CD40, a member of the TNF receptor family, is expressed on all mature B cells and on most B-cell lymphomas. Recently, we have shown that constitutive activation of CD40 signaling in B cells induced by a fusion protein consisting of the transmembrane part of the Epstein-Barr viral latent membrane protein 1 (LMP1) and the cytoplasmic part of CD40 (LMP1/CD40) drives B-cell lymphoma development in transgenic mice. Because LMP1/ CD40-expressing B cells showed an upregulation of CD19, we investigated CD19's function in CD40-driven B-cell expansion and lymphomagenesis. Here, we demonstrate that ablation of CD19 in LMP1/CD40 transgenic mice resulted in a severe loss and reduced lifespan of mature B cells and completely abrogated development of B-cell lymphoma. CD19 is localized to lipid rafts and constitutively activated by the LMP1/CD40 fusion protein in B cells. We provide evidence that the improved survival and malignant transformation of LMP1/CD40-expressing B cells are dependent on activation of the MAPK Erk that is mediated through CD19 in a PI3K-dependent manner. Our data suggest that constitutively active CD40 is dependent on CD19 to transmit survival and proliferation signals. Moreover, we detected a similarly functioning prosurvival pathway involving phosphorylated CD19 and PI3K-dependent Erk phosphorylation in human diffuse large B-cell lymphoma cell lines. Our data provide evidence that CD19 plays an important role in transmitting survival and proliferation signals downstream of CD40 and therefore might be an interesting therapeutic target for the treatment of lymphoma undergoing chronic CD40 signaling. Cancer Res; 74(16); 4318-28. Ó2014 AACR.

The non-canonical NF-κB pathway is highly conserved, can be activated by TNF-receptors e.g. CD40 and is dysregulated in several lymphomas (Krappmann and Vincendeau 2016). In human DLBCL hyper-activation of the non-canonical NF-kB signaling pathway could be detected in about 15% (Zhang et al. 2015). Upon activation of the non-canonical NF-κB pathway RelB is translocated to the nucleus and acts as transcription factor. RelB is important to maintain viability of a Hodgkin Lymphoma cell line (Ranuncolo et al. 2012), but interestingly B cell specific inactivation of RelB in mature B cells of transgenic mice have only a minor effect on B cell maintenance and activation (De Silva et al. 2016). Therefore it is still elusive, whether RelB-regulated genes contribute to lymphoma development. To study the contribution of RelB-regulated genes to lymphoma development we used a mouse model expressing a constitutively active CD40 receptor in B cells (LMP1/CD40stopflmice). LMP1/CD40 is a fusion protein comprising the signaling domain of human CD40 and the transmembrane domain of LMP1 and promotes a persistently activation of CD40 signaling. In LMP1/CD40stopfl//CD19-Cre (LMP1/CD40) mice the constitutive CD40 activation leads to the selective activation of the non-canonical NF-kB pathway and the MAP kinase pathways JNK and ERK. LMP1/CD40-expression positively affects cell survival and proliferation of B cells resulting in a B cell expansion and accumulation of B cells in follicles of young mice. Mice older than 12 months develop lymphomas with a high incidence. In most cases the lymphoma was monoclonal and had the phenotype B220low, CD21- and CD23- (Hojer et al. 2014; Hömig-Hölzel et al. 2008). Recently, we observed that this aberrant population appears at around 6 months of age and increases continuously during the aging process. To unveil the contribution of the non-canonical NF-κB pathway to the phenotype and lymphoma development of constitutively CD40-activated B cells we crossed conditional RelBfl/fl mice to LMP1/CD40stopfl//CD19-Cre mice (RelBKO//LMP1/CD40 mice). In young mice we were able to observe that LMP1/CD40 B cells without functional RelB were less expanded in the spleen and lymph nodes, but had elevated B cell numbers in the blood and a higher number of recirculating B cells in the bone marrow. This indicates that RelB-regulated genes are important for the retention of CD40-activated B cells in B cell follicles of secondary lymphoid organs. In addition, the survival of ex vivo isolated LMP1/CD40-expressing RelB-deficient B cells was diminished in comparison to LMP1/CD40-expressing RelB-proficent B cells, but RelBKO//LMP1/CD40 B cells still survived better than control B cells. Furthermore, we found that RelBKO//LMP1/CD40 B cells were less activated than LMP1/CD40 B cells, but had a higher expression of the tested activation markers than control B cells. In order to characterize the influence of RelB-regulated genes on lymphoma development in LMP1/CD40 mice we analyzed LMP1/CD40-expressing mice at different time points. The aberrant population, arising in LMP1/CD40 mice, was also detectable in aged RelBKO//LMP1/CD40 mice, though showed a delayed progression and always had a lower expansion than in age-matched LMP1/CD40 mice. Both transgenic mice developed monoclonal lymphomas. However, the double mutant mice had a lower tumor incidence than LMP1/CD40 mice with functional RelB. Nevertheless, the risk of lymphoma development in RelBKO//LMP1/CD40 was still higher than in wildtype mice. In the last part of this study we performed a RNA-Seq. analysis to see which genes are RelB-dependent in chronically CD40-activated B cells. We identified several RelB-dependent genes and 21 of these had been correlated with Non-Hodgkin Lymphoma. Some of these candidates could be promising new therapy targets and are under further investigation. In conclusion we provide evidence that RelB-regulated genes contribute to the survival and activation of B cells, which are chronically activated by CD40 signaling. Furthermore, we found that CD40 signaling enhances the retention of B cells in B cell follicles through activation of the non-canonical NF-kB pathway. Moreover, we demonstrated that RelB-regulated genes contribute to B cell lymphomagenesis and identified several RelB-regulated genes that may contribute to the expansion and lymphomagenesis of B cells receiving a chronic CD40 signal. Disclosures Weigert: Roche: Research Funding; Novartis: Research Funding.

<div>Abstract<p>CD40, a member of the TNF receptor family, is expressed on all mature B cells and on most B-cell lymphomas. Recently, we have shown that constitutive activation of CD40 signaling in B cells induced by a fusion protein consisting of the transmembrane part of the Epstein–Barr viral latent membrane protein 1 (LMP1) and the cytoplasmic part of CD40 (LMP1/CD40) drives B-cell lymphoma development in transgenic mice. Because LMP1/CD40-expressing B cells showed an upregulation of CD19, we investigated CD19's function in CD40-driven B-cell expansion and lymphomagenesis. Here, we demonstrate that ablation of CD19 in LMP1/CD40 transgenic mice resulted in a severe loss and reduced lifespan of mature B cells and completely abrogated development of B-cell lymphoma. CD19 is localized to lipid rafts and constitutively activated by the LMP1/CD40 fusion protein in B cells. We provide evidence that the improved survival and malignant transformation of LMP1/CD40-expressing B cells are dependent on activation of the MAPK Erk that is mediated through CD19 in a PI3K-dependent manner. Our data suggest that constitutively active CD40 is dependent on CD19 to transmit survival and proliferation signals. Moreover, we detected a similarly functioning prosurvival pathway involving phosphorylated CD19 and PI3K-dependent Erk phosphorylation in human diffuse large B-cell lymphoma cell lines. Our data provide evidence that CD19 plays an important role in transmitting survival and proliferation signals downstream of CD40 and therefore might be an interesting therapeutic target for the treatment of lymphoma undergoing chronic CD40 signaling. <i>Cancer Res; 74(16); 4318–28. ©2014 AACR</i>.</p></div>

<p>PDF file - 364K, Suppl. Figure 1. BM B cell development is comparable in LMP1/CD40//CD19+/- and LMP1/CD40//CD19-/- mice. Suppl. Figure 2. CD40 employs CD19 to mediate survival signals. Suppl. Figure 3. Loss of CD19 abrogates lymphoma development in LMP1/CD40 mice. Suppl. Figure 4A. Stimulation of B cells with anti-IgM in the presence of Dasatinib. Suppl. Figure 4B. Titration of the PI3K inhibitor LY294004 Suppl. Figure 5A. Sorafenib does not affect Erk phosphorylation in LMP1/CD40+ B cells. Suppl. Figure 5B. CD19 expression on different DLBCL and HL cell lines. Suppl. Figure 6A. CD40 expression in different DLBCL cell lines. Suppl. Figure 6B. CD19 phosphorylation after stimulation with a CD40 ligand.</p>

<p>PDF file - 364K, Suppl. Figure 1. BM B cell development is comparable in LMP1/CD40//CD19+/- and LMP1/CD40//CD19-/- mice. Suppl. Figure 2. CD40 employs CD19 to mediate survival signals. Suppl. Figure 3. Loss of CD19 abrogates lymphoma development in LMP1/CD40 mice. Suppl. Figure 4A. Stimulation of B cells with anti-IgM in the presence of Dasatinib. Suppl. Figure 4B. Titration of the PI3K inhibitor LY294004 Suppl. Figure 5A. Sorafenib does not affect Erk phosphorylation in LMP1/CD40+ B cells. Suppl. Figure 5B. CD19 expression on different DLBCL and HL cell lines. Suppl. Figure 6A. CD40 expression in different DLBCL cell lines. Suppl. Figure 6B. CD19 phosphorylation after stimulation with a CD40 ligand.</p>