32 These results arein agreement with Pighi et al. who described a weak phos-phorylation of SYK (Y525/526) in Jeko-1 cells and variablelevel of phosphorylation in patients’ MCL cells.9 In addition,in MCL cells the highest expressed phosphopeptides identi-fied by a PhosphoScan approach correspond to kinases
Figure 6. Treatment with Ibrutinib and R406 in short-term (24 hr) and long-term co-culture (15 days) blocked and disrupted the adhesion of
MCL cells and induced apoptosis. (a) Primary MCL cells of three leukaemic patients were co-cultured on a HMSC layer for 24 hr. The cells
were pretreated or not with Ibrutinib (5 mM) or R406 (2 mM) for 90 min before BCR stimulation or not with anti-IgM antibody for 24 hr.
Supernatants containing non-adherent (suspension) MCL cells were collected. Numbers of adherent/under cells were obtained after trypsini-
sation of the co-culture and CD191 cells were counted by flow cytometry for 10,000 recorded events. Mean 6 SEM values of the three inde-
pendent experiments are represented on the graph. (b) Apoptosis was measured in parallel in MCL cells cultured alone (no HMSC) and co-
cultured with HMSC separately in non-adherent CD191 MCL cells collected in the supernatants (suspension), and in adherent/under
CD191 MCL cells after trypsinisation. Mean apoptosis 6 SEM values of the three independent experiments are represented. (c) Primary MCL
cells of three patients were cultured alone (no HMSC) or co-cultured on HMSC for 15 days before treatment with Ibrutinib (5 mM) or R406
(2 mM) for 24 hr. Apoptosis was analysed by flow cytometry on CD191 cells in MCL cells cultured alone (no HMSC) or co-cultured with
HMSC. Apoptosis in non-adherent MCL cells collected in the supernatants (suspension) and in adherent/under CD191 MCL cells after tryp-
sinisation are reported separately. Mean apoptosis 6 SEM values of the three experiments are represented. (d) In parallel, % of CD191
adherent/under cells count is reported for untreated (control) and Ibrutinib- and R406-treated co-culture, *p<0.02.
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Int. J. Cancer: 00, 00–00 (2014) VC 2014 UICC
involved in the BCR signalling pathway (e.g., SYK, LYN,HCK, BTK and PKC-delta).9 Interestingly, similar phospho-SYK (Y525/526) profile has been described in DLBCL. Inthese cells, R406 cytotoxicity was due to the specific inhibi-tion of SYK autophosphorylation without an effect on SYKY352 that does not belong to the kinase domain.33 Moreover,marked increase of SYK (Y525/526) phosphorylation follow-ing BCR stimulation was described as predictive to R406sensitivity in DLBCL. The constitutive phosphorylation ofBTK Y223 and ERK (Y42/44) with a marked increase ofSYK (Y525/526) phosphorylation following BCR triggeringsuggests a tonic BCR signalling as it is described forDLBCL.33
One of the effects of BCR activation is the secretion ofprosurvival factors. We previously reported that BCR acti-vation inhibited spontaneous apoptosis in MCL cellsthrough an autocrine NF-kB-dependent loop of secretedIL-6 and/or IL-10.27 With this in mind, we selected seventarget proteins of the NF-kB pathway, i.e., IL-6, IL-8, IL-10,IL-1b, CCL5, TNFa and VEGF known to promote cell sur-vival in MCL and CLL and checked for their secretionupon BCR stimulation. In CLL cells, IL-1b, IL-6 and IL-8are upregulated after IgM-triggering,34 therefore promotingcell survival. Importantly, several studies have uncoveredpotential mechanisms by which SYK mediates both theproduction and the maturation of the proinflammatory fac-tor IL-1b.32 Moreover, higher plasma levels of IL-6 or IL-8,in CLL, were correlated with advanced stages and higherrisk of disease progression.35,36 These observations are alsoconsistent with studies on DLBCL.37 Indeed, plasma levelsof TNFa and IL-10 appear indicative prognostic factors tothe outcome of the disease in patients with DLBCL.38 InMCL, CCL5 was already shown upregulated and importantfor cell survival.39 In addition, VEGF may act as an auto-crine growth factor in lymphoma, because aggressive lym-phomas often secrete VEGF and express VEGF receptors.40
Overexpression of VEGF in MCL tumours also correlateswith worse overall survival and increased disease aggres-siveness in patients.41 Moreover, VEGF signalling is knownto regulate survival signals in CLL cells and the interrup-tion of this autocrine pathway results in caspase activationand subsequent leukaemic cell death.42 In our study, wereport a basal and BCR-mediated secretion of IL-1b, IL-6,IL-8, CCL5, TNFa, IL-10 and VEGF by MCL patients’ cells.This autocrine secretion of chemokines, cytokines andgrowth factors, due to a constitutive BCR signalling, leadsto an important survival signal for leukaemic IGHV MCLthat maintains tumour cells in the blood circulation. Nosignificant difference was found between IGHV groups atthe level of basal secretion but we observed a noticeableBCR-dependent increase of the secretion of IL-1b, IL-6,TNFa and IL-10 in the mutated IGHV group. A largernumber of cases should be analysed to confirm this differ-ence, but the data suggest an important role for BCR sig-nalling related to soluble factor-dependent B-cell survival in
mutated IGHV cells. Similarly, the BAFF/SYK/PI3K axismediates B-cell survival and requires intact BCR andCD79A that allows sustained signal and production of sur-vival factors.43 Interestingly, in the bone marrow environ-ment, a paracrine secretion of IL-6, IL-8 and VEGFa bystromal cells was previously shown to generate a strongsurvival signal in MCL cells.44,45 We suggest that someBCR-induced secreted factors such as IL-1b, CCL5 andTNFa that do not originate from HMSC could stimulatethe B cells to upregulate genes encoding chemokines oradhesion molecules like VCAM1 facilitating the adhesionof MCL cells to HMSC.34,46–48
One expected consequence of the inhibition of BCR sig-nalling is the downregulation of the secretion of survival fac-tors and the blockade of signalling pathways involved in cellsurvival, such as STAT3 signalling pathway. The latter isknown constitutively activated in MCL cells, as a result of anautocrine loop of NF-jB-dependent secretion of IL-6 or IL-10. However, STAT3 is also substrate of both SYK and BTKin oxidative stress conditions and in a cytokines-independentfashion.49 Our results on the suppression of phosphorylationof STAT3 signal in the presence of SYK inhibitor (R406) butnot with BTK inhibitor (Ibrutinib) suggest that, also in MCL,STAT3 might be a direct target of SYK. Moreover, weobserve that BTK inhibition by Ibrutinib leads to a decreasein the secretion of prosurvival factors that can contribute toincreased apoptosis and cell death. The effect of BTK inhibi-tor on cytokines secretion is in agreement with the findingthat Ibrutinib decreases IL-10 and TNFa secretion in MCLcells in vitro following BCR activation.31 It is demonstratedthat BTK is also required for IL-10 production and phospho-rylation of NF-jB p65RelA in response to CpG in B cells.50
Finally, BTK is essential for synergistic IL-6 production inresponse to CpG and anti-IgM in primary murine andhuman B cells.51 Importantly, we observed that BTK andSYK inhibitors affect not only the secretion profiles but alsoblock the adhesion of MCL cells to BMSCs. Thus, we pro-pose that BCR signalling promotes adhesion to stromal cellsby the secretion of cytokines and growth factors able toenhance the contact with integrins such as VLA4/VCAM1.Disrupting this contact by Ibrutinib or Fostamatinib (R406)might cause cells to return to the flow and die. Indeed, auto-crine VEGF was shown important for chemokine-inducedCLL cell motility. The importance of autocrine VEGF wasfurther confirmed by the fact that blocking VEGF inhibitedthe chemokine-induced motility of CLL cells on purifiedVCAM-1.52 As specific antibodies and therapies against thesesecreted factors and integrins are becoming available for clin-ical use, the combination of these agents might have thera-peutic potential in patients with MCL who present tissueinvasion.
AcknowledgementS.B. was supported by ARC (Fondation ARC pour la Recherche sur leCancer).
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