A20 Tumor Suppressor Deletion and BCL6 Oncogene Activation Cooperate In Deregulating B Cell Differentiation In Vivo

Abstract 148 Diffuse large B-cell lymphoma (DLBCL), the most common type of non-Hodgkin lymphoma, is a heterogeneous disease comprising multiple biologically and clinically distinct subgroups, including germinal center B cell-like (GCB) and activated B cell-like (ABC) DLBCL. Numerous genetic alterations segregate with ABC-DLBCL, namely translocations of the BCL6 proto-oncogene, BLIMP1 inactivation and constitutive NF-κB activation. We recently reported that A20 , a negative regulator of NF-κB signaling, is biallelically inactivated by mutations and deletions in one-third of ABC-DLBCL (Compagno et al, Nature, 2009), indicating a tumor suppressor role in this disease. Notably, A20 inactivation is commonly associated with chromosomal translocations deregulating BCL6 (n=11/20 DLBCL cases). Furthermore, the two genes are linked in the same pathway, where NF-κB induced activation of IRF4 leads to BCL6 down-regulation (Saito et al. Cancer Cell 2007) and consequent release of the BCL6 target BLIMP1 , a master regulator of plasma cell differentiation. These observations suggest that A20 inactivation and BCL6 translocations cooperate in DLBCL pathogenesis. In order to examine the individual and combined contribution of these two lesions in vivo , we have generated an A20 conditional knockout allele in which a loxP-flanked exon 3 of the A20 gene can be deleted upon Cre-mediated recombination. The resulting mice were crossed with both a Cγ1-Cre deletor strain, which expresses the Cre recombinase in germinal centre (GC) B cells, and the lymphoma-prone Iμ HABCL6 mouse model (Cattoretti et al. , Cancer Cell, 2005), which mimics a BCL6 translocation to the immunoglobulin heavy chain locus. When analyzed at 3 months of age, GC B-cell conditional heterozygous (A20 Cγ1HET ) and homozygous (A20 Cγ1KO ) A20 knockout mice showed a significant increase in the B220 dim CD138 + plasma cell population (0.6% and 0.5%, respectively, versus 0.3% for wild-type littermates) and a corresponding 2-fold increase in IgG1 serum immunoglobulin levels after immunization with sheep red blood cells. Furthermore, A20 knockout splenic B cells had increased proliferative capacity and survival after stimulation ex vivo with lipopolysaccharides, B-cell receptor cross-linking or CD40 activation, consistent with enhanced NF-κB activity in these cells. Interestingly, the increase in plasma cells was not observed in compound Iμ HABCL6/ A20 Cγ1HET and Iμ HABCL6/ A20 Cγ1KO animals, presumably due to the known role of BCL6 in blocking plasma cell differentiation (Tunyaplin et al., J. of Immunol., 2004). In contrast, these animals displayed a marked increase in the B220 + PNA hi GC B cell compartment, as compared to both A20 knockout and Iμ HABCL6 mice (2.5% and 3.9% respectively, versus 1.9% for wild-type littermates). Overall, these findings document that A20 acts as a negative regulator of B cell proliferation and survival as well as of plasma cell differentiation in vivo , and support a model by which loss of A20 synergizes with BCL6 deregulation to promote the expansion of GC B cells while preventing terminal differentiation. Long-term follow-up of these cohorts will provide critical information on the role of A20 as a tumor suppressor gene in vivo and on its cooperative activity with BCL6 deregulation in the pathogenesis of DLBCL. Disclosures: No relevant conflicts of interest to declare.