Extrachromosomal DNA Gives Cancer a New Evolutionary Pathway

Extrachromosomal DNA Gives Cancer a New Evolutionary Pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Extrachromosomal DNA Gives Cancer a New Evolutionary Pathway Elizabeth Brunk, Yue Wang, Oliver Cope, Jingting Chen, Aarav Mehta, and 17 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9477622/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract During tumor progression, it has been assumed that individual cells that have acquired advantageous mutations overtake the population. Cancers driven by extrachromosomal DNA (ecDNA) do not follow this paradigm. Instead, these tumors have a spectrum of oncogene copy numbers across cells, and graded ecDNA variation may function as a form of bet-hedging that equips tumors with a broad range of phenotypes. Using imaging, single-cell multiomics, and multiplexed proteomics, we systematically characterized ecDNA levels across thousands of single cells. Higher ecDNA dosage produces proportional changes in transcript abundance, chromatin accessibility, protein levels, cell-cycle progression, and proliferation. Genes amplified on ecDNA exhibit distinct transcriptional scaling regimes that shift when the same genes are reintegrated into chromosomal homogeneous staining regions. When we experimentally disrupted the continuum of ecDNA dosage by sorting cells into low- and high-copy number states, the population rapidly recovered its original, continuous distribution. Our time-course data, live-cell imaging, and stochastic models collectively show that restoring this spectrum is an active, deterministic process rather than the passive outcome of random segregation. Together, these findings position ecDNA-mediated expression as a distinct evolutionary mechanism that endows tumors with rapid, population-level adaptability. These findings offer insight into why ecDNA-driven cancers are among the most aggressive and treatment-resistant. Biological sciences/Genetics/Gene regulation Biological sciences/Systems biology/Systems analysis Biological sciences/Cancer/Oncogenes Biological sciences/Genetics/Functional genomics/Gene expression profiling Biological sciences/Genetics/Cytogenetics Extrachromosomal DNA ecDNA single cell single-cell multi-omics sequencing population heterogeneity evolution Full Text Additional Declarations There is NO Competing Interest. Supplementary Files 2025FACSFISHFULLSUPP.pdf Supplementary Information Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9477622","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":627395072,"identity":"5599a11b-06a5-4800-a95f-8d21e1f31552","order_by":0,"name":"Elizabeth Brunk","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-8578-8658","institution":"University of North Carolina at Chapel Hill","correspondingAuthor":true,"prefix":"","firstName":"Elizabeth","middleName":"","lastName":"Brunk","suffix":""},{"id":627395073,"identity":"df163034-1a89-4a3a-b14a-f9d59745d666","order_by":1,"name":"Yue Wang","email":"","orcid":"","institution":"UNC Chapel Hill","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Wang","suffix":""},{"id":627395074,"identity":"a139e9e9-fe11-4b12-af8c-72fe4588130f","order_by":2,"name":"Oliver Cope","email":"","orcid":"","institution":"UNC Chapel Hill","correspondingAuthor":false,"prefix":"","firstName":"Oliver","middleName":"","lastName":"Cope","suffix":""},{"id":627395075,"identity":"bf1def19-20e1-4562-9cf8-48e31baba1e9","order_by":3,"name":"Jingting Chen","email":"","orcid":"","institution":"UNC Chapel Hill","correspondingAuthor":false,"prefix":"","firstName":"Jingting","middleName":"","lastName":"Chen","suffix":""},{"id":627395076,"identity":"00055162-20ba-4dc8-b6c2-7ac82c150d3a","order_by":4,"name":"Aarav Mehta","email":"","orcid":"","institution":"UNC Chapel Hill","correspondingAuthor":false,"prefix":"","firstName":"Aarav","middleName":"","lastName":"Mehta","suffix":""},{"id":627395077,"identity":"7d439539-db0e-4c5f-8242-85354ce52250","order_by":5,"name":"Dalia Fleifel","email":"","orcid":"","institution":"UNC Chapel Hill","correspondingAuthor":false,"prefix":"","firstName":"Dalia","middleName":"","lastName":"Fleifel","suffix":""},{"id":627395078,"identity":"17300670-5613-494a-a358-1104a3c72a61","order_by":6,"name":"Christina Gutierrez-Ford","email":"","orcid":"","institution":"UNC Lineberger Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Christina","middleName":"","lastName":"Gutierrez-Ford","suffix":""},{"id":627395079,"identity":"bbd3d374-1b07-432d-ad7e-1b5e00eeefa4","order_by":7,"name":"Poorya Benhamie","email":"","orcid":"","institution":"UNC Chapel Hill","correspondingAuthor":false,"prefix":"","firstName":"Poorya","middleName":"","lastName":"Benhamie","suffix":""},{"id":627395080,"identity":"694d796a-1ee8-424e-8925-242ccfc01321","order_by":8,"name":"Santiago Haase","email":"","orcid":"","institution":"UNC Chapel Hill","correspondingAuthor":false,"prefix":"","firstName":"Santiago","middleName":"","lastName":"Haase","suffix":""},{"id":627395081,"identity":"83128103-3fa9-4ef3-b3d9-7eeb9626e080","order_by":9,"name":"Saygin Gulec","email":"","orcid":"","institution":"UNC Chapel Hill","correspondingAuthor":false,"prefix":"","firstName":"Saygin","middleName":"","lastName":"Gulec","suffix":""},{"id":627395082,"identity":"8ae725be-44e1-46d3-92fc-f736ce699996","order_by":10,"name":"Tim Elston","email":"","orcid":"","institution":"UNC Chapel Hill","correspondingAuthor":false,"prefix":"","firstName":"Tim","middleName":"","lastName":"Elston","suffix":""},{"id":627395083,"identity":"ea0f9a36-afd4-4732-892c-eb7c7dcc4167","order_by":11,"name":"Philip Spanheimer","email":"","orcid":"","institution":"UNC Chapel Hill","correspondingAuthor":false,"prefix":"","firstName":"Philip","middleName":"","lastName":"Spanheimer","suffix":""},{"id":627395084,"identity":"8112ce7b-1ca3-4e89-ad68-2bd0fbe2ea0c","order_by":12,"name":"Caroline Tomblin","email":"","orcid":"","institution":"Bioskryb Genomics","correspondingAuthor":false,"prefix":"","firstName":"Caroline","middleName":"","lastName":"Tomblin","suffix":""},{"id":627395085,"identity":"c7d6a6ad-13c8-4a95-ab7c-2c9142c7189c","order_by":13,"name":"Alison Rojas","email":"","orcid":"","institution":"Bioskryb Genomics","correspondingAuthor":false,"prefix":"","firstName":"Alison","middleName":"","lastName":"Rojas","suffix":""},{"id":627395086,"identity":"b073acc1-61b9-41bf-adac-8c7e3b90c1b0","order_by":14,"name":"Tia Tate","email":"","orcid":"","institution":"Bioskryb Genomics","correspondingAuthor":false,"prefix":"","firstName":"Tia","middleName":"","lastName":"Tate","suffix":""},{"id":627395087,"identity":"cf4eb188-22de-4a5b-ae08-2fcd9ff83b97","order_by":15,"name":"Jeremy Purvis","email":"","orcid":"","institution":"University of North Carolina at Chapel Hill","correspondingAuthor":false,"prefix":"","firstName":"Jeremy","middleName":"","lastName":"Purvis","suffix":""},{"id":627395088,"identity":"434dbac4-a10b-4032-a40d-a560edf78ab5","order_by":16,"name":"Jeremy Wang","email":"","orcid":"","institution":"UNC Chapel Hill","correspondingAuthor":false,"prefix":"","firstName":"Jeremy","middleName":"","lastName":"Wang","suffix":""},{"id":627395089,"identity":"1b4a80c1-aca9-481f-99f5-4a8cfc0bb278","order_by":17,"name":"Joeseph Dahl","email":"","orcid":"","institution":"Bioskyrb Genomics","correspondingAuthor":false,"prefix":"","firstName":"Joeseph","middleName":"","lastName":"Dahl","suffix":""},{"id":627395090,"identity":"5aad3561-ac8c-4a7c-bf00-56f5d17ad464","order_by":18,"name":"Samuel Wolff","email":"","orcid":"","institution":"University of North Carolina at Chapel Hill","correspondingAuthor":false,"prefix":"","firstName":"Samuel","middleName":"","lastName":"Wolff","suffix":""},{"id":627395091,"identity":"60ef8252-1650-4547-8674-502fa1e89e8f","order_by":19,"name":"Jeanette Cook","email":"","orcid":"https://orcid.org/0000-0003-0849-7405","institution":"University of North Carolina at Chapel Hill","correspondingAuthor":false,"prefix":"","firstName":"Jeanette","middleName":"","lastName":"Cook","suffix":""},{"id":627395092,"identity":"bb212aac-8c06-4140-8dc1-6550ebc61116","order_by":20,"name":"Molly Murray","email":"","orcid":"","institution":"UNC Chapel Hill","correspondingAuthor":false,"prefix":"","firstName":"Molly","middleName":"","lastName":"Murray","suffix":""},{"id":627395093,"identity":"6f19d5fe-a292-4e8e-a85a-144db423a7c9","order_by":21,"name":"Logan Slade","email":"","orcid":"","institution":"UNC Chapel Hill","correspondingAuthor":false,"prefix":"","firstName":"Logan","middleName":"","lastName":"Slade","suffix":""}],"badges":[],"createdAt":"2026-04-21 02:25:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9477622/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9477622/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108181946,"identity":"000ab9d1-3b18-403e-ba7a-3c959e73cbe7","added_by":"auto","created_at":"2026-04-30 08:59:02","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8106927,"visible":true,"origin":"","legend":"Article File","description":"","filename":"2025FACSFISHFULLRevision1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9477622/v1_covered_ecbf4984-5fdd-4696-919f-fb90a01952ec.pdf"},{"id":108078099,"identity":"6f51bec7-48d8-4fde-ac30-d2a20566531a","added_by":"auto","created_at":"2026-04-29 07:14:38","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2380588,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"2025FACSFISHFULLSUPP.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9477622/v1/92ab6f95673f88e8d5c7b8e3.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Extrachromosomal DNA Gives Cancer a New Evolutionary Pathway","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Extrachromosomal DNA, ecDNA, single cell, single-cell multi-omics sequencing, population heterogeneity, evolution","lastPublishedDoi":"10.21203/rs.3.rs-9477622/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9477622/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"During tumor progression, it has been assumed that individual cells that have acquired advantageous mutations overtake the population. Cancers driven by extrachromosomal DNA (ecDNA) do not follow this paradigm. Instead, these tumors have a spectrum of oncogene copy numbers across cells, and graded ecDNA variation may function as a form of bet-hedging that equips tumors with a broad range of phenotypes. Using imaging, single-cell multiomics, and multiplexed proteomics, we systematically characterized ecDNA levels across thousands of single cells. Higher ecDNA dosage produces proportional changes in transcript abundance, chromatin accessibility, protein levels, cell-cycle progression, and proliferation. Genes amplified on ecDNA exhibit distinct transcriptional scaling regimes that shift when the same genes are reintegrated into chromosomal homogeneous staining regions. When we experimentally disrupted the continuum of ecDNA dosage by sorting cells into low- and high-copy number states, the population rapidly recovered its original, continuous distribution. Our time-course data, live-cell imaging, and stochastic models collectively show that restoring this spectrum is an active, deterministic process rather than the passive outcome of random segregation. Together, these findings position ecDNA-mediated expression as a distinct evolutionary mechanism that endows tumors with rapid, population-level adaptability. These findings offer insight into why ecDNA-driven cancers are among the most aggressive and treatment-resistant.","manuscriptTitle":"Extrachromosomal DNA Gives Cancer a New Evolutionary Pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-29 07:14:34","doi":"10.21203/rs.3.rs-9477622/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-genetics","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"ng","sideBox":"Learn more about [Nature Genetics](http://www.nature.com/ng/)","snPcode":"","submissionUrl":"","title":"Nature Genetics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5cab0d8b-4399-488f-aeba-770f535999c8","owner":[],"postedDate":"April 29th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-05-08T11:54:57+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-05-06T07:45:41+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-05-04T13:26:33+00:00","index":2,"fulltext":"This content is not available."}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":66775419,"name":"Biological sciences/Genetics/Gene regulation"},{"id":66775420,"name":"Biological sciences/Systems biology/Systems analysis"},{"id":66775421,"name":"Biological sciences/Cancer/Oncogenes"},{"id":66775422,"name":"Biological sciences/Genetics/Functional genomics/Gene expression profiling"},{"id":66775423,"name":"Biological sciences/Genetics/Cytogenetics"}],"tags":[],"updatedAt":"2026-04-29T07:14:34+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-29 07:14:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9477622","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9477622","identity":"rs-9477622","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}