Circadian variation in MGMT promoter methylation and expression predicts sensitivity to Temozolomide in glioblastoma

Circadian variation in MGMT promoter methylation and expression predicts sensitivity to Temozolomide in glioblastoma | 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 Research Article Circadian variation in MGMT promoter methylation and expression predicts sensitivity to Temozolomide in glioblastoma Maria F. Gonzalez-Aponte, Yitong Huang, William A. Leidig, Tatiana Simon, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7411649/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Oct, 2025 Read the published version in Journal of Neuro-Oncology → Version 1 posted 19 You are reading this latest preprint version Abstract Purpose Recent studies show that glioblastoma (GBM) is more sensitive to Temozolomide (TMZ) in the morning. In cells, inhibiting O6-Methylguanine-DNA-Methyltransferase (MGMT) abolished time-dependent TMZ efficacy, suggesting that circadian regulation of this DNA repair enzyme underlies daily TMZ sensitivity. Here, we tested the hypotheses that MGMT-promoter methylation and protein abundance vary with time-of-day in GBM, resulting in daily rhythms in TMZ efficacy. Methods We assessed daily rhythms in Mgmt -promoter methylation in GBM in vitro and retrospectively analyzed MGMT methylation status in human GBM biopsies collected at different times of day. Next, we measured MGMT and BMAL1 protein abundances in GBM cells collected at 4-hour intervals. To understand the therapeutic implications of circadian variations in MGMT, we incorporated its daily rhythms into an in vitro mathematical model capturing interactions between MGMT, TMZ, and GBM DNA. Results We found daily rhythms in Mgmt -promoter methylation and protein levels in GBM in vitro , and in patient biopsies peaking at midday. Further, MGMT protein levels peaked at CT4, corresponding to the time of maximal TMZ efficacy in vitro . When we incorporated cell-intrinsic circadian rhythms in MGMT protein into a mathematical model for GBM chemotherapy, we found that dosing when daily MGMT levels peaked and began to decline produced maximum DNA damage. Conclusion Our findings suggest that the likelihood of diagnosis of MGMT-promoter methylation varies with time of biopsy in GBM. Furthermore, we predict that efforts to deliver TMZ after the daily peak of MGMT activity, with exact time being dose-dependent, will significantly enhance its therapeutic efficacy. GBM TMZ MGMT expression and promoter methylation Chronotherapy Chronodiagnosis Full Text Additional Declarations Competing interest reported. Dr. Olivia Walch is the CEO and founder of Arcascope, a circadian rhythms software company. Dr. Albert Kim is a consultant for Monteris Medical and has research funding from Stryker to study a dural substitute, both of which have no direct relation to this study. All other authors declare no competing interests. Supplementary Files SupplementaryMaterials.docx Cite Share Download PDF Status: Published Journal Publication published 22 Oct, 2025 Read the published version in Journal of Neuro-Oncology → Version 1 posted Editorial decision: Revision requested 12 Sep, 2025 Reviews received at journal 09 Sep, 2025 Reviews received at journal 06 Sep, 2025 Reviews received at journal 04 Sep, 2025 Reviews received at journal 04 Sep, 2025 Reviews received at journal 04 Sep, 2025 Reviews received at journal 03 Sep, 2025 Reviewers agreed at journal 31 Aug, 2025 Reviewers agreed at journal 30 Aug, 2025 Reviewers agreed at journal 27 Aug, 2025 Reviewers agreed at journal 25 Aug, 2025 Reviewers agreed at journal 25 Aug, 2025 Reviewers agreed at journal 25 Aug, 2025 Reviewers agreed at journal 25 Aug, 2025 Reviewers agreed at journal 25 Aug, 2025 Reviewers invited by journal 20 Aug, 2025 Editor assigned by journal 20 Aug, 2025 Submission checks completed at journal 20 Aug, 2025 First submitted to journal 19 Aug, 2025 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-7411649","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":506461466,"identity":"e4ef417d-708b-4ac4-9c1d-7809aa50051f","order_by":0,"name":"Maria F. 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All other authors declare no competing interests.","formattedTitle":"Circadian variation in MGMT promoter methylation and expression predicts sensitivity to Temozolomide in glioblastoma","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-neuro-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"neon","sideBox":"Learn more about [Journal of Neuro-Oncology](https://www.springer.com/journal/11060)","snPcode":"11060","submissionUrl":"https://submission.nature.com/new-submission/11060/3","title":"Journal of Neuro-Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"GBM, TMZ, MGMT expression and promoter methylation, Chronotherapy, Chronodiagnosis","lastPublishedDoi":"10.21203/rs.3.rs-7411649/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7411649/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e\u003cp\u003eRecent studies show that glioblastoma (GBM) is more sensitive to Temozolomide (TMZ) in the morning. In cells, inhibiting O6-Methylguanine-DNA-Methyltransferase (MGMT) abolished time-dependent TMZ efficacy, suggesting that circadian regulation of this DNA repair enzyme underlies daily TMZ sensitivity. Here, we tested the hypotheses that MGMT-promoter methylation and protein abundance vary with time-of-day in GBM, resulting in daily rhythms in TMZ efficacy.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eWe assessed daily rhythms in \u003cem\u003eMgmt\u003c/em\u003e-promoter methylation in GBM \u003cem\u003ein vitro\u003c/em\u003e and retrospectively analyzed MGMT methylation status in human GBM biopsies collected at different times of day. Next, we measured MGMT and BMAL1 protein abundances in GBM cells collected at 4-hour intervals. To understand the therapeutic implications of circadian variations in MGMT, we incorporated its daily rhythms into an \u003cem\u003ein vitro\u003c/em\u003e mathematical model capturing interactions between MGMT, TMZ, and GBM DNA.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eWe found daily rhythms in \u003cem\u003eMgmt\u003c/em\u003e-promoter methylation and protein levels in GBM \u003cem\u003ein vitro\u003c/em\u003e, and in patient biopsies peaking at midday. Further, MGMT protein levels peaked at CT4, corresponding to the time of maximal TMZ efficacy \u003cem\u003ein vitro\u003c/em\u003e. When we incorporated cell-intrinsic circadian rhythms in MGMT protein into a mathematical model for GBM chemotherapy, we found that dosing when daily MGMT levels peaked and began to decline produced maximum DNA damage.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eOur findings suggest that the likelihood of diagnosis of MGMT-promoter methylation varies with time of biopsy in GBM. Furthermore, we predict that efforts to deliver TMZ after the daily peak of MGMT activity, with exact time being dose-dependent, will significantly enhance its therapeutic efficacy.\u003c/p\u003e","manuscriptTitle":"Circadian variation in MGMT promoter methylation and expression predicts sensitivity to Temozolomide in glioblastoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-29 20:34:27","doi":"10.21203/rs.3.rs-7411649/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-12T11:55:18+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-10T02:21:05+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-07T02:54:48+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-04T21:30:05+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-04T21:05:20+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-04T16:48:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-03T21:08:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"206540629849314550434994736444033316150","date":"2025-08-31T16:23:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"91778760083144532971337535941811202334","date":"2025-08-30T19:35:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"298622447699942890929535630552137345322","date":"2025-08-27T12:17:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"153465454995544053735943055133794553285","date":"2025-08-25T18:15:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"279521492810074125355267847255471428822","date":"2025-08-25T16:31:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"325637673405600978688858634616313653421","date":"2025-08-25T15:54:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"260286633977965161908322514270269942650","date":"2025-08-25T14:57:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"128220135454577717801756550249470818390","date":"2025-08-25T13:42:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-20T15:31:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-20T13:34:50+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-20T13:34:29+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Neuro-Oncology","date":"2025-08-19T20:30:26+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-neuro-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"neon","sideBox":"Learn more about [Journal of Neuro-Oncology](https://www.springer.com/journal/11060)","snPcode":"11060","submissionUrl":"https://submission.nature.com/new-submission/11060/3","title":"Journal of Neuro-Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"39316cd7-f001-4e55-a097-b2938eb6ebde","owner":[],"postedDate":"August 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-27T16:27:38+00:00","versionOfRecord":{"articleIdentity":"rs-7411649","link":"https://doi.org/10.1007/s11060-025-05242-3","journal":{"identity":"journal-of-neuro-oncology","isVorOnly":false,"title":"Journal of Neuro-Oncology"},"publishedOn":"2025-10-22 16:17:11","publishedOnDateReadable":"October 22nd, 2025"},"versionCreatedAt":"2025-08-29 20:34:27","video":"","vorDoi":"10.1007/s11060-025-05242-3","vorDoiUrl":"https://doi.org/10.1007/s11060-025-05242-3","workflowStages":[]},"version":"v1","identity":"rs-7411649","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7411649","identity":"rs-7411649","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}