Regulation of the Mutation Threshold During Immune Diversification by Activation Induced Cytidine Deaminase,

Abstract 3244

Activation-induced cytidine deaminase (AID) is expressed in germinal center B cells and is required for somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin (Ig) genes. AID converts cytosine to uracil and resulting U-G mismatches are subsequently processed by low-fidelity base excision and mismatch repair pathways to yield point mutations (for SHM) and DNA strand breaks (for CSR). Under normal conditions, genotoxic stressors activate DNA damage response pathways that result in DNA repair or cell cycle arrest and apoptosis. However, in normal germinal center B cells, key DNA damage checkpoint factors such as ATR, p53 and p21 are repressed by the germinal center transcriptional repressor BCL6. BCL6 thus creates a permissive environment to allow the accumulation of mutations within the Ig loci. However, in addition to this fundamental role in immune diversification, aberrant targeting of AID contributes to point mutations and translocations of proto-oncogenes associated with B cell malignancy. Indeed, the combined effect of BCL6 and AID poses a direct threat to genomic integrity but the mechanism responsible for regulating the mutation threshold in germinal center B cells is not understood. To determine if B cells have a mechanism for down-regulating AID activity in response to genotoxic stress, we subjected the Ramos-A23 cell line to continuous low-dose exposure to several genotoxic agents. Ramos-A23 is a Burkitt lymphoma line that constitutively expresses AID and mutates the Ig heavy chain variable region with high frequency. Mutation frequency can be monitored by flow cytometry through loss of surface expression of IgM (due to AID-dependent nonsense and missense mutations) and by direct sequencing of the variable region. IgM+ Ramos-A23 cells were maintained in continuous culture in the presence or absence of etoposide (100 nM), cytarabine (5 nM), 5-azacytidine (10 nM), or trichostatin A (15 nM). Drug concentrations were titrated to the highest dose that would minimally affect proliferation or survival when compared with untreated cells. After 4 weeks in culture, untreated clones were 20–22% IgM-, consistent with ongoing SHM. In contrast, clones treated with the DNA damaging agents etoposide or cytarabine were only 4–6% IgM- (p<0.0001). Cells treated with the epigenetic modifiers 5-azacytidine or trichostatin A were 15–19% IgM-. Sequence analysis of the Ig variable region (3 clones from each group) demonstrated 6.2 and 3.8-fold reductions in mutation frequency in cells treated with etoposide and cytarabine, respectively, when compared with untreated cells. The mutation frequency in the cells treated with epigenetic modifiers did not differ significantly from untreated controls. This suggests that while epigenetic stress has little effect on SHM, genotoxic stress leads to a significant reduction in AID activity. It has previously been demonstrated that genotoxic stress leads to an ATM-dependent down-regulation of BCL6, a finding that we also observed in our model. In addition, we found that genotoxic stress causes a dose and time-dependent decrease in AID protein and transcript levels, thus explaining the decrease in SHM in our system. Like BCL6, repression of AID by genotoxic stress was inhibited by pre-treating the cells with the ATM inhibitor KU-55933. Further analysis of markers of B cell differentiation revealed up-regulation of IRF4 and Blimp1 with complete down-regulation of PAX5 (a transcriptional regulator of AID) in response to genotoxic stress. Lentiviral expression of BCL6 shRNA in Ramos cells also lead to down-regulation of AID and SHM, consistent with the observation that BCL6-deficient B cells do not undergo SHM. It has also recently been demonstrated that DNA damage in germinal center B cells leads to ATM-dependent inactivation of the CREB transcriptional co-activator CRTC2 with subsequent down-regulation of CRTC2 target genes, including AID. Altogether, these data suggest that, during the germinal center response, a mutation threshold recognized by ATM leads to repression of the SHM machinery and reprogramming to facilitate B cell maturation. Additional investigation is needed to further define the critical steps in this regulatory pathway and how it might breakdown during the pathogenesis of B cell malignancy.