The Role of DNA Repair in the Pathogenesis of Activation Induced Cytidine Deaminase Dependent B Cell Lymphoma

Abstract 397 Upon antigenic stimulation of B cells, germinal centers (GCs) are formed where somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin ( Ig ) genes serve to diversify the immune response. SHM and CSR are initiated by the enzyme activation induced cytidine deaminase (AID) through the conversion of C/G base pairs to U-G mismatches. These mismatches are processed by UNG-dependent base excision repair (BER) and MSH2-dependent mismatch repair (MMR) pathways to yield mutations (for SHM) and DNA strand lesions (for CSR). Despite this essential role in immune diversification, the intrinsic activity of AID as a DNA mutator poses a threat to genomic integrity. Indeed, aberrant targeting of AID activity is associated with translocations and point mutations of proto-oncogenes associated with B cell malignancies. A specific dependence on AID in the pathogenesis of lymphomas of GC B cell origin is exemplified in Iμ-Bcl6 knock-in mice. These mice develop a diffuse large B cell lymphoma (DLBL) that resembles the human disease but are protected from development of this lymphoma when crossed onto an Aid-deficient background. To investigate the role of Aid-associated DNA repair in the pathogenesis of this disease, we crossed Iμ-Bcl6 mice onto a background deficient in BER ( Ung −/− ) and MMR ( Msh2 −/− ). Young healthy Iμ-Bcl6 and Iμ-Bcl6 Ung −/− Msh2 −/− mice displayed a normal number and distribution of B cells and normal architecture of lymphoid organs. Five of 28 Iμ-Bcl6 mice (17.9%) became sick starting at ∼12 months of age. Historically, median survival in these mice has not been reached and ∼80% survive to 15 months. In contrast, 21 of 28 Iμ-Bcl6 Ung −/− Msh2 −/− mice (75%) developed disease with an onset of ∼3 months and had a median survival of 6.2 months (p Iμ-Bcl6 mice and the majority of Iμ-Bcl6 Ung −/− Msh2 −/− mice developed B cell lymphoma with splenic involvement and variable nodal involvement. Five of the Iμ-Bcl6 Ung −/− Msh2 −/− mice developed other cancers (3 T cell lymphomas, 1 pre-B cell lymphoma and 1 colon adenocarcinoma). Tumors from both genotypes expressed a mature B cell phenotype (B220+ IgM+ Igκ+ CD138-) and morphology revealed loss of normal lymphoid architecture with infiltration by lymphoid blasts. Additional staining demonstrated expression of at least one GC marker (Fas, GL7 and/or PNA). Similar to Iμ-Bcl6 mice, while many of the Iμ-Bcl6 Ung −/− Msh2 −/− tumors had clonal mutated Ig heavy chain gene variable regions, two of the tumors were identified as oligoclonal, suggesting a preceding lymphoproliferative stage. In the absence of Ung and Msh2, Aid-generated U-G mismatches are not recognized and are simply replicated, causing only C/G to T/A transition mutations and no strand lesions. Thus, as expected, all Ig mutations in Iμ-Bcl6 Ung −/− Msh2 −/− mice were C/G to T/A transitions. Lymphomas from Iμ-Bcl6 mice have been found to harbor numerous chromosome translocations and aneuploidies. Although additional analyses are underway, spectral karyotyping of 3 Iμ-Bcl6 Ung −/− Msh2 −/− tumors revealed 2 with normal cytogenetics and 1 with a 40–41,XX,t(2;17),+15,+19. Surprisingly, sequence analysis of several known Aid target genes ( cMyc , Pim1 , RhoH , Pax5 , Cd79a , Fas , H2ax and OcaB ) in tumors from 3 Iμ-Bcl6 Ung −/− Msh2 −/− mice did not identify any clonal mutations. However, non-clonal C/T to T/A transition mutations in cMyc were present at a frequency of 1.2 × 10 −4 , suggestive of ongoing Aid activity. The presence of Aid activity but absence of off-target Aid-mediated clonal SHM suggests that either other genes are targeted by Aid or that Aid has a secondary role in lymphomagenesis such as epigenetic reprogramming, as has been shown in iPS cells. Nonetheless, the incidence of Aid-dependent lymphomagenesis in the absence of Aid-associated DNA repair is significantly increased and the latency is greatly shortened. Altogether, this data suggests that Aid-associated BER and MMR pathways afford a protective effect against the development of Aid-dependent GC B cell lymphomas such as DLBL. To investigate the role of the individual Aid-associated DNA repair pathways, we have also generated Iμ-Bcl6 Ung −/− and Iμ-Bcl6 Msh2 −/− single knockout mice. These studies are ongoing but preliminary results suggest that while the effect of Ung and Msh2 deficiency on lymphomagenesis may be synergistic, Msh2 might play a more critical role in preventing Aid-mediated genomic instability. Disclosures: No relevant conflicts of interest to declare.