Sunday, May 4, 2025
5/4/2025
Aberrant V(D)J recombination in B cells initiates lymphoid malignancy - PROJECT SUMMARY/ABSTRACT Chromosomal translocations occur in nearly all human lymphoid neoplasms, many the result of aberrant repair of DNA double-stand breaks (DSBs) generated in pre-B cells during V(D)J recombination. In this pathological process, unrepaired V(D)J DSBs induced at the IgH locus join with a second, non-IgH DSB to generate the translocation. The non-IgH DSBs occur by an unknown mechanism, representing a serious knowledge gap. The rationale for this project arose from creation of a sequencing database composed of DNA translocation breakpoint junctions mapped from over 2,000 human patients covering most B cell malignancies. This analysis uncovered 6 non-IgH human fragile zones (HFZs) ranging in size from 20-600 bp where DSBs are enriched up to 1,000-fold compared to the surrounding DNA. HFZ DSBs account for half of all human B cell cancers and break in the pre-B cell stage of development concurrent with V(D)J recombination. Building on a foundation of previous work, the central hypothesis is that three factors are critical for HFZ DSBs in lymphoid cells: 1) Formation of single-stranded DNA (ssDNA) structures at HFZs resulting from torsional stress, 2) Expression of activation-induced cytidine deaminase (AID) that targets ssDNA to create mismatched DNA lesions, and 3) Presence of an activated form of the structure-specific Artemis endonuclease that cleaves these lesions to generate the HFZ DSBs. The overall objectives for this proposal are: (1) Utilize digital PCR (dPCR) and DNA sequencing assays to quantify DNA DSB formation at HFZs under conditions that will elucidate the HFZ DSB mechanism and (2) show how loss of topoisomerase function through pharmacologic or genetic ablation enhances HFZ DSB formation. The central hypothesis will be tested by pursuing three specific aims: 1) To determine how torsional stress stabilizes ssDNA structures at HFZs, 2) To determine the role of AID in HFZ DSBs, and 3) To delineate how Artemis induces HFZ DSBs. The innovative aspects of this research are that human patient data was used as a source to demonstrate that many B cell malignances are the result of non- random DSBs at discreet 20-600 bp HFZs, human pre-B cell lines and primary human cells are used in the analysis, and the genetic assays used can quantify indels resulting from DSB formation and repair in human pre- B cell lines and patient material. This work is significant as it defines a unified mechanism for many B cell malignances involving multiple steps, each of which can be affected by an individual’s genetics and lifestyle. Results from this proposal would represent a significant advancement towards predicting disease in high-risk populations and tailoring treatments to individuals. Furthermore, our assays can be applied in the clinic to quickly diagnose diseases with a genome instability component and monitor a patient’s response to treatment and potential for relapse. Overall, the results will lead to early detection of primary cancers, prevention of secondary cancers, and an overall reduction in mortality.
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