Engineered extrachromosomal oncogene amplifications promote tumorigenesis.

Focal gene amplifications are among the most common cancer-associated mutations but have proven challenging to engineer in primary cells and model organisms. Here we describe a general strategy to engineer large (more than 1 Mbp) focal amplifications mediated by extrachromosomal DNAs (ecDNAs) in a spatiotemporally controlled manner in cells and in mice. By coupling ecDNA formation with expression of selectable markers, we track the dynamics of ecDNA-containing cells under physiological conditions and in the presence of specific selective pressures.

Enhancing transcription-replication conflict targets ecDNA-positive cancers.

Extrachromosomal DNA (ecDNA) presents a major challenge for cancer patients. ecDNA renders tumours treatment resistant by facilitating massive oncogene transcription and rapid genome evolution, contributing to poor patient survival. At present, there are no ecDNA-specific treatments.

Coordinated inheritance of extrachromosomal DNAs in cancer cells.

The chromosomal theory of inheritance dictates that genes on the same chromosome segregate together while genes on different chromosomes assort independently. Extrachromosomal DNAs (ecDNAs) are common in cancer and drive oncogene amplification, dysregulated gene expression and intratumoural heterogeneity through random segregation during cell division. Distinct ecDNA sequences, termed ecDNA species, can co-exist to facilitate intermolecular cooperation in cancer cells.

Origins and impact of extrachromosomal DNA.

Extrachromosomal DNA (ecDNA) is a major contributor to treatment resistance and poor outcome for patients with cancer. Here we examine the diversity of ecDNA elements across cancer, revealing the associated tissue, genetic and mutational contexts. By analysing data from 14,778 patients with 39 tumour types from the 100,000 Genomes Project, we demonstrate that 17.

Mapping extrachromosomal DNA amplifications during cancer progression.

To understand the role of extrachromosomal DNA (ecDNA) amplifications in cancer progression, we detected and classified focal amplifications in 8,060 newly diagnosed primary cancers, untreated metastases and heavily pretreated tumors. The ecDNAs were detected at significantly higher frequency in untreated metastatic and pretreated tumors compared to newly diagnosed cancers. Tumors from chemotherapy-pretreated patients showed significantly higher ecDNA frequency compared to untreated cancers.

Enhancer activation from transposable elements in extrachromosomal DNA.

Extrachromosomal DNA (ecDNA) is a hallmark of aggressive cancer, contributing to both oncogene amplification and tumor heterogeneity. Here, we used Hi-C, super-resolution imaging, and long-read sequencing to explore the nuclear architecture of -amplified ecDNA in colorectal cancer cells. Intriguingly, we observed frequent spatial proximity between ecDNA and 68 repetitive elements which we called ecDNA-interacting elements or EIEs.

Disparate Pathways for Extrachromosomal DNA Biogenesis and Genomic DNA Repair.

Our study harnesses a CRISPR-based method to examine ecDNA biogenesis, uncovering efficient circularization between double-strand breaks. ecDNAs and their corresponding chromosomal scars can form via nonhomologous end joining or microhomology-mediated end joining, but the ecDNA and scar formation processes are distinct. Based on our findings, we establish a mechanistic model of excisional ecDNA formation.

CoRAL accurately resolves extrachromosomal DNA genome structures with long-read sequencing.

Extrachromosomal DNA (ecDNA) is a central mechanism for focal oncogene amplification in cancer, occurring in ∼15% of early-stage cancers and ∼30% of late-stage cancers. ecDNAs drive tumor formation, evolution, and drug resistance by dynamically modulating oncogene copy number and rewiring gene-regulatory networks. Elucidating the genomic architecture of ecDNA amplifications is critical for understanding tumor pathology and developing more effective therapies.

Transcriptional immune suppression and up-regulation of double-stranded DNA damage and repair repertoires in ecDNA-containing tumors.

Extrachromosomal DNA is a common cause of oncogene amplification in cancer. The non-chromosomal inheritance of ecDNA enables tumors to rapidly evolve, contributing to treatment resistance and poor outcome for patients. The transcriptional context in which ecDNAs arise and progress, including chromosomally-driven transcription, is incompletely understood.

CoRAL accurately resolves extrachromosomal DNA genome structures with long-read sequencing.

Extrachromosomal DNA (ecDNA) is a central mechanism for focal oncogene amplification in cancer, occurring in approximately 15% of early stage cancers and 30% of late-stage cancers. EcDNAs drive tumor formation, evolution, and drug resistance by dynamically modulating oncogene copy-number and rewiring gene-regulatory networks. Elucidating the genomic architecture of ecDNA amplifications is critical for understanding tumor pathology and developing more effective therapies.

Circular extrachromosomal DNA promotes tumor heterogeneity in high-risk medulloblastoma.

Circular extrachromosomal DNA (ecDNA) in patient tumors is an important driver of oncogenic gene expression, evolution of drug resistance and poor patient outcomes. Applying computational methods for the detection and reconstruction of ecDNA across a retrospective cohort of 481 medulloblastoma tumors from 465 patients, we identify circular ecDNA in 82 patients (18%). Patients with ecDNA-positive medulloblastoma were more than twice as likely to relapse and three times as likely to die within 5 years of diagnosis.

Coordinated inheritance of extrachromosomal DNA species in human cancer cells.

The chromosomal theory of inheritance has dominated human genetics, including cancer genetics. Genes on the same chromosome segregate together while genes on different chromosomes assort independently, providing a fundamental tenet of Mendelian inheritance. Extrachromosomal DNA (ecDNA) is a frequent event in cancer that drives oncogene amplification, dysregulated gene expression and intratumoral heterogeneity, including through random segregation during cell division.

Immortalization and transformation of primary cells mediated by engineered ecDNAs.

Focal gene amplifications are among the most common cancer-associated mutations, but their evolution and contribution to tumorigenesis have proven challenging to recapitulate in primary cells and model organisms. Here we describe a general approach to engineer large (>1 Mbp) focal amplifications mediated by extrachromosomal circular DNAs (ecDNAs, also known as "double minutes") in a spatiotemporally controlled manner in cancer cell lines and in primary cells derived from genetically engineered mice. With this strategy, ecDNA formation can be coupled with expression of fluorescent reporters or other selectable markers to enable the identification and tracking of ecDNA-containing cells.

Epigenetic dysregulation from chromosomal transit in micronuclei.

Chromosomal instability (CIN) and epigenetic alterations are characteristics of advanced and metastatic cancers, but whether they are mechanistically linked is unknown. Here we show that missegregation of mitotic chromosomes, their sequestration in micronuclei and subsequent rupture of the micronuclear envelope profoundly disrupt normal histone post-translational modifications (PTMs), a phenomenon conserved across humans and mice, as well as in cancer and non-transformed cells. Some of the changes in histone PTMs occur because of the rupture of the micronuclear envelope, whereas others are inherited from mitotic abnormalities before the micronucleus is formed.

Transcriptional immune suppression and upregulation of double stranded DNA damage and repair repertoires in ecDNA-containing tumors.

Extrachromosomal DNA is a common cause of oncogene amplification in cancer. The non-chromosomal inheritance of ecDNA enables tumors to rapidly evolve, contributing to treatment resistance and poor outcome for patients. The transcriptional context in which ecDNAs arise and progress, including chromosomally-driven transcription, is incompletely understood.

Integrated analysis of single-cell chromatin state and transcriptome identified common vulnerability despite glioblastoma heterogeneity.

In 2021, the World Health Organization reclassified glioblastoma, the most common form of adult brain cancer, into isocitrate dehydrogenase (IDH)-wild-type glioblastomas and grade IV IDH mutant (G4 IDHm) astrocytomas. For both tumor types, intratumoral heterogeneity is a key contributor to therapeutic failure. To better define this heterogeneity, genome-wide chromatin accessibility and transcription profiles of clinical samples of glioblastomas and G4 IDHm astrocytomas were analyzed at single-cell resolution.

Extrachromosomal DNA in the cancerous transformation of Barrett's oesophagus.

Oncogene amplification on extrachromosomal DNA (ecDNA) drives the evolution of tumours and their resistance to treatment, and is associated with poor outcomes for patients with cancer. At present, it is unclear whether ecDNA is a later manifestation of genomic instability, or whether it can be an early event in the transition from dysplasia to cancer. Here, to better understand the development of ecDNA, we analysed whole-genome sequencing (WGS) data from patients with oesophageal adenocarcinoma (EAC) or Barrett's oesophagus.

Targeted profiling of human extrachromosomal DNA by CRISPR-CATCH.

Extrachromosomal DNA (ecDNA) is a common mode of oncogene amplification but is challenging to analyze. Here, we adapt CRISPR-CATCH, in vitro CRISPR-Cas9 treatment and pulsed field gel electrophoresis of agarose-entrapped genomic DNA, previously developed for bacterial chromosome segments, to isolate megabase-sized human ecDNAs. We demonstrate strong enrichment of ecDNA molecules containing EGFR, FGFR2 and MYC from human cancer cells and NRAS ecDNA from human metastatic melanoma with acquired therapeutic resistance.

FastViFi: Fast and accurate detection of (Hybrid) Viral DNA and RNA.

DNA viruses are important infectious agents known to mediate a large number of human diseases, including cancer. Viral integration into the host genome and the formation of hybrid transcripts are also associated with increased pathogenicity. The high variability of viral genomes, however requires the use of sensitive ensemble hidden Markov models that add to the computational complexity, often requiring > 40 CPU-hours per sample.

Mapping clustered mutations in cancer reveals APOBEC3 mutagenesis of ecDNA.

Clustered somatic mutations are common in cancer genomes and previous analyses reveal several types of clustered single-base substitutions, which include doublet- and multi-base substitutions, diffuse hypermutation termed omikli, and longer strand-coordinated events termed kataegis. Here we provide a comprehensive characterization of clustered substitutions and clustered small insertions and deletions (indels) across 2,583 whole-genome-sequenced cancers from 30 types of cancer. Clustered mutations were highly enriched in driver genes and associated with differential gene expression and changes in overall survival.

Plasticity of Extrachromosomal and Intrachromosomal BRAF Amplifications in Overcoming Targeted Therapy Dosage Challenges.

Unlabelled: Focal amplifications (FA) can mediate targeted therapy resistance in cancer. Understanding the structure and dynamics of FAs is critical for designing treatments that overcome plasticity-mediated resistance. We developed a melanoma model of dual MAPK inhibitor (MAPKi) resistance that bears BRAFV600 amplifications through either extrachromosomal DNA (ecDNA)/double minutes (DM) or intrachromosomal homogenously staining regions (HSR).