Somatic hypermutation (SHM) is a B cell-specific process that supports the affinity maturation of antibodies. The reaction targets the immunoglobulin loci (“on-target" activity) and is initiated by activation-induced cytidine deaminase (AID). AID is known to be able to mutate the genome more broadly (“off-target” activity) and hence was found to be linked to various types of B-cell malignancies. However, the mechanism by which AID is recruited and exerts its function is partially known. To unravel this puzzle, we have analyzed two types of diffuse large B-cell lymphoma (DLBCL) cell lines: germinal B cell like (GCB-DLBCL), in which AID is expressed and introduces mutations, and activated B cell like (ABC-DLBCL), where AID levels are typically increased but paradoxically, SHM is much reduced. To begin to accomplish this goal, we have taken advantage of a Diversification Activator (DIVAC)-GFP reporter assay to measure targeted SHM activity. We first confirmed that AID is expressed in ABC-DLBCL cell lines at levels up to fourfold higher than in GCB-DLBCL cell lines by RT-qPCR. In contrast to the high levels of AID seen in ABC-DLBCL cells, we have observed that SHM is reduced by as much as 10-fold in ABC versus GCB lines using the DIVAC–GFP assay. As this assay should provide a good surrogate for SHM activity at Ig loci, our data support the possibility that the Ig-locus-specific SHM targeting program is defective in ABC-DLBCL. To search for factors that might explain the differential activity of the SHM program in GCB versus ABC cells, we performed RNA-seq using cell lines from each group. Initial analysis revealed that expression of two cell surface markers, CD27 and CD38, is highly correlated with the high mutation phenotype. Flow cytometry analysis for these two markers along with the pan B cell marker CD20 revealed various subpopulations in some DLBCL lines. Isolation, characterization, and measurement of SHM activity using the DIVAC-GFP assay of these subpopulations further strengthened the correlation between CD27/CD28 expression and SHM activity. These analyses also uncovered SHM-permissive subpopulations in ABC lines and SHM-resistant subpopulations in GCB lines. This observation explains residual SHM activity in ABC lines and argues for heterogeneity in the activity of the SHM program in DLBCL cell lines and perhaps within tumor populations in patients. Future studies will include deletion and introduction of specific factors from GCB or ABC lines to validate phenotype and identify regulators of the SHM program. Finally, sequence-analysis will be done to quantify mutations with the broad goal of understanding how AID targeting and mis-targeting are regulated.

Citation Format: Huseyin Saribasak, Ravi Dinesh, Lizhen Wu, David G. Schatz. Somatic hypermutation is perturbed in ABC-DLBCL lymphoma cell lines expressing high levels of activation-induced deaminase [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-23.