{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,10,17]],"date-time":"2026-10-17T20:08:10Z","timestamp":1792267690260,"version":"4.4.0"},"posted":{"date-parts":[[2024,1,20]]},"group-title":"Cancer Biology","reference-count":23,"publisher":"openRxiv","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2024,1,19]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:sec>\n                  <jats:title>Purpose<\/jats:title>\n                  <jats:p>Despite the robust efficacy of ROS1 tyrosine kinase inhibitors (TKIs) in ROS1-positive non-small cell lung cancer, TKI resistance continues to hamper durability of the therapeutic response. The resistance liabilities of next-generation ROS1 TKI are sparsely characterized.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Design<\/jats:title>\n                  <jats:p>We compared the activity of type I TKIs (crizotinib, entrectinib, taletrectinib, lorlatinib, and repotrectinib) to the type II TKIs (cabozantinib and merestinib), and to the type I FLT3 inhibitor, gilteritinib, in CD74-ROS1 wildtype and F2004C, L2026M, G2032R, or L2086 mutant Ba\/F3 cells. The findings from the Ba\/F3 cell model were confirmed using NIH3T3 colony formation assays and in vivo tumor growth. CRISPR\/Cas9 gene editing was used to generate isogenic wildtype and L2086F mutant TPM3-ROS1 expressing patient-derived cell lines. These lines were used to further evaluate TKI activity using cell viability and immunoblotting methods. Molecular modeling studies enabled the characterization of structural determinants of TKI sensitivity in wildtype and mutant ROS1 kinase domains. We also report clinical cases of ROS1 TKI resistance that were treated with cabozantinib.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Results<\/jats:title>\n                  <jats:p>ROS1 L2086F mutant kinase is resistant to type I TKI including crizotinib, entrectinib, lorlatinib, repotrectinib, taletrectinib, while the type II TKI cabozantinib and merestinib retain activity. Additionally, we found that gilteritinib, a type I FLT3 inhibitor, inhibited wildtype and L2086F mutant ROS1, however ROS1 G2032R solvent front mutation imposed resistance. The specific binding poses adopted by cabozantinib in the DFG-out kinase conformation and gilteritinib in the DFG-in kinase, provide rationale for their activity in the ROS1 mutants. Clinical cases demonstrated response to cabozantinib in tumors developing TKI resistance due to the ROS1 L2086F mutation.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion<\/jats:title>\n                  <jats:p>Cabozantinib and gilteritinib effectively inhibit ROS1 L2086F. Clinical activity of cabozantinib is confirmed in patients with TKI-resistant, ROS1 L2086F mutant NSCLC. Gilteritinib may offer an alternative with distinct off-target toxicities, however further studies are required. Since cabozantinib and gilteritinib are multi-kinase inhibitors, there is a persistent unmet need for more selective and better-tolerated TKI to overcome ROS1 L2086F kinase-intrinsic resistance.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Translational relevance<\/jats:title>\n                  <jats:p>\n                    ROS1 L2086F is an emerging recurrent resistance mutation to type I ROS1 TKIs, including later generation TKIs. Here, we show corroborating preclinical and clinical evidence for the activity of the quinolone-based type II TKI, cabozantinib, in ROS1\n                    <jats:sup>L2086F<\/jats:sup>\n                    resistance setting. In addition, we show activity of the pyrazine carboxamide-based type I TKI, gilteritinib, in ROS1 L2086F resistance, suggesting that gilteritinib could be another option for ROS1 L2086F TKI-resistant patients. This study represents the first comprehensive report of ROS1 L2086F in the context of later-generation TKIs, including the macrocyclic inhibitors.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2024.01.16.575901","type":"posted-content","created":{"date-parts":[[2024,1,20]],"date-time":"2024-01-20T08:50:19Z","timestamp":1705740619000},"source":"Crossref","is-referenced-by-count":2,"title":["TKI Type Switching Overcomes ROS1 L2086F in ROS1 Fusion-Positive Cancers"],"prefix":"10.64898","author":[{"given":"Rajat","family":"Thawani","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matteo","family":"Repetto","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Clare","family":"Keddy","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katelyn","family":"Nicholson","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kristen","family":"Jones","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kevin","family":"Nusser","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Catherine Z.","family":"Beach","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guilherme","family":"Harada","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexander","family":"Drilon","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1802-7597","authenticated-orcid":false,"given":"Monika A.","family":"Davare","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2024012300400959000_2024.01.16.575901v1.1","doi-asserted-by":"crossref","first-page":"845","DOI":"10.1080\/1061186X.2022.2085730","article-title":"ROS1-positive non-small cell lung cancer (NSCLC): biology, diagnostics, therapeutics and resistance","volume":"30","year":"2022","journal-title":"J. Drug Target"},{"key":"2024012300400959000_2024.01.16.575901v1.2","doi-asserted-by":"publisher","DOI":"10.1158\/1535-7163.MCT-21-0395"},{"key":"2024012300400959000_2024.01.16.575901v1.3","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1515281112"},{"key":"2024012300400959000_2024.01.16.575901v1.4","doi-asserted-by":"crossref","first-page":"1321","DOI":"10.1093\/annonc\/mdz167","article-title":"Immune checkpoint inhibitors for patients with advanced lung cancer and oncogenic driver alterations: results from the IMMUNOTARGET registry","volume":"30","year":"2019","journal-title":"Ann. Oncol"},{"key":"2024012300400959000_2024.01.16.575901v1.5","doi-asserted-by":"crossref","first-page":"1121","DOI":"10.1093\/annonc\/mdz131","article-title":"Crizotinib in ROS1-rearranged advanced non-small-cell lung cancer (NSCLC): updated results, including overall survival, from PROFILE 1001","volume":"30","year":"2019","journal-title":"Ann. Oncol. Off. J. Eur. Soc. Med. Oncol"},{"key":"2024012300400959000_2024.01.16.575901v1.6","doi-asserted-by":"publisher","DOI":"10.1016\/S1470-2045(19)30690-4"},{"key":"2024012300400959000_2024.01.16.575901v1.7","doi-asserted-by":"publisher","DOI":"10.1158\/2159-8290.CD-18-0484"},{"key":"2024012300400959000_2024.01.16.575901v1.8","doi-asserted-by":"crossref","first-page":"123","DOI":"10.2217\/fon-2022-1059","article-title":"TRUST-II: a global phase II study of taletrectinib in ROS1-positive non-small-cell lung cancer and other solid tumors","volume":"19","year":"2023","journal-title":"Future Oncol"},{"key":"2024012300400959000_2024.01.16.575901v1.9","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-019-11496-z"},{"key":"2024012300400959000_2024.01.16.575901v1.10","doi-asserted-by":"publisher","DOI":"10.1158\/1078-0432.CCR-20-1630"},{"key":"2024012300400959000_2024.01.16.575901v1.11","doi-asserted-by":"crossref","first-page":"598","DOI":"10.1158\/2159-8290.CD-22-0968","article-title":"NVL-520 Is a Selective, TRK-Sparing, and Brain-Penetrant Inhibitor of ROS1 Fusions and Secondary Resistance Mutations","volume":"13","year":"2023","journal-title":"Cancer Discov"},{"key":"2024012300400959000_2024.01.16.575901v1.12","doi-asserted-by":"publisher","DOI":"10.1158\/1078-0432.CCR-21-0032"},{"key":"2024012300400959000_2024.01.16.575901v1.13","doi-asserted-by":"publisher","DOI":"10.1038\/s41571-020-0408-9"},{"key":"2024012300400959000_2024.01.16.575901v1.14","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-021-21396-w"},{"key":"2024012300400959000_2024.01.16.575901v1.15","doi-asserted-by":"publisher","DOI":"10.1158\/1535-7163.MCT-11-0264"},{"key":"2024012300400959000_2024.01.16.575901v1.16","doi-asserted-by":"crossref","first-page":"3032","DOI":"10.1111\/1759-7714.14656","article-title":"MET gene amplification is a mechanism of resistance to entrectinib in ROS1+ NSCLC","volume":"13","year":"2022","journal-title":"Thorac. Cancer"},{"key":"2024012300400959000_2024.01.16.575901v1.17","doi-asserted-by":"crossref","first-page":"4443","DOI":"10.1042\/BCJ20200735","article-title":"A-loop interactions in Mer tyrosine kinase give rise to inhibitors with two-step mechanism and long residence time of binding","volume":"477","year":"2020","journal-title":"Biochem. J"},{"key":"2024012300400959000_2024.01.16.575901v1.18","doi-asserted-by":"crossref","first-page":"6111","DOI":"10.18632\/oncotarget.27222","article-title":"Effect of Fms-like tyrosine kinase 3 (FLT3) ligand (FL) on antitumor activity of gilteritinib, a FLT3 inhibitor, in mice xenografted with FL-overexpressing cells","volume":"10","year":"2019","journal-title":"Oncotarget"},{"key":"2024012300400959000_2024.01.16.575901v1.19","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-021-21396-w"},{"key":"2024012300400959000_2024.01.16.575901v1.20","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1186\/s13045-022-01299-z","article-title":"Foretinib can overcome common on-target resistance mutations after capmatinib\/tepotinib treatment in NSCLCs with MET exon 14 skipping mutation","volume":"15","year":"2022","journal-title":"J. Hematol. Oncol.J Hematol Oncol"},{"key":"2024012300400959000_2024.01.16.575901v1.21","doi-asserted-by":"publisher","DOI":"10.1158\/1078-0432.CCR-17-2452"},{"key":"2024012300400959000_2024.01.16.575901v1.22","doi-asserted-by":"crossref","first-page":"2351","DOI":"10.1158\/1078-0432.CCR-15-2013","article-title":"A Novel Crizotinib-Resistant Solvent-Front Mutation Responsive to Cabozantinib Therapy in a Patient with ROS1-Rearranged Lung Cancer","volume":"22","year":"2016","journal-title":"Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res"},{"key":"2024012300400959000_2024.01.16.575901v1.23","doi-asserted-by":"crossref","unstructured":"Repasky, M. P. , Shelley, M. & Friesner, R. A. Flexible ligand docking with Glide. Curr. Protoc. Bioinforma. Chapter 8, Unit 8.12 (2007).","DOI":"10.1002\/0471250953.bi0812s18"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2024.01.16.575901","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T05:48:54Z","timestamp":1768456134000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2024.01.16.575901"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,1,20]]},"references-count":23,"URL":"https:\/\/doi.org\/10.1101\/2024.01.16.575901","relation":{"is-preprint-of":[{"id-type":"doi","id":"10.1038\/s41698-024-00663-1","asserted-by":"subject"}]},"subject":[],"published":{"date-parts":[[2024,1,20]]},"subtype":"preprint"}}