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Although ALK tyrosine kinase inhibitors provide substantial clinical benefits, acquired resistance-conferring mutations frequently emerge, leading to disease progression. Preclinical prediction of these mutations might help guide the development of more effective sequential treatment strategies prior to clinical application.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Objective<\/jats:title>\n            <jats:p>To predict the emergence of resistance mutations to the investigational ALK inhibitors zotizalkib (TPX-0131), gilteritinib (ASP2215), and neladalkib (NVL-655) following resistance to first-line alectinib and assess the potential of these drugs as second-line therapies.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Methods<\/jats:title>\n            <jats:p>A polymerase chain reaction (PCR)-based mutagenesis system was used to introduce random mutations into ALK cDNA harboring representative alectinib-resistant mutations. Mutant libraries were expressed in Ba\/F3 cells, which were exposed to each inhibitor. Drug-resistant clones were isolated, sequenced, and evaluated for drug sensitivity using viability assays and immunoblotting.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Results<\/jats:title>\n            <jats:p>Several resistance mutations against zotizalkib, gilteritinib, and neladalkib were identified. Sequential use of these agents effectively suppressed all predicted resistance patterns with G1202R or I1171N.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Conclusions<\/jats:title>\n            <jats:p>This PCR-based platform provides a valuable approach for anticipating resistance mutations and guiding the design of optimized sequential therapies. Zotizalkib, gilteritinib, and neladalkib might represent promising alternatives to lorlatinib as second-line treatments for ALK-positive NSCLC.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Key Points<\/jats:title>\n            <jats:p>\u2022 A PCR-based mutation prediction system was successfully applied to fourth-generation ALK inhibitors.<\/jats:p>\n            <jats:p>\u2022 Neladalkib showed efficacy against G1202R-positive relapses with minimal evidence of secondary resistance mutations.<\/jats:p>\n            <jats:p>\u2022 Sequential combinations of gilteritinib with either neladalkib or ensartinib may sustain efficacy and delay resistance in I1171N-positive relapses.<\/jats:p>\n          <\/jats:sec>","DOI":"10.1007\/s11095-025-03916-1","type":"journal-article","created":{"date-parts":[[2025,9,24]],"date-time":"2025-09-24T18:14:35Z","timestamp":1758737675000},"page":"1497-1509","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Preclinical Prediction of Resistance Mutations and Proposal of Sequential Treatment Strategies for ALK-positive Lung Cancer Using Next-generation ALK Inhibitors"],"prefix":"10.1007","volume":"42","author":[{"given":"Yuki","family":"Takei","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hirotaka","family":"Kuroiwa","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chisaki","family":"Arai","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuta","family":"Doi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7507-8214","authenticated-orcid":false,"given":"Kentaro","family":"Semba","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,9,24]]},"reference":[{"key":"3916_CR1","doi-asserted-by":"publisher","first-page":"495","DOI":"10.1158\/2159-8290.CD-12-0009","volume":"2","author":"H Mano","year":"2012","unstructured":"Mano H. 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