Once again, the DNA segments previously considered “junk” have been shown to possess a significant biological function, actively contributing to the recurrence of leukemia. This recent discovery focuses on non-coding DNA, specifically excised DNA circles (ESCs), which are tiny loops that are byproducts of the V(D)J recombination process. The dogma surrounding the presence of ESCs was that they were harmless, distinct from other oncogenic extrachromosomal DNAs (ecDNA), because ESCs were thought to be progressively lost after each replication.
In a recent study published in Nature, Zeqian Gao and their colleagues investigated how ESCs contribute to BCP-ALL relapse. Acute lymphoblastic leukemia (ALL) is the most common childhood cancer, with B-cell precursor ALL (BCP-ALL) representing the majority of pediatric cases. This resulted in reframing them from mere byproducts of V(D)J recombination to extrachromosomal elements that actively participate in disease progression.
To analyze whether ESCs are replicated and inherited like ecDNAs, they isolated the splenocytes from six-week-old mice and purified genomic DNA, IgM⁺ and IgG⁺ B cells, and pre-B-cells and IgM⁺ B cells from mouse bone marrow as well. To separate the circular DNA, the researchers have chosen to treat the DNA with exonuclease V (RecBCD), which is an enzyme that selectively cleaves linear DNA while leaving circular DNA intact. The persistence of ESCs despite low levels of RAG1, a crucial enzyme involved in V(D)J recombination, and the absence of IgG expression, indicates that the circular DNAs were not newly generated but had been replicated and endured in earlier B cell development. This provided direct evidence that ESCs actually behave like stable and heritable ecDNAs.
The research team also performed cDNA and transcriptomic analyses on samples from over 85 patients with BCP-ALL. They quantified the number of ESCs using PCR and found that their number, replication capacity, and stability were strikingly higher in the patients who relapsed compared to those who did not. To evaluate whether the ESCs were being replicated, the researchers compared the number of ESCs with their corresponding coding joints, since the number of coding joints reflects how many cell divisions have occurred after the V(D)J recombination.
In addition, whole-genome sequencing data from the BCP-ALL patients showed that those who later relapsed had a significantly higher number of mutations in relapse-associated genes at diagnosis. Moreover, the number of mutations increased further by the time of relapse, suggesting that RAG ESC complexes remain active and ESCs are capable of replication and persistence.
To uncover the mechanism behind these mutational activities, the researchers analyzed the RAG1 and RAG2 enzyme activity. The findings showed that the individuals with high levels of RAG and ESCs had notably more mutations than those with low ESC levels. This provided evidence that the presence of ESCs is a key driver of these mutations, not just RAG enzymes alone. These results raise questions regarding the structural factors that encourage RAG enzymes to get engaged in “cut-and-run” reactions with ESCs instead of the V(D)J recombination.
In conclusion, this discovery provides a crucial insight into overturning a long-term belief. By measuring the DNA copy number of ESCs at diagnosis, it may become possible to predict which patients are at higher risk of relapse, allowing their treatments to be personalized more efficiently. Furthermore, the association between the increased ESC levels and the expression of the proteins that play a role in DNA repair and recombination opens the door to further investigation, which may pave the way for new and effective targeted therapies.
Author: Sude Aras
Editor: Tuana Keskin
Reference: Gao, Z., Scott, J.N.F., Edwards, M.P. et al. Excised DNA circles from
V(D)J recombination promote relapsed leukaemia. Nature (2025).
https://doi.org/10.1038/s41586-025-09372-6