This study investigates the mechanisms of acquired resistance to the ALK tyrosine kinase inhibitor crizotinib in ALK-positive non-small cell lung cancers (NSCLCs). The authors analyzed 18 patients who relapsed on crizotinib and identified a diverse array of secondary mutations within the ALK tyrosine kinase domain, including new resistance mutations and amplification of the ALK fusion gene. Next-generation ALK inhibitors showed varying potencies against specific resistance mutations. Additionally, the study found aberrant activation of other kinases, such as KIT and EGFR, in drug-resistant tumors. The results highlight the unique features of TKI resistance in ALK-positive NSCLCs and suggest the need for combinatorial therapeutics tailored to the precise resistance mechanisms identified in each patient.This study investigates the mechanisms of acquired resistance to the ALK tyrosine kinase inhibitor crizotinib in ALK-positive non-small cell lung cancers (NSCLCs). The authors analyzed 18 patients who relapsed on crizotinib and identified a diverse array of secondary mutations within the ALK tyrosine kinase domain, including new resistance mutations and amplification of the ALK fusion gene. Next-generation ALK inhibitors showed varying potencies against specific resistance mutations. Additionally, the study found aberrant activation of other kinases, such as KIT and EGFR, in drug-resistant tumors. The results highlight the unique features of TKI resistance in ALK-positive NSCLCs and suggest the need for combinatorial therapeutics tailored to the precise resistance mechanisms identified in each patient.