Dissecting the whole trajectory of aid-induced genomic instability from mutational activity to chromosomal translocation formation Free

Chromosomal translocations are key genomic events that frequently occur in hematopoietic cancers and solid tumors. In lymphoma and leukemia, such as diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL), most DSBs that lead to oncogenic translocation are initiated by activation-induced cytidine deaminase (AID). AID is a B-cell-specific enzyme that targets immunoglobulin (Ig) genes, considered AID on-target genes, to initiate somatic hypermutation (SHM) and class switch recombination (CSR). However, AID also exhibits off-target activity at non-immunoglobulin regions, contributing to genomic instability by promoting oncogenic chromosomal translocations and mutations that drive the development and progression of B cell lymphomas and leukemias. The mechanisms that govern AID's selective targeting of a limited subset of genomic regions remain poorly understood and represent a longstanding, fundamental question in the field. By utilizing high-throughput genome-wide translocation sequencing (HTGTS) in B cells, we previously demonstrated that PI3Kδ inhibition upregulates AID expression, thereby increasing genomic instability and partially elucidating these mechanisms.

To further delineate the mechanistic cascade of AID activity as a cytidine deaminase, we now developed dU-seq to map genome-wide AID-induced mutations and applied sBLISS to detect genome-wide AID-induced DNA double-strand breaks (DSBs) in both primary B cells and CH12F3 cells. By these approaches, we successfully captured AID-mediated mutations and AID-dependent DSBs at most known translocation hotspots in the whole genome, including AID on-targets and off-targets, validating the technical robustness of our approach. Surprisingly, we identified thousands of previously unrecognized AID-mediated mutations and DSB hotspots besides those known AID target regions, revealing a much broader genomic footprint of AID activity than previously appreciated.

To provide a potential clinical relevance of these discovery approaches, we tested whether emerging epigenetic therapies could impact the patterns and distribution of AID-mediated translocations in B cell lymphoma. EZH2 inhibitors, such as tazemetostat and valemetostat, are epigenetic regulators approved by the FDA for the treatment of follicular lymphoma and adult T-cell leukemia/lymphoma, respectively. We found that EZH2 inhibitors alone did not significantly alter AID expression or AID-mediated translocation frequency in CH12F3 mouse B cells or MEC-1 human B cells. However, when combined with PI3Kδ inhibition, EZH2 inhibition markedly enhanced the frequency of chromosomal translocations compared to either treatment alone in both cell models. EZH2 inhibition also further enhanced translocation formation in mouse B cells that were DNA repair deficient, such as Ligase4 knock-out cells. Mechanistically, EZH2 inhibition in B cells depletes the repressive histone modification H3K27me3 while concurrently enhancing the active histone modification H3K27ac, thereby selectively increasing transcriptional activity and facilitating chromosomal translocation formation in the presence of high AID activity or Ligase4 deficiency.

Overall, to our knowledge this work represents the most comprehensive mapping of AID-induced mutational and genotoxic activity, shedding light on the whole trajectory of AID activity from the very early initiation steps of cytidine deamination to the formation of DSB intermediates up to the final outcome of chromosomal translocations. The described approach can be exploited to functionally dissect the impact of novel drugs on AID-mediated genomic instability in B cell lymphoma.