: Targeted therapy changed the standard of care in ALK-dependent tumors. 
However, resistance remains a major challenge. Lorlatinib is a third-generation 
ALK inhibitor that inhibits most ALK mutants resistant to current ALK 
inhibitors. In this study, we utilize lorlatinib-resistant anaplastic large cell 
lymphoma (ALCL), non-small cell lung cancer (NSCLC), and neuroblastoma cell 
lines in vitro and in vivo to investigate the acquisition of resistance and its 
underlying mechanisms. ALCL cells acquired compound ALK mutations G1202R/G1269A 
and C1156F/L1198F in vitro at high drug concentrations. ALCL xenografts selected 
in vivo showed recurrent N1178H (5/10 mice) and G1269A (4/10 mice) mutations. 
Interestingly, intracellular localization of NPM/ALKN1178H skewed toward the 
cytoplasm in human cells, possibly mimicking overexpression. RNA sequencing of 
resistant cells showed significant alteration of PI3K/AKT and RAS/MAPK pathways. 
Functional validation by small-molecule inhibitors confirmed the involvement of 
these pathways in resistance to lorlatinib. NSCLC cells exposed in vitro to 
lorlatinib acquired hyperactivation of EGFR, which was blocked by erlotinib to 
restore sensitivity to lorlatinib. In neuroblastoma, whole-exome sequencing and 
proteomic profiling of lorlatinib-resistant cells revealed a truncating NF1 
mutation and hyperactivation of EGFR and ErbB4. These data provide an extensive 
characterization of resistance mechanisms that may arise in different 
ALK-positive cancers following lorlatinib treatment. SIGNIFICANCE: 
High-throughput genomic, transcriptomic, and proteomic profiling reveals various 
mechanisms by which multiple tumor types acquire resistance to the 
third-generation ALK inhibitor lorlatinib.