The role of BLIMP1 and A20 inactivation in the pathogenesis of diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease composed of at least two distinct subtypes: germinal centre B-cell like (GCB) DLBCL and activated B-cell like (ABC) DLBCL. These phenotypic subtypes segregate with multiple genetic lesions, suggesting the involvement of different pathogenetic mechanisms. Biallelic inactivation of the BLIMP1 gene, the master regulator of plasma cell differentiation, has been found exclusively in the ABC subtype (∼24% of cases), however, the precise mechanism by which these lesions contribute to lymphoma development has yet to be fully elucidated. Similar to BLIMP1, the A20 gene, a negative regulator of NF-κB signaling, is inactivated by mutations and biallelic deletions in ABC-DLBCL (∼30% of cases), suggesting that A20 also plays a tumor suppressor role in this disease. The role of A20 inactivation and constitutive NF-κB activation in lymphomagenesis in vivo, however, has yet to be addressed. Here we investigated the role of BLIMP1 and A20 inactivation in the pathogenesis of DLBCL by using a multi-faceted approach, combining human genetic data, in vitro functional assays and in vivo mouse models.;To address the role of A20 inactivation in lymphomagenesis, we generated A20 conditional knockout mice and crossed them to the Cγ1-Cre deleter strain, to delete A20 specifically in GC B cells (A20Cγ1KO). A20 Cγ1HET and A20Cγ1KO mice generated increased plasma cells after immunization, suggesting that A20 regulates plasma cell differentiation and gene dosage is important in GC B-cells. Furthermore, A20 knockout splenic B cells had increased proliferative capacity and survival after mitogenic stimulation. Finally, we crossed the mice to the lymphoma-prone IμHA BCL6 mouse model to assess the potential cooperation between these two lesions in DLBCL pathogenesis. Addition of the IμHABCL6 transgene abrogated the increase in plasma cell formation and cooperated with A20 deletion to increase the GC B cell compartment. 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.;With regards to BLIMP1 inactivation, we demonstrated that BLIMP1 is disrupted by truncating and missense mutations and biallelic deletions in one-third of ABC-DLBCL. We performed a variety of functional assays to validate that a subset of missense mutations impaired BLIMP1 function through protein instability or reduced transrepression activity. Additionally, translocations of BCL6, a master regulator of the germinal centre reaction, actively suppressed BLIMP1 expression in additional ∼22% of ABC-DLBCL. Finally, Blimp1 B-cell conditional knockout mice developed ABC-DLBCL, thus providing definitive in vivo proof that BLIMP1 is a tumor suppressor gene.