Preclinical Prediction of Resistance and Optimization of Sequential Therapy for ALK-positive Lung Cancer Using Next-generation ALK Inhibitors

Abstract

BackgroundAnaplastic lymphoma kinase (ALK) gene rearrangements occur in approximately 5% of non-small cell lung cancers (NSCLCs). 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. ObjectiveTo 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. MethodsA 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. ResultsSeveral resistance mutations against zotizalkib, gilteritinib, and neladalkib were identified. Sequential use of these agents effectively suppressed all predicted resistance patterns with G1202R or I1171N. ConclusionsThis 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. Key PointsO_LIPCR-based mutation prediction system was successfully applied to fourth-generation ALK inhibitors. C_LIO_LIshowed efficacy against G1202R-positive relapses with minimal evidence of secondary resistance mutations. C_LIO_LIcombinations of gilteritinib with either neladalkib or ensartinib may sustain efficacy and delay resistance in I1171N-positive relapses. C_LI