Reciprocal Regulatory Link Between ecDNA Maintenance and DNA Damage Response Revealed

Extrachromosomal DNA (ecDNA) is a unique form of circular DNA that exists outside of normal chromosomes. Found exclusively in tumor cells, it often carries cancer-promoting genes (oncogenes) and is detectable in most human cancers.

Recent studies highlight ecDNA's critical role in tumor progression, including enhancing genetic heterogeneity, promoting tumor adaptation, and worsening patient prognosis. However, the molecular mechanisms governing ecDNA replication and maintenance-and their functional implications-have remained poorly understood.

Now, a research team led by Prof. GAN Haiyun from the Shenzhen Institutes of Advanced Technology (SIAT) of the Chinese Academy of Sciences has uncovered a reciprocal regulatory relationship between ecDNA maintenance and the DNA damage response (DDR), increasing our understanding of these mechanisms. The study was published in Cell on April 28.

A major obstacle in ecDNA research has been the lack of well-controlled cell line models. To overcome this problem, Prof. GAN's team engineered two ecDNA-positive (ecDNA+) cell models using CRISPR technology, creating matched pairs of cell lines for rigorous comparison. Using these models, the team produced the first direct evidence that ecDNA actively replicates and is stably maintained in ecDNA+ cells. They also identified key molecules involved in ecDNA replication, offering a valuable resource for future research.

Moreover, the team discovered that ecDNA replication triggers the ataxia telangiectasia mutated (ATM)-mediated DNA damage response (DDR) pathway. In ecDNA+ cells, there was an increase in both replication and transcription activity, significantly raising the likelihood of encounters between replisomes or transcription complexes and topoisomerase-DNA complexes (TOPCCs). These encounters led to the formation of abortive TOPCCs and double-strand breaks (DSBs), ultimately resulting in the activation of DDR within ecDNA+ cells.

This study also enhances our understanding of how ecDNA is maintained. The team found that the alternative non-homologous end joining (alt-NHEJ) pathway is essential for repairing ecDNA-associated DNA damage, particularly DSBs. Inhibiting key alt-NHEJ factors, like LIG3, disrupted ecDNA circularization and significantly reduced ecDNA levels in tumor cells. These findings indicate that ecDNA maintenance heavily relies on the alt-NHEJ pathway.

Furthermore, the team explored the translational implications of the findings. They found that inhibiting DDR components specifically harmed ecDNA+ cells, which require ecDNA for growth and survival. This suggests that targeting DDR or alt-NHEJ pathways may offer a novel treatment strategy against cancers driven by ecDNA.

Our work provides insights into DDR's role in ecDNA dynamics and evolution. It shows that ATM-mediated DDR and alt-NHEJ are critical for ecDNA persistence in some cancers although more research is needed to understand ecDNA's effects on tumor heterogeneity, progression, and drug resistance. Targeting these pathways may offer new strategies for the diagnosis, prognosis, and treatment of ecDNA-driven tumors."

Prof. GAN Haiyun, Shenzhen Institutes of Advanced Technology (SIAT), Chinese Academy of Sciences