Circles with a Point: New Insights into Oncogenic Extrachromosomal DNA
Authors: Christopher J. Ott
Journal: Cancer Cell (2020)

For decades, scientists have observed small extrachromosomal DNA fragments in tumor cells, yet comprehensive examination of their structure and function has remained difficult. Three recent studies, published in Nature, Cell, and Nature Genetics, have now shed important light on the architecture, regulatory capacity, and oncogenic nature of tumor-associated extrachromosomal DNA. For decades, scientists have observed small extrachromosomal DNA fragments in tumor cells, yet comprehensive examination of their structure and function has remained difficult. Three recent studies, published in Nature, Cell, and Nature Genetics, have now shed important light on the architecture, regulatory capacity, and oncogenic nature of tumor-associated extrachromosomal DNA. In 1965, two reports described a curious observation: visual inspection of karyotyped chromosomes obtained from neuroblastoma and pediatric brain tumor cells revealed clear evidence of numerous small—"minute"—extrachromosomal DNA fragments (Cox et al., 1965Cox D. Yuncken C. Spriggs A.I. Minute chromatin bodies in malignant tumours of childhood.Lancet. 1965; 1: 55-58Abstract PubMed Scopus (126) Google Scholar, Lubs and Salmon, 1965Lubs Jr., H.A. Salmon J.H. The chromosomal complement of human solid tumors.J. Neurosurg. 1965; 22: 160-168Crossref PubMed Scopus (28) Google Scholar). Since this discovery, we have gained significant insights into tumor-associated extrachromosomal DNA (ecDNA) and its contribution to malignancy. ecDNA can be a primary source of extreme gene amplification in tumors, enabling a single cell to harbor up to hundreds of copies of oncogenes such as EGFR and MYC (Von Hoff et al., 1988Von Hoff D.D. Needham-VanDevanter D.R. Yucel J. Windle B.E. Wahl G.M. Amplified human MYC oncogenes localized to replicating submicroscopic circular DNA molecules.Proc. Natl. Acad. Sci. USA. 1988; 85: 4804-4808Crossref PubMed Scopus (96) Google Scholar, Kohl et al., 1983Kohl N.E. Kanda N. Schreck R.R. Bruns G. Latt S.A. Gilbert F. Alt F.W. Transposition and amplification of oncogene-related sequences in human neuroblastomas.Cell. 1983; 35: 359-367Abstract Full Text PDF PubMed Scopus (461) Google Scholar). They can arise via selective pressures such as drug treatment, enabling cells to ramp up expression of drug resistance genes (Haber and Schimke, 1981Haber D.A. Schimke R.T. Unstable amplification of an altered dihydrofolate reductase gene associated with double-minute chromosomes.Cell. 1981; 26: 355-362Abstract Full Text PDF PubMed Scopus (62) Google Scholar). They are atelomeric, acentromeric, and presumed to be circular, enabling efficient replication and asymmetric partitioning following mitosis (Wahl, 1989Wahl G.M. The importance of circular DNA in mammalian gene amplification.Cancer Res. 1989; 49: 1333-1340PubMed Google Scholar). Yet while ecDNA has been readily observable for some time, higher-resolution imaging, genomics, and epigenomic technologies have only recently been deployed to study them. A recent comprehensive analysis of ecDNA frequency in tumors was performed by Turner et al. in 2017 and revealed that ecDNA can be detected in nearly half of human tumors (Turner et al., 2017Turner K.M. Deshpande V. Beyter D. Koga T. Rusert J. Lee C. Li B. Arden K. Ren B. Nathanson D.A. et al.Extrachromosomal oncogene amplification drives tumour evolution and genetic heterogeneity.Nature. 2017; 543: 122-125Crossref PubMed Scopus (175) Google Scholar). Motivated by this apparent prevalence, a collaborative team led by Howard Chang, Bing Ren, Vineet Bafna, and Paul Mischel have now reported in Nature the use of a combination of electron microscopy, new computational tools for ecDNA sequence construction from whole-genome sequencing and long-read optical sequencing, and chromatin accessibility analysis to structurally resolve tumor ecDNA at high resolution (Wu et al., 2019Wu S. Turner K.M. Nguyen N. Raviram R. Erb M. Santini J. Luebeck J. Rajkumar U. Diao Y. Li B. et al.Circular ecDNA promotes accessible chromatin and high oncogene expression.Nature. 2019; 575: 699-703Crossref PubMed Scopus (61) Google Scholar). Their efforts demonstrate conclusively the circular configuration of ecDNA, with sizes ranging from several hundred kilobases to five megabases. Using quantitative, allele-specific RNA transcript detection, they were also able to show that ecDNA-derived oncogene expression is typically higher than linearly amplified chromosomal expression. Much of this increased gene expression could be attributed to increased copy number: ecDNA-derived oncogenes exist at far higher copy numbers than linearly amplified oncogenes. Yet in some cases, increased expression could not be explained by copy number increases alone. To further understand this, Wu et al. deployed chromatin accessibility assays (the assay for transposase accessible chromatin, ATAC-seq; micrococcal nuclease digestion and sequencing, MNase-seq) to measure nucleosome density at both ecDNA and linearly amplified chromosomal DNA. Comparing the two structures showed that ecDNA chromatin is more accessible with lower-order nucleosome compaction. With proximity ligation-assisted ChIP-seq (PLAC-seq), a method to identify direct physical contacts within chromatin fractions purified by immunoprecipitation, the authors find that ecDNA species are highly organized, with distal active chromatin regions directly interacting with oncogene loci at high frequencies. The nature of ecDNA oncogenic transcriptional regulation, specifically with respect to enhancer architecture, was the subject of another study reported in Cell by a team led by Jeremy Rich and Peter Scacheri (Morton et al., 2019Morton A.R. Dogan-Artun N. Faber Z.J. MacLeod G. Bartels C.F. Piazza M.S. Allan K.C. Mack S.C. Wang X. Gimple R.C. et al.Functional enhancers shape extrachromosomal oncogene amplifications.Cell. 2019; 179: 1330-1341Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Using EGFR ecDNA from glioblastoma as a primary model, the authors carefully resolve amplicon patterns from primary tumor sequencing in the TCGA database. Correlating these patterns to enhancer features (acetylated histone H3 lysine 27, H3K27ac), they observed non-random "skewing" of amplified segments toward non-coding regions that include enhancers. In fact, local enhancer elements were almost always included in the ecDNA structures they observed, strongly suggesting that enhancers are co-selected together with the coding regions of oncogenes during the formation of ecDNA. Notably, these enhancers seem to be established in the tumor cell of origin yet can exist outside the topologically associated domain (TAD) structures of the original chromosome. Chromosome conformation experiments confirmed that the ecDNA-amplified EGFR locus established contacts with distal enhancer elements that were typically insulated from each other on chromosomes. Using CRISPRi technology to ablate individual enhancer activity, they show that these enhancers are "rewired" in ecDNA to enforce EGFR expression and maintain the proliferative potential of the tumor cell. Thus, this extends our understanding of enhancer "hijacking" to include extrachromosomal structures and gives further mechanistic understanding to how genes on oncogene-associated ecDNA are so highly transcribed. While high-copy-number ecDNA has long been thought to contribute to malignancy via amplified oncogene expression, tumor cells may also benefit from dynamic interactions between ecDNA and chromosomal DNA. Reporting in Nature Genetics, a team led by Richard Koche, Elias Rodriguez-Fos, Konstantin Helmsauer, David Torrents, Johannes Schulte, and Anton Henssen describe a deep dive into the genomic structures of circular DNA in neuroblastoma, a tumor type often characterized by ecDNA-amplified MYCN (Koche et al., 2020Koche R.P. Rodriguez-Fos E. Helmsauer K. Burkert M. MacArthur I.C. Maag J. Chamorro R. Munoz-Perez N. Puiggròs M. Garcia H. et al.Extrachromosomal circular DNA drives oncogenic genome remodeling in neuroblastoma.Nat. Genet. 2020; 52 (Published online December 16, 2019): 29-34Crossref PubMed Scopus (34) Google Scholar). Using an analysis framework that leverages WGS datasets combined with sequencing of circular DNA structures directly purified from tumor cells (Circle-seq), the authors identify the comprehensive set of both neuroblastoma ecDNAs (sized on the order of hundreds to thousands of kilobases in length, corresponding to the findings of Wu et al.) and also smaller circular structures (termed eccDNA, typically between 1 and 10 kb). While total numbers of eccDNA in individual tumors are greater than those of ecDNA, their smaller size typically precludes the inclusion of full genes. Intriguingly, both ecDNA and eccDNA structures included many genomic regions beyond MYCN, including coding but also vast swaths of non-coding regions. The authors astutely hypothesize that in addition to fostering oncogene amplification, circular DNA structures may also play a role in remodeling the chromosomal genome. By focusing on sites of interchromosomal and intrachromosomal rearrangements, they found that a majority coincided with circularized DNA structures. Careful inspection of WGS, Circle-seq, and long reads generated by Nanopore sequencing revealed that these chromosomal rearrangements are formed first through chimeric circular DNA generation followed by segmented reintegration into chromosomal genomic loci. One can imagine that this widespread reintegration of circular DNA structures could have profound effects on chromosomal gene expression, including the disruption of tumor suppressor expression and augmentation of oncogenes. Several instances were detected that suggest this, including circle fragment integration into the tumor suppressor DCLK1 locus, associated with decreased expression, and an integration site nearby the TERT oncogene, associated with increased expression. These observations are also clinically relevant, as patients for which these circle-associated rearrangements were detected had significantly worse overall survival. The cancer genome continues to reveal itself in fascinating ways, and these unprecedented glimpses into ecDNA open up new questions about their roles in malignancy (Figure 1). Further insights into the dynamics of ecDNA formation and genomic reintegration are now required, including the instigating signals and mutagenic or epigenetic factors responsible for this genomic shuffle. This will lead to a greater understanding of tumor heterogeneity and therapy response and potentially offer us new therapeutic targets for ecDNA-driven cancers. C.J.O. is supported by funding from the NIH /NCI ( CA190861 ), The V Foundation for Cancer Research , and the Leukemia Research Foundation . Functional Enhancers Shape Extrachromosomal Oncogene AmplificationsMorton et al.CellNovember 21, 2019In BriefExtrachromosomal oncogene amplification is a common occurrence across a broad range of cancers, yet the chromatin landscape of these high-level amplifications is poorly understood. Using a combination of approaches to explore their chromatin topology and enhancer landscape, Morton et al. observed that these oncogene amplifications are shaped by regulatory dependencies in the non-coding genome. Full-Text PDF Open Archive