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. 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.

Disparate pathways for extrachromosomal DNA biogenesis and genomic DNA repair.

Oncogene amplification on extrachromosomal DNA (ecDNA) is a pervasive driver event in cancer, yet our understanding of how ecDNA forms is limited. Here, we couple a CRISPR-based method for induction of ecDNA with extensive characterization of newly formed ecDNA to examine ecDNA biogenesis. We find that DNA circularization is efficient, irrespective of 3D genome context, with formation of a 1 Mb and 1.8 Mb ecDNA both reaching 15%. We show non-homologous end joining and microhomology mediated end joining both contribute to ecDNA formation, while inhibition of DNA-PKcs and ATM have opposing impacts on ecDNA formation. EcDNA and the corresponding chromosomal excision scar form at significantly different rates and respond differently to DNA-PKcs and ATM inhibition. Taken together, our results support a model of ecDNA formation in which double strand break ends dissociate from their legitimate ligation partners prior to joining of illegitimate ends to form the ecDNA and excision scar.

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. A subset of tumors harbored multiple ecDNA lineages, each containing distinct amplified oncogenes. Multimodal sequencing, imaging and CRISPR inhibition experiments in medulloblastoma models reveal intratumoral heterogeneity of ecDNA copy number per cell and frequent putative 'enhancer rewiring' events on ecDNA. This study reveals the frequency and diversity of ecDNA in medulloblastoma, stratified into molecular subgroups, and suggests copy number heterogeneity and enhancer rewiring as oncogenic features of ecDNA.

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.

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.

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. Targeted enrichment of ecDNA versus chromosomal DNA enabled phasing of genetic variants, identified the presence of an EGFRvIII mutation exclusively on ecDNAs and supported an excision model of ecDNA genesis in a glioblastoma model. CRISPR-CATCH followed by nanopore sequencing enabled single-molecule ecDNA methylation profiling and revealed hypomethylation of the EGFR promoter on ecDNAs. We distinguished heterogeneous ecDNA species within the same sample by size and sequence with base-pair resolution and discovered functionally specialized ecDNAs that amplify select enhancers or oncogene-coding sequences.

Targeting glioblastoma signaling and metabolism with a re-purposed brain-penetrant drug.

The highly lethal brain cancer glioblastoma (GBM) poses a daunting challenge because the blood-brain barrier renders potentially druggable amplified or mutated oncoproteins relatively inaccessible. Here, we identify sphingomyelin phosphodiesterase 1 (SMPD1), an enzyme that regulates the conversion of sphingomyelin to ceramide, as an actionable drug target in GBM. We show that the highly brain-penetrant antidepressant fluoxetine potently inhibits SMPD1 activity, killing GBMs, through inhibition of epidermal growth factor receptor (EGFR) signaling and via activation of lysosomal stress. Combining fluoxetine with temozolomide, a standard of care for GBM, causes massive increases in GBM cell death and complete tumor regression in mice. Incorporation of real-world evidence from electronic medical records from insurance databases reveals significantly increased survival in GBM patients treated with fluoxetine, which was not seen in patients treated with other selective serotonin reuptake inhibitor (SSRI) antidepressants. These results nominate the repurposing of fluoxetine as a potentially safe and promising therapy for patients with GBM and suggest prospective randomized clinical trials.

ecDNA hubs drive cooperative intermolecular oncogene expression.

Extrachromosomal DNA (ecDNA) is prevalent in human cancers and mediates high expression of oncogenes through gene amplification and altered gene regulation1. Gene induction typically involves cis-regulatory elements that contact and activate genes on the same chromosome2,3. Here we show that ecDNA hubs-clusters of around 10-100 ecDNAs within the nucleus-enable intermolecular enhancer-gene interactions to promote oncogene overexpression. ecDNAs that encode multiple distinct oncogenes form hubs in diverse cancer cell types and primary tumours. Each ecDNA is more likely to transcribe the oncogene when spatially clustered with additional ecDNAs. ecDNA hubs are tethered by the bromodomain and extraterminal domain (BET) protein BRD4 in a MYC-amplified colorectal cancer cell line. The BET inhibitor JQ1 disperses ecDNA hubs and preferentially inhibits ecDNA-derived-oncogene transcription. The BRD4-bound PVT1 promoter is ectopically fused to MYC and duplicated in ecDNA, receiving promiscuous enhancer input to drive potent expression of MYC. Furthermore, the PVT1 promoter on an exogenous episome suffices to mediate gene activation in trans by ecDNA hubs in a JQ1-sensitive manner. Systematic silencing of ecDNA enhancers by CRISPR interference reveals intermolecular enhancer-gene activation among multiple oncogene loci that are amplified on distinct ecDNAs. Thus, protein-tethered ecDNA hubs enable intermolecular transcriptional regulation and may serve as units of oncogene function and cooperative evolution and as potential targets for cancer therapy.

Overexpression of iASPP is required for autophagy in response to oxidative stress in choriocarcinoma.

BACKGROUND: Gestational trophoblastic disease (GTD) is a heterogeneous group of diseases developed from trophoblasts. ASPP (Ankyrin-repeat, SH3-domain and proline-rich region containing protein) family proteins, ASPP1 and ASPP2, have been reported to be dysregulated in GTD. They modulate p53 activities and are responsible for multiple cellular processes. Nevertheless, the functional role of the ASPP family inhibitory member, iASPP, is not well characterized in GTD. METHODS: To study the functional role of iASPP in GTD, trophoblastic tissues from normal placentas, hydatidiform mole (HM) and choriocarcinoma were used for immunohistochemistry, whereas siRNAs were used to manipulate iASPP expression in choriocarcinoma cell lines and study the subsequent molecular changes. RESULTS: We demonstrated that iASPP was overexpressed in both HM and choriocarcinoma when compared to normal placenta. Progressive increase in iASPP expression from HM to choriocarcinoma suggests that iASPP may be related to the development of trophoblastic malignancy. High iASPP expression in HM was also significantly associated with a high expression of autophagy-related protein LC3. Interestingly, iASPP silencing retarded the growth of choriocarcinoma through senescence instead of induction of apoptosis. LC3 expression decreased once iASPP was knocked down, suggesting a downregulation on autophagy. This may be due to iASPP downregulation rendered decrease in Atg5 expression and concomitantly hindered autophagy in choriocarcinoma cells. Autophagy inhibition per se had no effect on the growth of choriocarcinoma cells but increased the susceptibility of choriocarcinoma cells to oxidative stress, implying a protective role of iASPP against oxidative stress through autophagy in choriocarcinoma. CONCLUSIONS: iASPP regulates growth and the cellular responses towards oxidative stress in choriocarcinoma cells. Its overexpression is advantageous to the pathogenesis of GTD. (266 words).