Unfolding ecDNA as a pan-cancer therapeutic target

ecDNA induces aberrant gene expression

Cells containing ecDNA maintain certain proteins (such as tumor-related genes) at higher levels. Compared to the same genes amplified in the genome, the expression levels of genes on ecDNA are generally higher [1]. This phenomenon primarily stems from two factors: the high copy number of ecDNA and its inherently open structural features. ecDNA is characterized by fewer repressive histone marks and a significant enrichment of activating histone modifications [3], 4]. Furthermore, ecDNA molecules tend to form ecDNA hub structures, which may facilitate coordinated expression of genes on ecDNA and further elevate gene expression levels [3]. During the formation of ecDNA, fragments derived from chromosomal DNA may recombine to form fusion genes, which also drive the expression of tumor-related genes [3].

ecDNA promotes cancer evolution

The aberrant gene expression driven by ecDNA can promote tumor initiation and progression. Additionally, other ecDNA-related factors contribute to tumor development, including copy number variations of ecDNA and sequence variations in the DNA carried by ecDNA. DNA sequences carried on ecDNA are more prone to genetic damage [5], and the error-prone DNA repair process can introduce mutations, resulting in sequence variations in tumor cells. These copy number and sequence variations further drive tumor evolution.

ecDNA induces DNA damage and DNA damage response

In cancer cells, ecDNA exhibits markedly elevated DNA damage levels, primarily driven by two key biological processes: aberrantly active replication and transcription. These processes potently activate the ATM-mediated DNA damage response (DDR) pathway [5]. Our study revealed the abnormal accumulation of topoisomerase cleavage complexes (TOPCCs) on ecDNA. This phenomenon stems from increased conflicts between the replication/transcription machinery and TOPCCs on ecDNA, ultimately leading to the accumulation of DNA double-strand breaks (DSBs).

ecDNA biogenesis

While DNA damage repair pathways are widely implicated in the generation of circular DNA, the specific pathways involved are a subject of debate. Multiple studies support the critical role of alternative non-homologous end joining repair (alt-NHEJ) in circular DNA formation. Through systematic knockout experiments, Paulsen found that circular DNA formation depends on the alt-NHEJ pathway, while classical non-homologous end joining (NHEJ) pathway inhibits its formation [6]. This is corroborated by the findings that knockout of the key alt-NHEJ factor LIG3 significantly suppresses circular DNA generation, whereas knockout of LIG1 or LIG4 has no significant effect [7].

However, there is also evidence suggesting that the NHEJ pathway may play an important role. For instance, inhibition of DNA-PK significantly reduce circular DNA formation [8]. These apparently conflicting findings indicate that the mechanism of circular DNA formation may be highly complex and likely context-dependent, with the contributions of different repair pathways potentially regulated by cell type or microenvironmental factors.

The replication of ecDNA

Early models proposed that ecDNA replicates strictly during S-phase and only once per cell cycle, similar to chromosomal DNA [1]. However, recent research has revealed unique characteristics in ecDNA replication behavior.

Cells carrying ecDNA exhibit a significantly slower overall DNA replication rates compared to normal cells and sequences on ecDNA replicate more slowly than their chromosomal counterparts [9]. Even within the same cell, ecDNA replicates at a slower pace compared to chromosomal DNA [10]. Notably, the distribution of replication origins differ markedly from those on chromosomes and is susceptible to changes induced by replication stress [10].

These distinct replication patterns of ecDNA imply unique replication mechanisms. Haiyun Gan’s team employed an improved procedure to isolate proteins on nascent DNA and systematically identified the ecDNA replication-associated proteome, providing critical insights into its molecular regulation [5]. Their work revealed that during ecDNA replication, core DNA replication machinery, epigenetic regulators, and other factors are highly enriched on newly synthesized DNA strands [5]. A particularly striking observation was the abnormal enrichment of DDR proteins at ecDNA replication sites [5]. Although this study did not fully elucidate the precise replication mechanism of ecDNA, the established methodology and the identified protein candidates provide a crucial foundation for future research.

The maintenance of ecDNA

The abundance of ecDNA within tumors shows a strong correlation with tumor malignancy. The stable maintenance of ecDNA critically depends on specific DNA damage repair pathways. Through systematic screening, Gan’s team found that individual or combined blockade of HR and NHEJ pathways had limited effects on ecDNA levels, whereas specific inhibition of alt-NHEJ significantly reduced the abundance of ecDNA in cells [5].

In-depth mechanistic studies revealed that the absence of alt-NHEJ impedes DSB repair during specific cell cycle phases, thereby interfering with the circularization process of ecDNA [5]. This disruption causes ecDNA to either reintegrate into chromosomes or be eliminated by unclear cellular clearance mechanisms.