Birgitte Regenberg

Extrachromosomal circular DNA (eccDNA) are molecules that originate from chromosomal DNA but exist independently. While large eccDNA (ecDNA) contributes to tumorigenesis, the role of smaller eccDNA (CXCL5 exemplified this phenomenon, showing substantial copy-number increase and upregulation when present on eccDNA. Functional validation in cell lines showed that CXCL5 eccDNA enhanced transcriptional output and immune cell recruitment function. The recurrence and overexpression of CRC-related genes on eccDNA indicate their selection in tumors, suggest that eccDNA can serve as an additional mechanism for dynamically influencing gene expression and is capable of conferring cancer phenotypes on cells. Analysis of chromatin landscapes revealed that eccDNA preferentially forms at sites of open chromatin and active transcription, with architectural boundaries marked by CTCF protein. Clinically, higher eccDNA levels correlated with poorer relapse-free survival in a small patient cohort. These findings suggest that circular DNA elements across the entire size spectrum participate in cancer evolution and warrant further investigation in larger cohorts.

Over 40% of human cancers harbor extrachromosomal DNA (ecDNA), which correlates with therapy resistance and poor prognosis. Yet research is limited by a lack of robust de novo ecDNA models. We present a CRISPR-Cas9 based approach for generation of ecDNA in cell lines and mouse liver. In HEK293 cells, CRISPR-induced MYC-bearing ecDNA ([MYCcircle]) promoted cancer hallmarks, including enhanced proliferation, increased migration, and reduced apoptosis. To model ecDNA in mice, we combined Cas9 knock in mice with lipid-nanoparticle delivery of sgRNAs in the liver targeting the Myc–Pvt1 locus. When combined with Trp53 or Pten loss, the resultant [Myccircle] caused hepatocellular carcinomas with amplified levels of [Myccircle] and elevated, heterogenous Myc expression, supporting evidence of ecDNA involvement in tumorigenesis. Our approach offers a fast and versatile tool, where target-sequence flexibility allows rapid generation of animal and cell models with any investigator-specified ecDNA, thereby enabling study of the direct role of ecDNA in cancer progression.

In this Perspective article, we follow the journey of a gene that breaks free from its stringent chromosomal transmission dynamics to become an independently multiplying agent on so-called extrachromosomal circular DNA (ecDNA/eccDNA). We discuss how the release of a gene from its chromosomal anchor can affect its dosage, regulatory context, and potential evolutionary trajectory before examining the phenotypic implications for unicellular and multicellular eukaryotes that transmit genes on circular DNA. We also briefly explore the fundamental difference between circularized genes in flowering plants (angiosperms) and mammals (primarily cancer) concerning somatic and germline inheritance.

Table_S1. Study cohort. Table_S2. The sample composition of each dataset. Table_S3. Patient information. Table_S4. Whole genome sequencing (WGS) datasets. 1,411 UC whole genomes. Table_S5. Whole exome sequencing (WES) datasets. Table_S6. RNA sequencing (RNA-seq) datasets. Table_S7. Circle-seq datasets. Table_S8. Single-cell RNA-seq (scRNA-seq) datasets. Table_S9. Amplicon classification. Table_S10. Amplicon similarity scores. Table_S11. ecDNA prevalence in human cancers. Table_S12. Genomic and sequence features of amplicons. Table_S13. Oncogene list. Table_S14. Genes encoded on all focal amplifications. Table_S15. ecDNA status and CNV ITH in tumor multi-region sampling cohort. Table_S16. Diver gene alterations in ecDNA- and ecDNA+ tumors. Table_S17. Summary of Single-cell RNA-seq (scRNA-seq) data. Table_S18. IHC evaluation of CD45 in 19 tumors with scRNA-seq data. Table_S19. Signature genes of 50 robust NMF programs. Table_S20. Pathway enriched in each NMF program. Table_S21. Top 30 signature genes of tumor meta-program. Table_S22. Differential pathways of malignant cells between ecDNA- and ecDNA+ tumors. Table_S23. Gene signature matrix for deconvolution analysis of bulk RNA-seq data. Table_S24. Software.

Supplementary Figure 1. Prevalence of ecDNA in human cancers. Supplementary Figure 2. Characteristics of ecDNA. Supplementary Figure 3. ecDNA drives massive oncogene expression in UC. Supplementary Figure 4. ecDNA can arise in flat urothelial lesions. Supplementary Figure 5. ecDNA sequence is rearranged during tumor progression. Supplementary Figure 6. Clonal ecDNA. Supplementary Figure 7. Subclonal ecDNA. Supplementary Figure 8. Timing analysis of ecDNA and driver genes. Supplementary Figure 9. Clinical and genetic features associated with ecDNA. Supplementary Figure 10. Tumor immune microenvironment associated with ecDNA. Supplementary Figure 11. Transcriptional programs associated with ecDNA. Supplementary Figure 12. Urinary ecDNA.

Extrachromosomal DNA (ecDNA) presents a promising target for cancer therapy; however, its spatial–temporal diversity and influence on tumor evolution and the immune microenvironment remain largely unclear. We apply computational methods to analyze ecDNA from whole-genome sequencing data of 595 patients with urothelial carcinoma. We demonstrate that ecDNA drives clonal evolution through structural rearrangements during malignant transformation and recurrence of urothelial carcinoma. This supports a model wherein tumors evolve via the selective expansion of ecDNA-bearing cells. Through multiregional sampling of tumors, we demonstrate that ecDNA contributes to the evolution of multifocality and increased intratumoral heterogeneity. ecDNA is present in 36% of urothelial carcinoma tumors and correlates with an immunosuppressive phenotype and poor prognosis. Single-cell RNA sequencing analyses reveal that ecDNA+ malignant cells exhibit diminished expression of MHC class I molecules, enabling them to evade T-cell immunity. Finally, we show that sequencing of urinary sediment–derived DNA has excellent specificity in detecting ecDNA. Significance: Our comprehensive analysis of ecDNA in urothelial carcinoma reveals its crucial role in driving the evolution and heterogeneity of multifocal cancer, as well as its early involvement in tumorigenesis. Moreover, this study sheds light on immune evasion mechanisms associated with ecDNA and offers valuable insights for developing targeted therapeutic strategies.

Recent research reveals that eukaryotic genomes form circular DNA from all parts of their genome, some large enough to carry whole genes. In organisms like yeast and in human cancers, it is often observed that extrachromosomal circular DNA (eccDNA) benefits the individual cell by providing resources for rapid cellular growth. However, our comprehension of eccDNA remains incomplete, primarily due to their transient nature. Early studies suggest they arise when DNA breaks and is subsequently repaired incorrectly. In this review, we provide an overview of the evidence for molecular mechanisms that lead to eccDNA formation in human cancers and yeast, focusing on nonhomologous end joining, alternative end joining, and homologous recombination repair pathways. Furthermore, we present hypotheses in the form of molecular eccDNA formation models and consider cellular conditions which may affect eccDNA generation. Finally, we discuss the framework for future experimental evidence.