Reversal of Acquired Resistance to EGFR–TKI in T790M-negative Patients With Non–small-cell Lung Cancer Using Anlotinib

Reversal of Acquired Resistance to EGFR–TKI in T790M-negative Patients With Non–small-cell Lung Cancer Using Anlotinib | 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 Reversal of Acquired Resistance to EGFR–TKI in T790M-negative Patients With Non–small-cell Lung Cancer Using Anlotinib Chen Zhang, Honggang Cao, Shidai Jin, Wen Gao, Chenjun Huang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-445996/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Treatment options for epidermal growth factor receptor (EGFR) T790M-negative patients with non–small-cell lung cancer (NSCLC) and acquired resistance (AR) to EGFR–tyrosine kinase inhibitor (EGFR–TKI) are limited. The efficacy of EGFR–TKI and anti-angiogenic drug combination therapy in these patients is known. We investigated the effectiveness of EGFR–TKI+anlotinib combination therapy in patients with T790M-negative NSCLC. Method: We evaluated the antitumor effects of gefitinib combined with anlotinib in gefitinib-resistant lung adenocarcinoma cells. We also investigated the treatment effect and absence of adverse events of EGFR–TKI+anlotinib therapy in 22 T790M-negative patients after EGFR–TKI treatment failure between January 2018 and August 2020. Results: Anlotinib reversed gefitinib resistance in the gefitinib-resistant cell line, PC9/GR, by enhancing anti-proliferative and pro-apoptotic effects of gefitinib. The gefitinib+anlotinib treatment exerted a synergistic antitumor effect by downregulating the activation of VEGFR2 and downstream effectors, Akt and ERK. The EGFR–TKI+anlotinib therapy exhibited an objective response rate of 18.2% and a disease control rate of 95.5%. The median progression-free survival (PFS) was 11.53 ± 1.94 months, whereas the median overall survival was not reached. The median PFS was longer in patients exhibiting gradual progression (13.30 ± 1.69 months) than in patients with dramatic progression (8.60 ± 5.39 months, p = 0.041). One Grade 3 adverse event was noted (diarrhea, n = 2, 9.1%), and Grade 4 or 5 adverse events were absent. Conclusion: EGFR–TKI combined with anlotinib demonstrated powerful antitumor activity in vitro and excellent treatment effect in T790M-negative NSCLC patients after AR. Cancer Biology Oncology Non-small-cell lung cancer EGFR VEGFR Gefitinib Anlotinib Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Full Text Due to technical limitations, full-text HTML conversion of this manuscript could not be completed. However, the manuscript can be downloaded and accessed as a PDF. Supplementary Files FigureS1.jpg Supplementary Figure 1. Effect of gefitinib and anlotinib on key signal transduction proteins in PC9/GR cells. PC9/GR cells were treated with gefitinib, anlotinib, or gefinib plus anlotinib for 48 h, Western blot analysis was performed to detect the expression of key signal transduction proteins. Significance levels determined by the t test are indicated (ns: not significant compared with the control group. *P < 0.05, **P < 0.01 compared with the control group. #P < 0.05, ###P < 0.01 compared with the gefitinib group). FigureS2.jpg Supplementary Figure 2. Diagram of the possible mechanism of reversing gefitinib resistance by anlotinib. Gefitinib and anlotinib synergistically promoted PC9/GR cells proliferation and inhibited PC9/GR cells apoptosis through the inhibition of EGFR phosphorylation, VEGFR2 phosphorylation and the downregulation of ERK and Akt signaling. Cite Share Download PDF Status: Posted 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-445996","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":23148811,"identity":"b36639f6-9ca7-4145-94c8-705230bdc2b4","order_by":0,"name":"Chen Zhang","email":"","orcid":"","institution":"Jiangsu Province Hospital and Nanjing Medical University First Affiliated Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chen","middleName":"","lastName":"Zhang","suffix":""},{"id":23148812,"identity":"ee49d99d-eab7-4875-b013-654c45fb31d4","order_by":1,"name":"Honggang Cao","email":"","orcid":"","institution":"Jiangsu Province Hospital and Nanjing Medical University First Affiliated Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Honggang","middleName":"","lastName":"Cao","suffix":""},{"id":23148813,"identity":"35515dbe-e53b-4287-af53-5eb5a8987e24","order_by":2,"name":"Shidai Jin","email":"","orcid":"","institution":"Jiangsu Province Hospital and Nanjing Medical University First Affiliated Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shidai","middleName":"","lastName":"Jin","suffix":""},{"id":23148814,"identity":"62d24861-4ba8-4f3e-8b55-0b0942638791","order_by":3,"name":"Wen Gao","email":"","orcid":"","institution":"Jiangsu Province Hospital and Nanjing Medical University First Affiliated Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wen","middleName":"","lastName":"Gao","suffix":""},{"id":23148815,"identity":"e6155e45-929c-43c0-80e0-f71394128bfc","order_by":4,"name":"Chenjun Huang","email":"","orcid":"","institution":"Jiangsu Province Hospital and Nanjing Medical University First Affiliated Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chenjun","middleName":"","lastName":"Huang","suffix":""},{"id":23148816,"identity":"d7fe7904-fdb1-49ae-a3ec-e09dc12aae1a","order_by":5,"name":"Renhua Guo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAu0lEQVRIiWNgGAWjYDACCRBRwcAM5vAQr+UMyVoY26AcorSYz26/JvFx3h123RkJjA/etjHImxPSInPnTJnkzG3PmM1uJDAbzm1jMNzZQMhdEjlp0rzbDoO0sEnztjEkGBwgSsscsBb230RqST8mzdsAsYWZWFuYLWccA2o587BZcs45CcMNRNjy8MaHmsPJZseTD354U2YjT9AWYFwYgMhkYOw0MEBjlhBgfwAi7YhROgpGwSgYBSMUAAAxWjw32lbxgQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-4475-8617","institution":"Jiangsu Province Hospital and Nanjing Medical University First Affiliated Hospital","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Renhua","middleName":"","lastName":"Guo","suffix":""}],"badges":[],"createdAt":"2021-04-21 10:06:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-445996/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-445996/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":8451833,"identity":"979cd205-101a-4813-9c3f-edd49e30272c","added_by":"auto","created_at":"2021-04-26 15:01:59","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":79752,"visible":true,"origin":"","legend":"Anlotinib resensitized gefitinib-resistant PC9/GR cells to gefitinib. Parental lung adenocarcinoma PC9 cells and gefitinib-resistant PC9/GR cells were treated with indicated concentrations of gefitinib (A), anlotinib (B), or gefinib plus anlotinib (C) for 72 h. Cell viability was measured by the CCK8 assay. D, The combination index of gefitinib plus anlotinib was calculated using CompuSyn software. E, Half maximal inhibitory concentration (IC50) and combination index (CI) values of gefitinib alone or combined with anlotinib in PC9/GR cells.","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-445996/v1/3e70a316040354d09a05ebaa.jpg"},{"id":8451834,"identity":"3a947da6-8744-42b8-a6ac-9e8435f4238b","added_by":"auto","created_at":"2021-04-26 15:01:59","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":133053,"visible":true,"origin":"","legend":"Treatment with gefitinib plus anlotinib synergistically inhibited proliferation of PC9/GR cells. A and B, EdU proliferation assays were performed 48 h after treatment with gefitinib, anlotinib, and gefinib plus anlotinib. C and D, Colony-formation assays were performed to analyze the colony-formation efficiency of PC9/GR cells in the different treatment groups. Results exhibited as mean ± SD of three independent experiments performed in triplicates. Significance levels determined using the t test are indicated (ns: not significant compared with the control group. *P \u003c 0.01, **P \u003c 0.001 compared with the control group. #P \u003c 0.01, ##P \u003c 0.001 compared with the gefitinib group).","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-445996/v1/3dbfce6e4c5393f250608fcd.jpg"},{"id":8452048,"identity":"01822052-d940-4f0d-ac82-2fa4d1753b7f","added_by":"auto","created_at":"2021-04-26 15:04:59","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":81791,"visible":true,"origin":"","legend":"Treatment with gefitinib plus anlotinib synergistically promoted apoptosis of PC9/GR cells. A and C, After treatment with gefitinib, anlotinib, and gefinib plus anlotinib for 48 h, annexin V-FITC/PI staining was used to determine the apoptosis rate of PC9/GR cells. B and D, The apoptotic marker protein, cleaved-caspase 3, was analyzed by western blot analysis. Significance levels determined by the t test are indicated (ns: not significant compared with the control group. *P \u003c 0.05, **P \u003c 0.01 compared with the control group. #P \u003c 0.05, ##P \u003c 0.01 compared with the gefitinib group).","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-445996/v1/6215aa0833318beb5b93a089.jpg"},{"id":8451848,"identity":"a11e216f-c313-45f0-bb11-f895d429a1e1","added_by":"auto","created_at":"2021-04-26 15:01:59","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":84315,"visible":true,"origin":"","legend":"Gefitinib and anlotinib synergistically inhibited the Akt and ERK signaling pathways. After 48-h treatment with gefitinib, anlotinib, and gefinib plus anlotinib, the key proteins involved in the EGFR and VEGFR2 downstream signaling pathways such as VEGFR2, p-VEGFR2, EGFR, p-EGFR, Akt, p-Akt, ERK1/2, and p-ERK1/2, were analyzed by Western blot analysis.","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-445996/v1/0422f79a87f0641bdd8abf56.jpg"},{"id":8452049,"identity":"bc8d564b-503f-4334-8ef3-acef30761bc3","added_by":"auto","created_at":"2021-04-26 15:04:59","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":91472,"visible":true,"origin":"","legend":"Anlotinib reversed the EGFR–TKI resistance in patients. A, The maximal tumor shrinkage of patients receiving EGFR–TKI and anlotinib therapy. B, The changes of tumor burden in individual patients after combination therapy. C, Kaplan–Meier PFS curve. D, Kaplan-–Meier PFS curves stratification based on EGFR mutation types. E, Kaplan–Meier PFS curves stratification based on prior EGFR–TKI failure modes.","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-445996/v1/bc7eb61d80aff4cc67c75d88.jpg"},{"id":13624764,"identity":"caa950c2-fdad-47e8-ac0a-6e0a7cb1b985","added_by":"auto","created_at":"2021-09-17 07:23:52","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1398634,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-445996/v1_covered.pdf"},{"id":9904127,"identity":"40477447-59f8-4142-ab31-a69fdfb665ea","added_by":"auto","created_at":"2021-06-02 18:59:54","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1395099,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-445996/v1_covered.pdf"},{"id":8452433,"identity":"cb12ccb4-fe9e-4217-8a33-ae5f58b3d021","added_by":"auto","created_at":"2021-04-26 15:08:05","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1099631,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-445996/v1_stamped.pdf"},{"id":8451531,"identity":"71ecefbe-f71e-42ee-8642-6f900ae8780b","added_by":"auto","created_at":"2021-04-26 14:58:59","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":66805,"visible":true,"origin":"","legend":"Supplementary Figure 1. Effect of gefitinib and anlotinib on key signal transduction proteins in PC9/GR cells. PC9/GR cells were treated with gefitinib, anlotinib, or gefinib plus anlotinib for 48 h, Western blot analysis was performed to detect the expression of key signal transduction proteins. Significance levels determined by the t test are indicated (ns: not significant compared with the control group. *P \u003c 0.05, **P \u003c 0.01 compared with the control group. #P \u003c 0.05, ###P \u003c 0.01 compared with the gefitinib group).","description":"","filename":"FigureS1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-445996/v1/b2c89fa97771e2dfe201a42e.jpg"},{"id":8451532,"identity":"b19e9334-f534-48d6-9ae9-0809ea66bd16","added_by":"auto","created_at":"2021-04-26 14:58:59","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":29300,"visible":true,"origin":"","legend":"Supplementary Figure 2. Diagram of the possible mechanism of reversing gefitinib resistance by anlotinib. Gefitinib and anlotinib synergistically promoted PC9/GR cells proliferation and inhibited PC9/GR cells apoptosis through the inhibition of EGFR phosphorylation, VEGFR2 phosphorylation and the downregulation of ERK and Akt signaling.","description":"","filename":"FigureS2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-445996/v1/487d12511d7ec2f4faa43a60.jpg"}],"financialInterests":"","formattedTitle":"\u003cp\u003eReversal of Acquired Resistance to EGFR–TKI in T790M-negative Patients With Non–small-cell Lung Cancer Using Anlotinib\u003c/p\u003e","fulltext":[{"header":"Full Text","content":"\u003cp\u003eDue to technical limitations, full-text HTML conversion of this manuscript could not be completed. However, the manuscript can be downloaded and accessed as a PDF.\u003c/p\u003e "}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Non-small-cell lung cancer, EGFR, VEGFR, Gefitinib, Anlotinib","lastPublishedDoi":"10.21203/rs.3.rs-445996/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-445996/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eTreatment options for epidermal growth factor receptor (EGFR) T790M-negative patients with non–small-cell lung cancer (NSCLC) and acquired resistance (AR) to EGFR–tyrosine kinase inhibitor (EGFR–TKI) are limited. The efficacy of EGFR–TKI and anti-angiogenic drug combination therapy in these patients is known. We investigated the effectiveness of EGFR–TKI+anlotinib combination therapy in patients with T790M-negative NSCLC. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethod:\u003c/strong\u003e We evaluated the antitumor effects of gefitinib combined with anlotinib in gefitinib-resistant lung adenocarcinoma cells. We also investigated the treatment effect and absence of adverse events of EGFR–TKI+anlotinib therapy in 22 T790M-negative patients after EGFR–TKI treatment failure between January 2018 and August 2020. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Anlotinib reversed gefitinib resistance in the gefitinib-resistant cell line, PC9/GR, by enhancing anti-proliferative and pro-apoptotic effects of gefitinib. The gefitinib+anlotinib treatment exerted a synergistic antitumor effect by downregulating the activation of VEGFR2 and downstream effectors, Akt and ERK. The EGFR–TKI+anlotinib therapy exhibited an objective response rate of 18.2% and a disease control rate of 95.5%. The median progression-free survival (PFS) was 11.53 ± 1.94 months, whereas the median overall survival was not reached. The median PFS was longer in patients exhibiting gradual progression (13.30 ± 1.69 months) than in patients with dramatic progression (8.60 ± 5.39 months, p = 0.041). One Grade 3 adverse event was noted (diarrhea, n = 2, 9.1%), and Grade 4 or 5 adverse events were absent.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e EGFR–TKI combined with anlotinib demonstrated powerful antitumor activity in vitro and excellent treatment effect in T790M-negative NSCLC patients after AR.\u003c/p\u003e","manuscriptTitle":"Reversal of Acquired Resistance to EGFR–TKI in T790M-negative Patients With Non–small-cell Lung Cancer Using Anlotinib","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-04-26 14:58:57","doi":"10.21203/rs.3.rs-445996/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"407688bd-815e-43ca-a16f-bda91f7d72b3","owner":[],"postedDate":"April 26th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":3901764,"name":"Cancer Biology"},{"id":3901765,"name":"Oncology"}],"tags":[],"updatedAt":"2021-06-02T18:59:39+00:00","versionOfRecord":[],"versionCreatedAt":"2021-04-26 14:58:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-445996","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-445996","identity":"rs-445996","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}