Immunoglobulin (Ig) diversification by somatic hypermutation in germinal center B cells is instrumental for maturation of the humoral immune response, but also bears the risk of excessive or aberrant genetic changes. Thus, introduction of DNA damage by activation-induced cytidine deaminase as well as DNA repair by multiple pathways need to be tightly regulated during the germinal center response to prevent lymphomagenesis. In the present study, we show that DNA damage checkpoint signaling via checkpoint kinase 1 (Chk1) negatively regulates somatic hypermutation. Chk1 inhibition in human B cell lymphoma lines as well as inactivation of Chk1 alleles by gene targeting in DT40 B cells leads to increased somatic hypermutation. This is apparently due to changes in DNA repair pathways regulated by Chk1, such as a decreased homologous recombination efficiency that also leads to decreased Ig gene conversion in DT40. Our data show that Chk1 signaling plays a crucial role in regulation of Ig diversification and sheds unexpected light on potential origins of aberrant somatic hypermutation in B cell lymphomagenesis.

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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.

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