Several studies have recently suggested that the anti-apoptotic Bcl-2 family member Bfl-1/A1 could be involved in the long-term survival of normal mature bone marrow PC but there is no evidence that Bfl-1/A1 is actually highly expressed at the late stage of PC-differentiation. In addition, the expression of Bfl-1/A1 in malignant PC is still a matter of controversy (Jourdan, et al 2003
, Mitsiades, et al 2002a
, Mitsiades, et al 2002b
). The aim of this study was thus to definitively clarify the role of Bfl-1/A1 in the biology of normal PC and in the pathogenesis of MM.
We demonstrated here using a combination of microarray experiments and quantitative RT-PCR that Bfl-1/A1 mRNA level was very low in normal PC compared to normal B cells and was not statistically different between short-lived plasmablasts and long-lived BMPC. The downregulation of Bfl-1/A1
during B-cell differentiation into PC is in good agreement with previous studies showing that PRDI-BF1/Blimp-1 is expressed in all PC subsets (Angelin-Duclos, et al 2000
, Tarte, et al 2003
) and strongly represses Bfl-1/A1 expression (Knödel, et al 1999
, Shaffer, et al 2002
). Moreover, our current work suggests that the prolonged survival of BMPC within specialized microenvironment niches is not due to an up-regulation of Bfl-1/A1, but probably to other anti-apoptotic proteins, such as A20, c-IAP2, and Bcl-2, that were all recently described as overexpressed in BMPC compared to PPC (Tarte, et al 2003
). Concerning malignant PC, we found a similarly low expression of Bfl-1/A1
in 16/16 HMCL and 26/26 purified primary myeloma cell samples. This confirms our previous results obtained using ribonuclease protection assay (Jourdan, et al 2003
) and Affymetrix microarrays (Tarte, et al 2002
, Tarte, et al 2003
, Zhan, et al 2002
). In particular, in our gene expression profiling of 74 newly diagnosed MM patients Bfl-1/A1
was not identified as a “spiked gene” i.e.
a gene overexpressed in a small subset of patients (Zhan, et al 2002
). Interestingly, Bfl-1/A1
levels evaluated using quantitative RT-PCR were very heterogeneous in MM samples. This could reflect the heterogeneity of myeloma disease where the tumor cell compartment comprised different subpopulations based on growth and maturation criteria (Bataille, et al 2003
). Of note, there was no difference in Bfl-1/A1 levels between primary myeloma samples collected at diagnosis and HMCL obtained from patients at the terminal stage of the disease. Collectively, these data suggest that Bfl-1/A1 is not a major anti-apoptotic protein in the survival of myeloma cells and plays no crucial role in the emergence of myeloma disease.
It was previously reported that the MM1-S myeloma cell line constitutively expressed Bfl-1/A1, cIAP-2, FLIP, survivin, and XIAP and that further activation of NF-κB pathway by insulin-like growth factor-1 (IGF-1) is associated with an up-regulation of these four anti-apoptotic proteins in this cell line (Mitsiades, et al 2002a
). The NF-κB pathway plays probably a key role in the survival of normal and malignant PC, as it is the case for normal and malignant B cells. Interestingly, we have previously detected a NF-κB signature, i.e
. an up-regulation of several NF-κB family members and target genes, in fully mature long-lived BMPC unlike in PPC that are characterized by their short survival (Tarte, et al 2003
). In addition, NF-κB blockade, using the proteasome inhibitors PS-341/Bortezomib, the IκB kinase inhibitor PS-1145, or SN50, a synthetic peptide that specifically inhibits NF-κB nuclear translocation, induces apoptosis in HMCL and primary myeloma cells (Hideshima, et al 2002
, Mitsiades, et al 2002b
). Recently, a phase II clinical trial has demonstrated that Bortezomib is active in vivo
in patients with relapsed refractory MM (Richardson, et al 2003
). BAFF and APRIL, two members of the TNF superfamily, strongly induce NF-κB activation in MM cells, in association with an induction of Bcl-2 and Mcl-1, and protect them against apoptosis induced by IL-6 deprivation and dexamethasone (Moreaux, et al 2003
). We thus tested them for Bfl-1/A1
induction. However, we show here that neither BAFF nor APRIL is able to upregulate Bfl-1/A1 in XG-13 HMCL.
The low level of Bfl-1/A1 found in BMPC despite hallmarks of NF-κB activation, in association with data showing that Bfl-1/A1 was poorly expressed and not inducible in MM cells in response to potent NF-κB activators, suggests that the active repression of Bfl-1/A1 by PRDI-BF1/Blimp-1 in normal and malignant PC is fairly strong to overcome NF-κB signaling. In addition, the apoptotic effect of Bortezomib and SN50 on MM cells is probably not essentially due to a downregulation of Bfl-1/A1, but rather to the reported inhibition of expression of Bcl-2, XIAP, cIAP-2, or survivin.
Overexpression of Bfl-1/A1 through retroviral transduction protects B cells from antigen receptor-mediated (Craxton, et al 2000
, Kuss, et al 1999
) and PRDI-BF1/Blimp-1-mediated (Knödel, et al 1999
) apoptosis. We thus evaluated if Bfl-1/A1 could also protect MM cells from apoptotic stimuli. We showed here that transduction with Bfl-1/A1 resulted in the survival of an IL-6-dependent HMCL in the absence of IL-6, making it possible its long-term autonomous growth. It also conferred resistance to dexamethasone-induced apoptosis. Until now, only Mcl-1 and Bcl-2 overexpression were reported to protect MM cells from apoptosis induced respectively by IL-6 deprivation (Jourdan, et al 2000
, Jourdan, et al 2003
) and dexamethasone (Feinman, et al 1999
). The survival of Bfl-1/A1-transduced HMCL was not due to the induction of an IL-6 autocrine loop since it was not abrogated by B-E8 anti-IL-6 antibody whereas the IL-6-dependent survival of parental and mock-transduced HMCL was completely blocked by B-E8. Interestingly, Bfl-1/A1 constitutive expression protected only partially MM cells from IL-6 deprivation, at a level similar to Mcl-1 constitutive expression (Jourdan, et al 2003
In conclusion, Bfl-1/A1 expression is strongly repressed at all stages of normal PC differentiation as well as in malignant PC. Ectopic expression of Bfl-1/A1 promotes myeloma cell survival, as it was previously reported for normal B cells committed in vitro
to a PC fate (Knödel, et al 1999
). A protection of malignant PC from apoptosis by Bfl-1/A1 overexpression might be of potential interest, especially to promote the development of immunotherapy strategies in MM. Indeed, myeloma cell lines can only be obtained from patients with secondary extramedullary proliferation and fulminant disease (Zhang, et al 1994
). At the early stages of myeloma disease, malignant PC can be harvested only in small amount, do not proliferate and rapidly dye in vitro
, even in the presence of exogenous cytokines such as IL-6. In this context, Mcl-1 transduction would not be sufficient to promote prolonged primary myeloma cell growth since a high level of Mcl-1 is already achieved in these cells through addition of exogenous cytokines, without promoting their survival. On the contrary, Bfl-1/A1 ectopic expression could be a first step in the generation of myeloma cell lines at diagnostic. In addition, generation of long-lived, antibody-secreting, normal PC is an essential goal for the development of protective humoral vaccination strategies. We recently demonstrated that peripheral blood memory B cells can be reproducibly induced to differentiate into short-lived plasmablastic cells producing somatically mutated immunoglobulin (Tarte, et al 2002
). Transduction of Bfl-1/A1 in these human in vitro
derived plasmablastic cells might prevent them from apoptosis, as previously described in the context of murine in vitro
generated plasmablasts, and help obtaining PC lines producing human antibodies.