Oncogene (2007) 26, 5828–5832
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SHORT COMMUNICATION
Downregulation of Bfl-1 protein expression sensitizes malignant B cells to apoptosis G Brien1,2,3,4,5, M-C Trescol-Biemont1,2,3,4,5 and N Bonnefoy-Be´rard1,2,3,4 1 4
Inserm, U503, Lyon, F-69007, France; 2Universite´ de Lyon, Lyon, F-69003, France; 3Universite´ Lyon 1, Lyon, F-69003, France and IFR128, Lyon, F-69007, France
Elevated expression of the antiapoptotic protein Bfl-1 (A1) was previously reported in several cancer cell lines. Recently, molecular profiling of large B-cell lymphoma identified Bfl-1 as a gene signature in ‘OxPhos’ diffuse large B-cell lymphoma subtype and in primary mediastinal large B-cell lymphoma, suggesting that in addition to Bcl-2, Bcl-xL and Mcl-1, Bfl-1 may be a relevant target in the design of new strategies for cancer therapy. Using short hairpin RNA strategy, we show here that Bfl-1 silencing in one lymphoblastoid B-cell line and in two diffuse large B-cell lymphoma cell lines potently induces their apoptosis and sensitizes those cell lines to anti-CD20 (Rituximab)-mediated cell death as well as to apoptosis induced by chemotherapeutic molecules such as doxorubicin, vincristine, cisplatin and fludarabine. These results demonstrate for the first time that Bfl-1 is an essential protein for survival of malignant B cells and suggest Bfl-1 may represent a potential target for future drug development against B cell lymphoma. Oncogene (2007) 26, 5828–5832; doi:10.1038/sj.onc.1210363; published online 12 March 2007 Keywords: Bcl-2; Bfl-1; apoptosis; B lymphoma
Apoptosis is a highly regulated process, which serves to remove superfluous, damaged or infected cells throughout the lifespan of an organism and therefore to maintain cellular homeostasis. A delicate balance between proapoptotic and antiapoptotic regulators of apoptosis pathways ensures the proper survival of cells in a variety of tissues, including the bone marrow, thymus and peripheral lymphoid organs. Imbalance between proapoptotic and antiapoptotic proteins occurs in diseases such as cancer where an overexpression of antiapoptotic proteins endows cells with a selective survival advantage that promotes malignancy. Bcl-2 family members are essential regulators of the intrinsic apoptotic pathway, which centers on mitochondria as Correspondence: Dr N Bonnefoy-Berard, Laboratoire d’Home´ostasie Lymphocytaire, Inserm U503, 21 avenue Tony Garnier, Lyon 69007, France. E-mail:
[email protected] 5 These two authors contributed equally to this work. Received 8 November 2006; revised 14 December 2006; accepted 24 January 2007; published online 12 March 2007
initiator of cell death. The Bcl-2 protein and its closest relatives (Bcl-xL, Bcl-w, A1/Bfl-1 and Mcl-1) promote cell survival. Instead, structurally similar proteins Bax and Bak promote cell death. The life and death balance is displaced in favor to cell death by BH3-only proteins of the Bcl-2 family, such as Bim, Bad, Bid, Puma and Noxa, which interact with Bcl-2-like proteins and inactivate their functions (Willis and Adams, 2005). Imbalance in the levels or activities of several antiapoptotic Bcl-2 proteins has been documented in various lymphocytic malignancies, as for example nonHodgkin’s lymphoma in which Bcl-2 was discovered by virtue of the involvement of its encoding gene in chromosomal translocation (Tsujimoto et al., 1985). Molecular profiling of large B-cell lymphoma recently identified Bfl-1 as a gene signature in ‘OxPhos’ diffuse large B-cell lymphoma (DLBCL) subtype and in primary mediastinal large B-cell lymphoma (MLBCL) (Feuerhake et al., 2005; Monti et al., 2005; Dave et al., 2006), suggesting that Bfl-1 might also be a relevant target in the treatment of lymphocytic malignancies. Bfl-1 is a direct transcriptional target of nuclear factor-kappa B (NF-kB) (Grumont et al., 1999; Wang et al., 1999; Zong et al., 1999), and its overexpression in cell lines is associated with an increased resistance to apoptotic stimuli such as tumor-necrosis factor (TNF)a, anti-CD95, TNF-related apoptosis-inducing ligand or chemotherapeutic drugs (Wang et al., 1999; Zong et al., 1999; Werner et al., 2002). However, whether specific inhibition of Bfl-1 expression could sensitize cell lines to apoptosis has never been tested. We and others reported that the expression of A1, the murine homolog of Bfl-1, is controlled by B-cell survival signals such as CD40, Baff receptor or B-cell receptor (BCR) engagement (Hsu et al., 2002; Hatada et al., 2003; Trescol-Biemont et al., 2004). A1/Bfl-1 induction through CD40/NF-kB pathway is associated with an increased resistance to BCRmediated cell death in B cell lines and mature B cells (Grumont et al., 1999; Kuss et al., 1999; Lee et al., 1999). In DLBCL (Lam et al., 2005), inhibition of the activated NF-kB pathway leads to tumor cell apoptosis in association with downregulation of the expression of several antiapoptotic genes such as A20, c-IAP2, GADD45b and Bfl-1. Whether one of these genes is the crucial determinant of cell survival is still unclear. Interestingly, among these genes, Bfl-1 is the only one identified by molecular profiling as a gene signature in
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DLBCL, suggesting that Bfl-1 expression might be critical for survival of malignant B cells such as DLBCL. Among several B cell lines tested, we identified two DLBCL cell lines, BJAB and BP3 and one B lymphoblastoid cell line, IM9, that highly expressed Bfl-1 mRNA and protein (Figure 1). IM9 cells also expressed Bcl-2 and Bcl-xL proteins and very low level of Mcl-1, whereas BJAB and BP3 cells did not express Bcl-2, but expressed Mcl-1 and Bcl-xL proteins. We therefore directly evaluated consequences of downregulation of Bfl-1 expression on cell death resistance of these three cell lines, using short hairpin (sh) RNA strategy. We cloned Bfl-1 specific sequences into the lentivirus-based vector pLL3.7. This vector was engineered to co-express shRNA and enhanced green fluorescent protein (GFP) as a reporter gene, permitting infected cells to be tracked by flow cytometry. One shRNA, over the three designed, very efficiently inhibited Bfl-1 expression whereas
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control (Ctl) shRNA did not affect Bfl-1 expression (data not shown and Figure 3). Pilot experiments, realized on IM9 cells, indicated that multiplicity of infection (MOI) comprised between 4 and 16 resulted in the transduction of 70–95% of cells 2 days after infection and that the percentage of transduced cells remained over 70% for at least 10 days in viable cells (data not shown and Figure 2b). However, we observed that the level of expression of GFP in Bfl-1 shRNA transduced IM9 cells but not in Ctl shRNA transduced cells decreased over time (Figure 2a). Such observation indicated that only IM9 cells expressing low level of Bfl-1 shRNA (MFI below 100) survived 1 week after infection, and suggested that IM9 cells do not survive to efficient Bfl-1 silencing. We therefore directly assessed consequences of Bfl-1 inhibition on survival of IM9 cells and the two DLBCL cell lines, BJAB and BP3. As a control, we used the leukemic cell line RS4;11.
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Figure 1 Gene and protein expression of Bcl-2 family members in several B cell lines. (a) Real-time RT–PCR was performed using Syber Green method as described previously (Cottalorda et al., 2006). Relative expression of specific mRNA was normalized using housekeeping genes HPRT and GusB. The data are the mean of triplicates from one experiment representative of two independent experiments. (b) Protein levels for Bfl-1, Bcl-2, Bcl-xL and Mcl-1 in cell lines were determined by Western blot. Cells were lysed in NP40 lysis buffer (200 mM NaCl, 40 mM Tris-HCl pH 8, 2 mM EDTA, 1% NP-40) supplemented with Protease Inhibitor Cocktail (Sigma-Aldrich, France). Protein extracts (50 mg) were separated on a NuPage 4–12% Bis-Tris gel (Invitrogen, France) and transferred to nitrocellulose membrane. Protein expression was analysed by Western blotting using specific antibodies against Bfl-1 (kindly provided by J Borst, The Netherlands Cancer Institute, Amsterdam, The Netherlands), Bcl-2 (Dako, France), Bcl-xL (BD Pharmingen, France), Mcl-1 (Santa Cruz Biotechnology, France) or actin (Sigma, France). Protein–antibody complexes were visualized by chemoluminescence (Western Lighting Chemiluminescence Reagent Plus, PerkinElmer, France).
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Figure 2 Effect of downregulation of Bfl-1 expression in IM9 cells. The RNA interference sequence AAggAgTTTgAAgACggC ATC, specific for human Bfl-1 was used. Scrambled sequence from Bfl-1 (AAggTCACAgATAgATTggC) was used to produce Ctl shRNA. The corresponding oligonucleotides were cloned into the lentiviral vector pLL3.7 (LentiLox 3.7, Van Parijs Laboratory, MA, USA) according to Van Parijs Laboratory’s instruction. IM9 cells were transduced with Ctl or Bfl-1 shRNA (MOI 16). (a) MFI of transduced cells (GFP þ cells) was followed on viable cells from day 3 to day 10 after transduction. Data are representative of three independent experiments. (b) Histograms plots comparing the level of transduced cells (GFP þ ) measured by FACS at day 3 and 10 are shown. (c) Cells were non-infected (NI) or transduced with Ctl or Bfl-1 shRNA. Percentage of dead cells (PI þ ) was measured by FACS 5 days after infection. Results are expressed as mean7s.e.m. from triplicates of three independent experiments. Oncogene
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Figure 3 Effect of Bfl-1 silencing on susceptibility of IM9 and BP3 cells to cell death. Cell lines were NI or transduced with Ctl or Bfl-1 or Bcl-2 shRNA. The RNA interference sequence AACCgggAgA TAgTgATgAAg, specific for human Bcl-2 was used. Three days after infection, cells were treated with rituximab (50 mg/ml, Roche), doxorubicin (100 nM, Pfizer, France), vincristine (10 nM, Lilly, France), cisplatin (5 mg/ml for BP3, 1 mg/ml for IM9, Sigma) or fludarabine (10 mM, Sigma). Cell death was measured by FACS after PI labelling at 36 h for IM9 (a) and 48 h for BP3 (b). Data are the mean7s.e.m. of duplicates from one experiment representative of at least three independent experiments. The knockdown of Bfl-1 or Bcl-2 by the specific shRNA in IM9 and BP3 was controlled by Western blot 3 days after infection and is shown in insets of Figure 3(a) and (b) (for details, see experimental procedure in Figure 1b).
This cell line does not express Bfl-1 but expresses high level of Bcl-2 (Figure 1). Cells were transduced with Ctl and Bfl-1 shRNA and 5 days later cell viability was determined. We observed that Bfl-1 shRNA but not Ctl shRNA potently induced cell death in IM9, BJAB and BP3 but not in RS4;11 cell lines (Figure 2c). These results directly implicate Bfl-1 in survival of malignant B cells. As overexpression of Bfl-1 in cell lines has previously been associated with increased resistance to apoptotic stimuli (Wang et al., 1999; Zong et al., 1999; Werner et al., 2002), we next tested whether Bfl-1 silencing would affect chemotherapeutic agents-induced apoptosis of IM9 and BP3 cell lines. IM9 or BP3 cells were first transduced with Ctl or Bfl-1 shRNA. After 72 h, cells were treated with suboptimal concentrations of antiCD20, doxorubicin, vincristine, cisplatin or fludarabine for 36 or 48 h and cell death was measured by flow cytometry. As shown in Figure 3, we observed that downregulation of Bfl-1 increased efficacy to anti-CD20 and chemosensitivity to doxorubicin, cisplatin and fludarabine in both cell lines. Bfl-1 silencing also increased vincristine-induced cell death in IM9 but not in BP3 cells. These results show for the first time that specific downregulation of Bfl-1 protein increases cell death Oncogene
induced by anti-CD20 antibodies, but also chemosensitivity to doxorubicin, fludarabine and cisplatin, in malignant B cells. In agreement with previous results, demonstrating that high expression of Bfl-1 in cell lines or in primary cells from B-chronic lymphocytic leukemia patients is associated with increased resistance to apoptotic stimuli such as cisplatin, doxorubicin and fludarabine (Cheng et al., 2000; Morales et al., 2005), these results reinforce the potential role of Bfl-1 in chemoresistance. As IM9 cells expressed also Bcl-2 mRNA and protein, we compared the effect of Bfl-1 and Bcl-2 shRNA. We observed that downregulation of Bcl-2 in IM9 cells only slightly increased cell death of doxorubicin- and cisplatin-treated cells when compared with the effect of Bfl-1 shRNA. In contrast to Bfl-1 shRNA, treatment of IM9 cells with Bcl-2 shRNA had no effect on anti-CD20- and vincristine-induced cell death. However, we observed that Bcl-2 downregulation sensitized IM9 cells to fludarabine treatment. Altogether, these results clearly show that Bfl-1 silencing increases sensitivity of IM9 and BP3 cell lines to apoptosis induced by several chemotherapeutics agents and that the effect of Bfl-1 silencing is distinguishable from that of Bcl-2 silencing in cells that expressed both proteins. Upregulation of the antiapoptotic Bcl-2 family proteins, with resultant dysregulation of the intrinsic apoptotic pathway, has been established as one of the key pathways involved in chemotherapy resistance, aggressive clinical course and poor survival of various hematopoietic malignancies. For example, high Bcl-2 protein as a result of the t(14;18) chromosomal translocation is a key feature of low-grade follicular non-Hodgkin’s lymphoma (Reed and Pellecchia, 2005). Elevated Bcl-2 protein level, in association with t(14;18) translocation or gene amplification, has also been reported in approximately 30% of DLBCL patients. However, the correlation of Bcl-2 expression with survival remains controversial (Mounier et al., 2003; Iqbal et al., 2006). Among Bcl-2 proteins family, Bfl-1 has also been recently suggested to be a potent antiapoptotic factor in large B-cell lymphomas. Indeed, gene profiling analysis identified Bfl-1 as a gene signature in ‘OxPhos’ DLBCL subtype (Monti et al., 2005) and in MLBCL (Feuerhake et al., 2005). Moreover, in agreement with the earlier identification of Bfl-1 as a direct transcriptional target of NF-kB (Grumont et al., 1999; Wang et al., 1999; Zong et al., 1999), it has been demonstrated that the inhibition of NF-kB pathway in DLBCL (Davis et al., 2001; Lam et al., 2005) leads to tumor cell apoptosis in association with downregulation of Bfl-1 expression. These studies strongly suggest that Bfl-1 expression might be critical for survival of some malignant B cells such as large B-cell lymphoma. Reinforcing such hypothesis we provide here, for the first time, the direct evidence that downregulation of Bfl-1 sensitizes malignant DLBCL cell lines to apoptosis. The demonstration that Bfl-1 silencing increases apoptosis to anti-CD20, doxorubicin and cisplatin might be of considerable interest for treatment of large B-cell lymphoma. Improved clinical results in DLBCL
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patients were obtained owing to recent advances in empiric chemotherapy, including interval reduction of CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) and the introduction of rituximab (Coiffier, 2005; Feugier et al., 2005; Sehn et al., 2005). However, DLBCL is an heterogeneous disease with many subtypes and a significant proportion of patients are still resistant to treatment and die of their disease. Advances in the treatment of the disease may now come from the identification of novel therapeutic targets in specific disease subtypes. Results of the present study on the consequences of inhibition of Bfl-1 expression in DLBCL cell lines, and from recent gene expression profiling studies that highlighted overexpression of Bfl-1 in some DLBCL and in MLBCL, indicated that among Bcl-2 family members, Bfl-1 may be a rational therapeutic target in such disorders. However, whether failure to respond to current treatments might be related to Bfl-1 expression remains to be analysed in patients.
Abbreviations BCR, B-cell receptor; DLBCL, diffuse large B-cell lymphoma; GFP, green fluorescent protein; MLBCL, mediastinal large Bcell lymphoma; MOI, multiplicity of infection; shRNA, short hairpin RNA. Acknowledgements This work is support by institutional grants from INSERM and universite´ Lyon I, and additional support from the Association pour la Recherche sur le Cancer, the Ligue contre le Cancer (comite´ de Savoie et de la Droˆme) and Cance´ropole National. G Brien was supported by a fellowship from the re´gion Rhoˆne-Alpes and is now supported by a fellowship from the Ligue Nationale contre le Cancer. We thank Dr C Thieblemont for critical reading of the paper and Dr C Dumontet for helpful discussions, C Bella and O de Bouteiller for their expertise with cell sorting and D Ne`gre and B Boson for production of lentivirus particules.
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