Biology subjects

Wong, I. T.-L.

Publications and source records attributed to Wong, I. T.-L..

10 recordsLinked to original sources

bioRxiv2025

Genetic elements promote retention of extrachromosomal DNA in cancer cells

Extrachromosomal DNA (ecDNA) is a prevalent and devastating form of oncogene amplification in cancer1,2. Circular megabase-sized ecDNAs lack centromeres and segregate stochastically during cell division3-6 yet persist over many generations. EcDNAs were first observed to hitchhike on mitotic chromosomes into daughter cell nuclei over 40 years ago with unknown mechanism3,7. Here we identify a family of human genomic elements, termed retention elements, that tether episomes to mitotic chromosomes to increase ecDNA transmission to daughter cells. We develop Retain-seq, a genome-scale assay that reveals thousands of human retention elements conferring generational persistence to heterologous episomes. Retention elements comprise a select set of CpG-rich gene promoters and act additively. Live-cell imaging and chromatin conformation capture show that retention elements physically interact with mitotic chromosomes at regions which are mitotically bookmarked by transcription factors and chromatin proteins, intermolecularly recapitulating promoter-enhancer interactions. Multiple retention elements are co-amplified with oncogenes on individual ecDNAs in human cancers and shape their sizes and structures. CpG-rich retention elements are focally hypomethylated; targeted cytosine methylation abrogates retention activity and leads to ecDNA loss, suggesting that methylation-sensitive interactions modulate episomal DNA retention. These results highlight the DNA elements and regulatory logic of mitotic ecDNA retention. Amplifications of retention elements promote the maintenance of oncogenic ecDNA across generations of cancer cells, revealing the principles of episome immortality intrinsic to the human genome.

cancer biology↗

bioRxiv2025

Accurate Prediction of ecDNA in Interphase Cancer Cells using Deep Neural Networks

Oncogene amplification is a key driver of cancer pathogenesis and is often mediated by extrachromosomal DNA (ecDNA). EcDNA amplifications are associated with increased pathogenicity of cancer and poorer outcomes for patients. EcDNA can be detected accurately using fluorescence in situ hybridization (FISH) when cells are arrested in metaphase. However, the majority of cancer cells are non-mitotic and must be analyzed in interphase, where it is difficult to discern extrachromosomal amplifications from chromosomal amplifications. Thus, there is a need for methods that accurately predict oncogene amplification status from interphase cells. Here, we present interSeg, a deep learning-based tool to cytogenetically determine the amplification status as EC-amp, HSR-amp, or not amplified from interphase FISH images. We trained and validated interSeg on 652 images (40,446 nuclei). Tests on 215 cultured cell and tissue model images (9,733 nuclei) showed 89% and 97% accuracy at the nuclear and sample levels, respectively. The neuroblastoma patient tissue hold-out set (67 samples and 1,937 nuclei) also revealed 97% accuracy at the sample level in detecting the presence of focal amplification. In experimentally and computationally mixed images, interSeg accurately predicted the level of heterogeneity. The results showcase interSeg as an important method for analyzing oncogene amplifications.

cancer biology↗

bioRxiv2025

Oncogene Silencing via ecDNA Micronucleation

Extrachromosomal DNA (ecDNA) contributes to cancer genome instability by enabling high-copy oncogene amplification, intratumoural heterogeneity and rapid genetic change. Micronuclei (MN) are frequently observed in chromosomally unstable cancers, yet their origins and relevance in ecDNA-driven tumours remain incompletely understood. Here, we investigate the relationship between ecDNA segregation errors and MN formation. We find that ecDNA frequently localizes to MN and represents a prominent source of MN content in ecDNA-positive cancer cells. Mitotic clustering of oncogene-bearing ecDNAs is associated with asymmetric inheritance and mis-segregation into MN. Transfer of oncogenes from ecDNA into MN is accompanied by reduced transcriptional output. Single-MN sequencing shows that individual MN are enriched for ecDNA to a degree that exceeds expectations from stochastic mis-segregation and that in MN oncogenes originating from multiple distinct ecDNAs coalesce. Using live-cell imaging, we observe that cells inheriting ecDNA-positive MN show limited proliferative capacity and an increased likelihood of cell death. In neuroblastoma patients with MYCN-amplified ecDNA, higher frequencies of ecDNA-positive MN at diagnosis are associated with improved event-free and overall survival. Together, these findings link ecDNA mis-segregation to MN formation and reduced cellular fitness, suggesting that ecDNA-positive MN may reflect a state of impaired oncogenic ecDNA function with potential relevance for clinical outcome.

cancer biology↗

bioRxiv2025

EcDNA-borne PVT1 fusion stabilizes oncogenic mRNAs

Extrachromosomal DNA (ecDNA) amplifications are prevalent drivers of human cancers. We show that ecDNAs exhibit elevated structural variants leading to gene fusions that produce oncogene fusion transcripts. The long noncoding RNA (lncRNA) gene PVT1 is the most recurrent structural variant across cancer genomes, with PVT1-MYC fusions arising most frequently on ecDNA. PVT1 exon 1 is the predominant 5 partner fused to MYC or other oncogenes on the 3 end. Mechanistic studies demonstrate that PVT1 exon 1 confers enhanced RNA stability for fusion transcripts, which requires PVT1 exon 1 interaction with SRSF1 protein. Genetic rescue of MYC-addicted cancer models and isoform-specific single-cell RNA sequencing of tumors reveal that PVT1-MYC better supports MYC dependency and better activates MYC target genes in vivo. Thus, the mutagenic landscape of ecDNA contributes to genome instability and generates chimeric fusions of lncRNA and mRNA genes, selecting PVT1 5 region as a stabilizer of oncogene mRNAs.

cancer biology↗

bioRxiv2024

Extrachromosomal DNA driven oncogene spatial heterogeneity and evolution in glioblastoma

Oncogene amplification on extrachromosomal DNA (ecDNA) is strongly associated with treatment resistance and shorter survival for patients with cancer, including patients with glioblastoma. The non-chromosomal inheritance of ecDNA during cell division is a major contributor to intratumoral genetic heterogeneity. At present, the spatial dynamics of ecDNA, and the impact on tumor evolutionary trajectories, are not well understood. Here, we investigate the spatial-temporal evolution of ecDNA and its clinical impact by analyzing tumor samples from 94 treatment-naive human IDH-wildtype glioblastoma patients. We developed a spatial-temporal computational model of ecDNA positive tumors ( SPECIES) that integrates whole-genome sequencing, multi-region DNA FISH, and nascent RNAscope, to provide unique insight into the spatial dynamics of ecDNA evolution. Random segregation in combination with positive selection of ecDNAs induce large, predictable spatial patterns of cell-to-cell ecDNA copy number variation that are highly dependent on the oncogene encoded on the circular DNA. EGFR ecDNAs often reach high mean copy number (mean of 50 copies per tumor cell), are under strong positive selection (mean selection coefficient, s > 2) and do not co-amplify other oncogenes on the same ecDNA particles. In contrast, PDGFRA ecDNAs have lower mean copy number (mean of 15 copies per cell), are under weaker positive selection and frequently co-amplify other oncogenes on the same ecDNA. Evolutionary modeling suggests that EGFR ecDNAs often accumulate prior to clonal expansion. EGFR structural variants, including vIII and c-terminal deletions are under strong positive selection, are found exclusively on ecDNA, and are intermixed with wild-type EGFR ecDNAs. Simulations show EGFRvIII ecDNA likely arises after ecDNA formation in a cell with high wild-type EGFR copy number (> 10) before the onset of the most recent clonal expansion. This remains true even in cases of co-selection and co-amplification of multiple oncogenic ecDNA species in a subset of patients. Overall, our results suggest a potential time window in which early ecDNA detection may provide an opportunity for more effective intervention. HighlightsO_LIecDNA is the most common mechanism of focal oncogene amplification in IDHwt glioblastoma. C_LIO_LIEGFR and its variants on ecDNA are particularly potent, likely arising early in tumor development, providing a strong oncogenic stimulus to drive tumorigenesis. C_LIO_LIWild-type and variant EGFR ecDNA heteroplasmy (co-occurrence) is common with EGFRvIII or c-terminal deletions being derived from EGFR wild-type ecDNA prior to the most recent clonal expansion. C_LIO_LITumors with ecDNA amplified EGFR versus PDGFRA exhibit different evolutionary trajectories. C_LIO_LISPECIES model can infer spatial evolutionary dynamics of ecDNA in cancer. C_LIO_LIA delay between ecDNA accumulation and subsequent oncogenic mutation may give a therapeutic window for early intervention. C_LI

cancer biology↗

bioRxiv2024

Rampant transcription replication conflict creates therapeutic vulnerability in extrachromosomal DNA containing cancers

Extrachromosomal DNA (ecDNA) presents a major challenge for precision medicine, contributing to poor survival for patients with oncogene-amplified tumours. EcDNA renders tumours resistant to targeted treatments by facilitating massive transcription of oncogenes and rapid genome evolution. At present, there are no ecDNA- specific treatments. Here we show that enhancing transcription replication conflict enables targeted elimination of ecDNA-containing cancers, exposing an actionable vulnerability. Stepwise analyses of ecDNA transcription reveal landscapes of pervasive RNA transcription and associated single-stranded DNA, leading to excessive transcription replication conflicts and replication stress (RS) compared to chromosomal loci. Nucleotide incorporation onto growing DNA strands is markedly slower on ecDNA, and RS is significantly higher in ecDNA-containing tumours regardless of cancer type or oncogene cargo. Replication Protein A2 phosphorylated on serine 33, a mediator of DNA damage repair that binds single-stranded DNA, shows elevated localization on ecDNA in a transcription dependent manner, along with increased DNA double strand breaks, and activation of the S-phase checkpoint kinase, CHK1. Genetic or pharmacological CHK1 inhibition abrogates the DNA replication check point, causing extensive and preferential tumour cell death in ecDNA-containing tumours as they enter S-phase. To exploit this vulnerability, we develop a highly selective, potent, and bioavailable oral CHK1 inhibitor, BBI-2779, and demonstrate that it preferentially kills ecDNA-containing tumour cells. In a gastric cancer model containing FGFR2 on ecDNA, BBI-2779, suppresses tumour growth and prevents ecDNA-mediated acquired resistance to the pan-FGFR inhibitor infigratinib, resulting in potent and sustained tumour regression in mice. These results reveal transcription-replication conflict as an ecDNA-generated vulnerability that can be targeted as an ecDNA-directed therapy and suggest that synthetic lethality of excess can be exploited as a strategy for treating cancer.

cancer biology↗

bioRxiv2024

CoRAL accurately resolves extrachromosomal DNA genomestructures 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. Paired-end short-read (Illumina) sequencing and mapping have been utilized to represent ecDNA amplifications using a breakpoint graph, where the inferred architecture of ecDNA is encoded as a cycle in the graph. Traversals of breakpoint graph have been used to successfully predict ecDNA presence in cancer samples. However, short-read technologies are intrinsically limited in the identification of breakpoints, phasing together of complex rearrangements and internal duplications, and deconvolution of cell-to-cell heterogeneity of ecDNA structures. Long-read technologies, such as from Oxford Nanopore Technologies, have the potential to improve inference as the longer reads are better at mapping structural variants and are more likely to span rearranged or duplicated regions. Here, we propose CoRAL (Complete Reconstruction of Amplifications with Long reads), for reconstructing ecDNA architectures using long-read data. CoRAL reconstructs likely cyclic architectures using quadratic programming that simultaneously optimizes parsimony of reconstruction, explained copy number, and consistency of long-read mapping. CoRAL substantially improves reconstructions in extensive simulations and 9 datasets from previously-characterized cell-lines as compared to previous short-read-based tools. As long-read usage becomes wide-spread, we anticipate that CoRAL will be a valuable tool for profiling the landscape and evolution of focal amplifications in tumors.

cancer biology↗

bioRxiv2023

Breakage fusion bridge cycles drive high oncogene copy number, but not intratumoral genetic heterogeneity or rapid cancer genome change

Oncogene amplification is a major driver of cancer pathogenesis. Breakage fusion bridge (BFB) cycles, like extrachromosomal DNA (ecDNA), can lead to high copy numbers of oncogenes, but their impact on intratumoral heterogeneity, treatment response, and patient survival are not well understood due to difficulty in detecting them by DNA sequencing. We describe a novel algorithm that detects and reconstructs BFB amplifications using optical genome maps (OGMs), called OM2BFB. OM2BFB showed high precision (>93%) and recall (92%) in detecting BFB amplifications in cancer cell lines, PDX models and primary tumors. OM-based comparisons demonstrated that short-read BFB detection using our AmpliconSuite (AS) toolkit also achieved high precision, albeit with reduced sensitivity. We detected 371 BFB events using whole genome sequences from 2,557 primary tumors and cancer lines. BFB amplifications were preferentially found in cervical, head and neck, lung, and esophageal cancers, but rarely in brain cancers. BFB amplified genes show lower variance of gene expression, with fewer options for regulatory rewiring relative to ecDNA amplified genes. BFB positive (BFB (+)) tumors showed reduced heterogeneity of amplicon structures, and delayed onset of resistance, relative to ecDNA(+) tumors. EcDNA and BFB amplifications represent contrasting mechanisms to increase the copy numbers of oncogene with markedly different characteristics that suggest different routes for intervention.

cancer biology↗

bioRxiv2023

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. Distinct ecDNA sequences, herein termed ecDNA species, can co-exist to facilitate intermolecular cooperation in cancer cells. However, how multiple ecDNA species within a tumor cell are assorted and maintained across somatic cell generations to drive cancer cell evolution is not known. Here we show that cooperative ecDNA species can be coordinately inherited through mitotic co-segregation. Imaging and single-cell analyses show that multiple ecDNAs encoding distinct oncogenes co-occur and are correlated in copy number in human cancer cells. EcDNA species are coordinately segregated asymmetrically during mitosis, resulting in daughter cells with simultaneous copy number gains in multiple ecDNA species prior to any selection. Computational modeling reveals the quantitative principles of ecDNA co-segregation and co-selection, predicting their observed distributions in cancer cells. Finally, we show that coordinated inheritance of ecDNAs enables co-amplification of specialized ecDNAs containing only enhancer elements and guides therapeutic strategies to jointly deplete cooperating ecDNA oncogenes. Coordinated inheritance of ecDNAs confers stability to oncogene cooperation and novel gene regulatory circuits, allowing winning combinations of epigenetic states to be transmitted across cell generations.

cancer biology↗

bioRxiv2021

The landscape of extrachromosomal circular DNA in medulloblastoma

Extrachromosomal circular DNA (ecDNA) is an important driver of aggressive tumor growth, promoting high oncogene copy number, intratumoral heterogeneity, accelerated evolution of drug resistance, enhancer rewiring, and poor outcome. ecDNA has been reported in medulloblastoma (MB), the most common malignant pediatric brain tumor, but the ecDNA landscape and its association with specific MB subgroups, its impact on enhancer rewiring, and its potential clinical implications, are not known. We assembled a retrospective cohort of 468 MB patient samples with available whole genome sequencing (WGS) data covering the four major MB subgroups WNT, SHH, Group 3 and Group 4. Using computational methods for the detection and reconstruction of ecDNA1, we find ecDNA in 82 patients (18%) and observe that ecDNA+ MB patients are more than twice as likely to relapse and three times as likely to die of disease. In addition, we find that individual medulloblastoma tumors often harbor multiple ecDNAs, each containing different amplified oncogenes along with co-amplified non-coding regulatory enhancers. ecDNA was substantially more prevalent among 31 analyzed patient-derived xenograft (PDX) models and cell lines than in our patient cohort. By mapping the accessible chromatin and 3D conformation landscapes of MB tumors that harbor ecDNA, we observe frequent candidate "enhancer rewiring" events that spatially link oncogenes with co-amplified enhancers. Our study reveals the frequency and diversity of ecDNA in a subset of highly aggressive tumors and suggests enhancer rewiring as a frequent oncogenic mechanism of ecDNAs in MB. Further, these results demonstrate that ecDNA is a frequent and potent driver of poor outcome in MB patients.

cancer biology↗