Bortezomib Enhances CD70 CAR-T Cell Therapy in Sunitinib Resistant Renal Cell Carcinoma by Inducing Mitochondrial DNA Mediated cGAS-STING Activation

Bortezomib Enhances CD70 CAR-T Cell Therapy in Sunitinib Resistant Renal Cell Carcinoma by Inducing Mitochondrial DNA Mediated cGAS-STING Activation | 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 Bortezomib Enhances CD70 CAR-T Cell Therapy in Sunitinib Resistant Renal Cell Carcinoma by Inducing Mitochondrial DNA Mediated cGAS-STING Activation Bo Zhai, ZhaoRong Wu, Hongye Wang, Xiaomin Huang, Binbin Xia, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8078134/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 Chimeric antigen receptor (CAR) T-cell therapy has shown remarkable efficacy in hematologic malignancies, yet its application to solid tumors remains limited by poor tumor infiltration, an immunosuppressive microenvironment, and insufficient antigen presentation. Here, we identify a mechanism by which the proteasome inhibitor bortezomib (BTZ) enhances CAR-T efficacy in renal cell carcinoma (RCC), particularly in sunitinib-resistant tumors characterized by immune exclusion and metabolic adaptation. Screening of an FDA-approved compound library revealed BTZ as a potent sensitizer that restores CAR-T cytotoxicity against resistant RCC cells. Mechanistically, BTZ induces mitochondrial and endoplasmic reticulum stress, leading to increased reactive oxygen species and voltage-dependent anion channel (VDAC)-dependent release of mitochondrial DNA (mtDNA) via extracellular vesicles. Tumor-derived mtDNA is internalized by dendritic cells, activating the cGAS-STING signaling axis, enhancing glycolytic metabolism, and promoting type I interferon responses and antigen presentation. This, in turn, augments CAR-T cell persistence, effector cytokine secretion, and tumor infiltration. Pharmacological inhibition of mtDNA release or glycolysis abrogates these effects, confirming the essential role of mtDNA-mediated innate immune activation. In multiple RCC models, including patient-derived xenografts and orthotopic tumors, BTZ synergized with CD70 CAR-T therapy to induce robust tumor regression, durable immune memory, and improved survival without overt toxicity. Collectively, these findings define a previously unrecognized immunometabolic mechanism by which proteasome inhibition transforms the tumor microenvironment and potentiate cellular immunotherapy, offering a clinically actionable strategy to overcome therapeutic resistance in solid tumors. Biological sciences/Cancer/Cancer therapy/Cancer immunotherapy Biological sciences/Cancer/Urological cancer/Renal cancer/Renal cell carcinoma Renal Cell Carcinoma CD70 CAR-T Dendritic Cell Mitochondrial DNA 80 cGAS-STING Pathway Full Text Additional Declarations There is NO Competing Interest. Supplementary Files TableS3.xlsx Table S3 TableS2.xlsx Table S2 TableS1.xlsx Table S1 figureS3.pdf Figure S3 figureS1.pdf Figure S1 figureS2.pdf Figure S2 figureS4.pdf Figure S4 figureS5.pdf Figure S5 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-8078134","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":547749974,"identity":"24aefa34-c039-413f-9134-e0061acd4eb2","order_by":0,"name":"Bo Zhai","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYDACCQaGw0AqgY29geEAiVp4DpCghRmkhUEigUh3yc9ufni4oKIuj0/yOZBRwyDPL0bAMsY5xwwOzzhzuJhNOgfIOMZgOHM2AeuYJRIMDvO2HUhsk85hOMzDxpBgcJuAFjaJ9A9ALXWJbZLHHxzm+UeEFh6JHJAtzIltEgwgBhFaJCRyCkB+SWzjAentkyDsF/kZ6Zs/A0MscX778cefeb7ZyPNLE9CCYStpykfBKBgFo2AUYAcAMH9DACK777MAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-4337-7126","institution":"Shanghai East Hospital, Tongji University","correspondingAuthor":true,"prefix":"","firstName":"Bo","middleName":"","lastName":"Zhai","suffix":""},{"id":547749975,"identity":"59a77450-9482-47c9-8d3b-a46cff4ca74e","order_by":1,"name":"ZhaoRong Wu","email":"","orcid":"","institution":"Renji Hospital","correspondingAuthor":false,"prefix":"","firstName":"ZhaoRong","middleName":"","lastName":"Wu","suffix":""},{"id":547749976,"identity":"37b2a9bc-0375-4c22-b7fb-c783e7179adb","order_by":2,"name":"Hongye Wang","email":"","orcid":"https://orcid.org/0000-0002-6467-0316","institution":"Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hongye","middleName":"","lastName":"Wang","suffix":""},{"id":547749977,"identity":"ac6743ff-2a29-467c-a9c5-f2f8f1ae177c","order_by":3,"name":"Xiaomin Huang","email":"","orcid":"","institution":"Eye Institute and Department of Ophthalmology, Eye \u0026 ENT Hospital, Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Xiaomin","middleName":"","lastName":"Huang","suffix":""},{"id":547749978,"identity":"7bf4cf75-7e1f-4be9-be11-942183bd8d11","order_by":4,"name":"Binbin Xia","email":"","orcid":"","institution":"Central South University, Changsha, China","correspondingAuthor":false,"prefix":"","firstName":"Binbin","middleName":"","lastName":"Xia","suffix":""},{"id":547749979,"identity":"5e242312-39d1-4cba-89c3-3bdc994ac8a5","order_by":5,"name":"Linke Bian","email":"","orcid":"","institution":"Renji Hospital, Shanghai Jiao Tong University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Linke","middleName":"","lastName":"Bian","suffix":""},{"id":547749980,"identity":"385e56c9-dd07-4384-9f7e-cd30de436476","order_by":6,"name":"Zhigang Zheng","email":"","orcid":"","institution":"Department of Biliary-Pancreatic Surgery Renji Hospital Shanghai Jiao Tong University School of Medicine Shanghai China.","correspondingAuthor":false,"prefix":"","firstName":"Zhigang","middleName":"","lastName":"Zheng","suffix":""},{"id":547749981,"identity":"ec679bc0-cefd-42d7-b9db-d99f5fd921c6","order_by":7,"name":"Yihao Zhang","email":"","orcid":"","institution":"Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Yihao","middleName":"","lastName":"Zhang","suffix":""},{"id":547749982,"identity":"3e2008b9-6182-4859-b425-8cc1532d3307","order_by":8,"name":"Yuan Tian","email":"","orcid":"","institution":"Institute of Molecular Medicine and Shanghai Key Laboratory for Nucleic Acid Chemistry and Nanomedicine, Renji Hospital, School of Medicine Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Tian","suffix":""},{"id":547749983,"identity":"c2d5ba55-bad5-46c6-b440-e3b5ff6e3359","order_by":9,"name":"Jiufei Zhu","email":"","orcid":"","institution":"Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Jiufei","middleName":"","lastName":"Zhu","suffix":""},{"id":547749984,"identity":"cfa1bc8e-0ef5-46f8-85da-12af48ecabbc","order_by":10,"name":"Dan Cui","email":"","orcid":"","institution":"Renji Hospital, Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Dan","middleName":"","lastName":"Cui","suffix":""},{"id":547749985,"identity":"aa435623-b5b4-4ce1-87e0-3b461676939e","order_by":11,"name":"Ying Liu","email":"","orcid":"","institution":"Shanghai East Hospital, Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Liu","suffix":""},{"id":547749986,"identity":"54604270-aad8-4511-969a-de68f2ab935a","order_by":12,"name":"Jiachao Xiong","email":"","orcid":"","institution":"Shanghai East Hospital, Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Jiachao","middleName":"","lastName":"Xiong","suffix":""},{"id":547749987,"identity":"3e3d82b9-4153-4fad-831c-cb605c65ff9a","order_by":13,"name":"Haigang Geng","email":"","orcid":"","institution":"Department of Gastrointestinal Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.","correspondingAuthor":false,"prefix":"","firstName":"Haigang","middleName":"","lastName":"Geng","suffix":""},{"id":547749988,"identity":"d718a77c-7b62-4794-9346-75d8f8d2b7c1","order_by":14,"name":"Yuan Peng","email":"","orcid":"","institution":"College of Life Sciences, Xiamen University.","correspondingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Peng","suffix":""},{"id":547749989,"identity":"9853ffe4-2524-4eda-9c01-42422449f613","order_by":15,"name":"Jingtong Zhao","email":"","orcid":"","institution":"Shanghai Chest Hospital, Department of Oncology, Shanghai, People's Republic of China.","correspondingAuthor":false,"prefix":"","firstName":"Jingtong","middleName":"","lastName":"Zhao","suffix":""},{"id":547749990,"identity":"117101de-858b-4a8e-b4f1-e9cd64b66c94","order_by":16,"name":"Xiaorong Huang","email":"","orcid":"","institution":"Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiaorong","middleName":"","lastName":"Huang","suffix":""},{"id":547749991,"identity":"407f266f-d5e9-490f-be3f-6f41275593da","order_by":17,"name":"Yanfang Zheng","email":"","orcid":"","institution":"Fujian University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yanfang","middleName":"","lastName":"Zheng","suffix":""}],"badges":[],"createdAt":"2025-11-10 14:32:03","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8078134/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8078134/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":97135993,"identity":"f224350d-3a8c-4c25-b53b-40fd57e60ec0","added_by":"auto","created_at":"2025-12-01 09:54:55","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4509882,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8078134/v1_covered_68274260-166e-4a81-9aff-6cc3e718814f.pdf"},{"id":96735260,"identity":"6a9c8693-1773-4835-9475-02464623d176","added_by":"auto","created_at":"2025-11-25 14:12:01","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10006,"visible":true,"origin":"","legend":"Table S3","description":"","filename":"TableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8078134/v1/70f3439d7c01c71961a969ca.xlsx"},{"id":96735261,"identity":"a500ed82-372a-4d08-8983-408e57eeb5b8","added_by":"auto","created_at":"2025-11-25 14:12:01","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":71271,"visible":true,"origin":"","legend":"Table S2","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8078134/v1/47cfc37da746da5cbe36b4c0.xlsx"},{"id":96913746,"identity":"2fa8f6da-6fd4-4254-b3ac-96feca22bc26","added_by":"auto","created_at":"2025-11-27 14:04:10","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":611892,"visible":true,"origin":"","legend":"Table S1","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8078134/v1/d1933bd35ab03ddd54ecb304.xlsx"},{"id":96735266,"identity":"e51d2a4d-f39f-4acd-9ed2-0d582c390f87","added_by":"auto","created_at":"2025-11-25 14:12:01","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":734019,"visible":true,"origin":"","legend":"Figure S3","description":"","filename":"figureS3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8078134/v1/d46399825ee27d915908f403.pdf"},{"id":96913880,"identity":"6d01f5eb-8160-4beb-aa6c-805378e7ff7e","added_by":"auto","created_at":"2025-11-27 14:04:39","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":973930,"visible":true,"origin":"","legend":"Figure S1","description":"","filename":"figureS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8078134/v1/ffd48a874261bc4b4c57bcde.pdf"},{"id":96735262,"identity":"2135a747-9605-4777-a982-3d8b38245bd7","added_by":"auto","created_at":"2025-11-25 14:12:01","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":252108,"visible":true,"origin":"","legend":"Figure S2","description":"","filename":"figureS2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8078134/v1/a849142e72aa0026800f23e5.pdf"},{"id":96735263,"identity":"b8e0a706-8ccc-428e-a185-310010c07d18","added_by":"auto","created_at":"2025-11-25 14:12:01","extension":"pdf","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":389591,"visible":true,"origin":"","legend":"Figure S4","description":"","filename":"figureS4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8078134/v1/3311b763e5ca43fd055467aa.pdf"},{"id":96914556,"identity":"61c7fa59-0e9d-4b04-a2fc-97b3540e41fb","added_by":"auto","created_at":"2025-11-27 14:06:03","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":213727,"visible":true,"origin":"","legend":"Figure S5","description":"","filename":"figureS5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8078134/v1/a1ff0c1837851d46a5bd3fe4.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Bortezomib Enhances CD70 CAR-T Cell Therapy in Sunitinib Resistant Renal Cell Carcinoma by Inducing Mitochondrial DNA Mediated cGAS-STING Activation","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":"Renal Cell Carcinoma, CD70 CAR-T, Dendritic Cell, Mitochondrial DNA, 80 cGAS-STING Pathway","lastPublishedDoi":"10.21203/rs.3.rs-8078134/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8078134/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Chimeric antigen receptor (CAR) T-cell therapy has shown remarkable efficacy in hematologic malignancies, yet its application to solid tumors remains limited by poor tumor infiltration, an immunosuppressive microenvironment, and insufficient antigen presentation. Here, we identify a mechanism by which the proteasome inhibitor bortezomib (BTZ) enhances CAR-T efficacy in renal cell carcinoma (RCC), particularly in sunitinib-resistant tumors characterized by immune exclusion and metabolic adaptation. Screening of an FDA-approved compound library revealed BTZ as a potent sensitizer that restores CAR-T cytotoxicity against resistant RCC cells. Mechanistically, BTZ induces mitochondrial and endoplasmic reticulum stress, leading to increased reactive oxygen species and voltage-dependent anion channel (VDAC)-dependent release of mitochondrial DNA (mtDNA) via extracellular vesicles. Tumor-derived mtDNA is internalized by dendritic cells, activating the cGAS-STING signaling axis, enhancing glycolytic metabolism, and promoting type I interferon responses and antigen presentation. This, in turn, augments CAR-T cell persistence, effector cytokine secretion, and tumor infiltration. Pharmacological inhibition of mtDNA release or glycolysis abrogates these effects, confirming the essential role of mtDNA-mediated innate immune activation. In multiple RCC models, including patient-derived xenografts and orthotopic tumors, BTZ synergized with CD70 CAR-T therapy to induce robust tumor regression, durable immune memory, and improved survival without overt toxicity. Collectively, these findings define a previously unrecognized immunometabolic mechanism by which proteasome inhibition transforms the tumor microenvironment and potentiate cellular immunotherapy, offering a clinically actionable strategy to overcome therapeutic resistance in solid tumors.","manuscriptTitle":"Bortezomib Enhances CD70 CAR-T Cell Therapy in Sunitinib Resistant Renal Cell Carcinoma by Inducing Mitochondrial DNA Mediated cGAS-STING Activation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-25 14:11:56","doi":"10.21203/rs.3.rs-8078134/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a0fe66f8-da4e-4663-bc1c-61ad3e4bb03e","owner":[],"postedDate":"November 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":58281625,"name":"Biological sciences/Cancer/Cancer therapy/Cancer immunotherapy"},{"id":58281626,"name":"Biological sciences/Cancer/Urological cancer/Renal cancer/Renal cell carcinoma"}],"tags":[],"updatedAt":"2025-11-25T14:11:56+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-25 14:11:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8078134","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8078134","identity":"rs-8078134","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}