AICDA Introduces Epigenetic Plasticity in Germinal Center-Derived Lymphomas and Accelerates Lymphomagenesis

Diffuse large B-cell lymphomas (DLBCLs) are aggressive tumors derived from germinal center B cells (GC) B cells. Previous work from our group established that inferior outcome in DLBCL is associated with higher degrees of intra-tumor and inter-tumor cytosine methylation heterogeneity, although the molecules driving this epigenetic perturbation are unknown. We investigated the contribution of activation-induced cytidine deaminase (AICDA) to cytosine methylation heterogeneity in DLBCLs. AICDA is highly expressed in GC B cells where it drives somatic hypermutation (SHM) of immunoglobulin (Ig) and non-Ig genes. AICDA is also expressed in DLBCLs and high level of AICDA in CHOP-treated DLBCL patients is associated with unfavorable prognosis. In addition to mutagenesis, AICDA can cause DNA demethylation through the removal of modified cytosine residues. Indeed, we previously reported that GC B cells manifest a marked AICDA-mediated DNA hypomethylation signature as well as a high degree of epigenetic heterogeneity as compared to other B-cell subsets. Thus, we hypothesized that AICDA could contribute to the progression of GC-derived lymphomas by facilitating epigenetic plasticity through the redistribution of cytosine methylation. We overexpressed AICDA in VavP-Bcl2 transgenic mice -which develop B cell lymphomas of GC origin- using an AICDA-expressing retroviral vector (VavP-Bcl2+Aicda) vs empty vector (VavP-Bcl2) as control and transplanted them into lethally irradiated recipient mice. We observed more aggressive lymphomas based on greater disruption of the splenic architecture and higher degree of B cell infiltration in organs such as lung, liver and kidney in VavP-Bcl2+Aicda mice (n=7) compared to VavP-Bcl2 mice (n=6). Notably, the overexpression of AICDA reduced significantly the lifespan of the mice (Log-rank test p=0.0289) with a median survival of 214 days for VavP-Bcl2+Aicda mice (n=10) and 293 days for VavP-Bcl2 animals (n=9). Neoplastic B cells from VavP-Bcl2+Aicda and VavP-Bcl2 micedisplayed similar SHM rate in Ig genes (J_H4 and Sm) and AICDA off-targets (Bcl6, Cd83 and Pax5), suggesting that the more aggressive phenotype is not likely due to increased mutagenesis. We then profiled the DNA methylation landscape of these lymphomas using enhanced reduced representation bisulfite sequencing. We analyzed the DNA methylation level and heterogeneity of all represented CpGs by calculating the mean methylation difference and interquartile range (IQR) between VavP-Bcl2+Aicda and VavP-Bcl2 tumors. A principal component analysis of all CpGs, each one represented by its mean methylation and IQR differences across replicates, revealed methylation loss and increased intertumor heterogeneity within VavP-Bcl2+Aicda methylomes compared to VavP-Bcl2. AICDA-perturbed CpGs (n=49,750) in particular showed significant intratumor heterogeneity (p<1e-300) in AICDA overexpressing tumors. These altered CpGs were depleted in promoters and enriched in introns and intergenic regions. We observed a remarkably similar pattern of hypomethylation and increased methylation heterogeneity in Aicda^+/+ GC B cells compared to Aicda^-/- GC B cells (AICDA-perturbed CpGs n=64,323) and also in primary DLBCLs with high AID compared to low AID expression (AICDA-perturbed CpGs n=37,557), suggesting a conserved epigenetic function of AICDA in GC B cells and human and mouse GC-derived lymphomas. We performed RNA sequencing on BCL2-driven lymphomas and found that expressed genes containing AICDA-perturbed CpGs displayed significantly lower expression in VavP-Bcl2+Aicda compared to VavP-Bcl2 tumors (gene set enrichment analysis (GSEA) NES=-1.68, FDR<0.001). Pathway analysis revealed that these AICDA-epigenetic target genes were associated with MAPK- and KMT2D-signaling pathways, which are related to B cell activation and lymphomagenesis. Similarly to mouse tumors, genes enriched for AICDA-perturbed CpG in human lymphomas exhibited lower expression in AID high DLBCLs compared to AICDA low DLBCLs (GSEA NES=-1.65, FDR<0.001) and were associated with MAPK and KMT2D pathways. These results demonstrate that AICDA acts as a methylome modifier in GC-derived lymphomas, introducing epigenetic plasticity and leading to gene expression changes, thus contributing to clonal evolution and higher adaptability to an evolving environment.