Genomewide Detection of Genes Targeted by Aberrant Somatic Hypermutation in Lymphoma,

Abstract 3474

The processes of somatic hypermutation (SHM) and class switch recombination introduced by activation-induced deaminase (AID) are key steps for creating a pool of diversified antibodies in activated germinal center (GC) B cells (centroblasts; CBs) upon antigen dependent stimulation of resting naïve B cells (NBs) by T cells. Periodically, centroblasts enter the light zone of the GC where they interact with dendritic cells and T helper cells to undergo clonal selection for cells expressing high affinity antibodies. Unfortunately, AID can also accidentally introduce mutations at bystander loci, particularly within the promoter regions of a number of proto-oncogenes (BCL6, MYC, PAX5, PIM1, RhoH, S1PR₂, and SOCS1) in diffuse large B cell lymphomas (DLBCL), an aggressive non-Hodgkins lymphomas. Since current methods, such as Exon Capture and RNAseq, only target mutations in coding regions, to date aberrant promoter SHMs have been studied only in a handful of genes. Given the complexity of lymphomagenesis, there may be additional genes that are targeted aberrantly by SHM at their promoters in DLBCLs.

To identify genes that are targeted by SHM genomewide, we designed a novel approach to search for SHM hotspots in the genome by integrating bioinformatics tools with next generation sequencing technology. We developed a comprehensive software framework (called SHMseeqer) for discovery of single nucleotide variants (SNV) that have SHM features from ChIP-seq reads. We ran SHMseeqer on several histone H3 lysine 4 trimethylation (H3K4me3, preferentially enriched at promoters) ChIP-seq datasets obtained from two DLBCL cell lines and from normal primary NBs and CBs. We observed increased numbers of genes that harbor SHM at their promoters in DLBCL cells as compared to primary normal B cells. Moreover, in DLBCL cells, we observed promoter SHM hotspots in genes that have not been reported before, including BACH2, BTG2, EBF1, and TCL1A, etc., all of which have potential roles in B cell survival, differentiation, and malignant transformation. In addition, using BCL6 and BACH2 promoters as examples, we demonstrated that SHMs identified in these promoters greatly altered the promoter activities in a reporter assay. BACH2 is a B-cell specific transcription factor that plays an important role in gene expression regulation during B-cell activation and terminal differentiation. We observed that the SHM containing BACH2 promoter had increased transcription activity in the reporter assay as compared to widetype BACH2 promoter. We further showed that there was an elevated BACH2 level in DLBCL cells, and together with BCL6, BACH2 can block mature B-cell differentiation by repressing the expression of PRDM1.

Our approach provides the first cost-efficient, genome-wide method to screen promoter mutations and SHM hotspots; an area that has not been fully explored thus far. Furthermore, by using this approach we showed that there were increased aberrant SHMs in DLBCL cells, suggesting that the process of AID-induced SHM is impaired in DLBCL cells. Moreover, the AID-induced promoter SHMs may deregulate the expression of key B-cell genes that leads to DLBCL transformation ultimately.