The Role of the IRF4-NOTCH2 Interplay in B-Cell Development and Malignancy
Final Report Abstract
This research proposal was aimed at exploring the role of the IRF4-NOTCH2 interplay in mature B cells in order to understand the potential tumor-suppressor function of IRF4 and the putative oncogenic role of NOTCH2 in the pathogenesis of CLL. Because of experimental difficulties outlined in the report, we were not able to determine the molecular mechanism by which IRF4 controls trafficking of mature B cells through regulation of NOTCH2 activation. However, the alternatively performed research on the role of the canonical NF-κB subunits in the development and the maintenance of B cells, as well as their contribution to the development of B-cell malignancies, resulted in several interesting findings. The results described in Aim 1 showed that combined ablation of the canonical NF-κB subunits c-REL and RELA specifically in B cells in vivo strongly impaired the generation of mature B cells, indicating functional redundancy of the canonical subunits during this developmental stage. Specifically, fewer and smaller B-cell follicles developed in the spleens of relfl/flrelafl/flCD19-Cre mice, and a dramatic counter-selection against c-REL/RELA-deficient marginal zone B cells was observed. Interestingly, while the combined loss of c-REL and RELA had no effect on B-cell development in the bone marrow, it led to a developmental block in the transitional (T) T1 to T2 phase in the periphery, most likely by facilitating the developmental transition rather than merely mediating cell survival. We observed that rel/rela-deleted B-cells cultured with B-cell activating factor (BAFF; the principal survival factor for mature B cells) showed the same predisposition to undergo apoptosis as B cells lacking the non-canonical NF-κB factors RELB and NF-κB2, as recently published by the laboratory. Thus, the lack of the canonical subunits was not compensated by the non-canonical pathway in B cells stimulated with BAFF. These novel results support a model in which canonical and non-canonical NF-κB pathways regulate two independent survival programs in mature B cells and provide a rationale to identify the specific transcriptional programs regulated by the separate NF-B pathways upon BAFF stimulation. In Aim 2 we sought to define the dependency of germinal center (GC)-derived malignancies on the canonical NF-κB subunits RELA and c-REL. To this end, we transduced activated B-cell-type diffuse large B-cell lymphoma (ABC-DLBCL) and multiple myeloma (MM) cell lines with fluorescence-tagged shRNAs targeting RELA or c-REL. Interestingly, we found that cell growth and survival of a subset of ABC-DLBCL lines was specifically dependent on the RELA subunit; ablation of c-REL expression in these lines had no functional consequences. These results point towards a non-redundant role for RELA in the maintenance of these lymphoma cell lines. Interestingly, similar assays performed in two MM cell lines indicate that also MM cell lines are addicted to RELA. The apparent RELA dependency of certain GC-derived malignancies and the known essential role of RELA in the generation of GC-derived plasma cells led us to hypothesize that RELA is a central transduction node for the recently described NF-κB-induced kinase (NIK)-driven plasmablast hyperplasia in mice, as published by our collaborator. Indeed, in vivo data from NIKfl/+relafl/flCγ1-Cre and the corresponding control mice suggest that a RELA-mediated transcriptional program is essential for NIK-mediated plasmablast hyperplasia. This conditional transgenic mouse model is thus highly suited to identify the mechanism by which the canonical NF-κB subunit RELA contributes to the development of plasmacytosis and potentially to the transformation of plasmablasts. The differential requirements of c-REL and RELA in B-cell activation and differentiation may be exploited for the development of more specific and thus less toxic therapies aimed at inhibiting pathogenic NF-κB signalling in malignant and chronically activated B cells at the level of NF-κB subunits.
Publications
- (2017). Differential requirements for the canonical NF-κB transcription factors c-REL and RELA during the generation and activation of mature B-cells. Immunol Cell Biol. 95(3):261-271
Milanovic M, Heise N, De Silva NS, Anderson MM, Silva K, Carette A, Orelli F, Bhagat G, Klein U
(See online at https://doi.org/10.1038/icb.2016.95)
