P1.14-35 Epithelial-To-Mesenchymal Transition Is a Mechanism of ALK Inhibitor Resistance in Lung Cancer Independent of ALK Mutation Status

ALK rearrangement, most commonly EML4-ALK, is detected in approximately 3%–5% of NSCLC. ALK tyrosine kinase inhibitor (TKI), shows dramatic clinical efficacy, however, almost all patients acquire resistance over time. The most defined mechanism of crizotinib resistance is secondary ALK mutations. A recent study reported that epithelial-to-mesenchymal transition (EMT) and ALK resistance mutation were simultaneously detected in a single tumor lesion in patients with ALK-rearranged lung cancer who were resistant to ALK-TKIs. However, it is still unknown whether ALK-TKI resistant tumor cells combine mesenchymal phenotype with ALK resistance mutation, or each of the mesenchymal type tumor cells and ALK resistance mutation–positive cells coexist in a single lesion. In any of these cases, no therapy for EMT-associated targeted drug resistance has yet been established. Specimens from a patient with ALK-rearranged lung adenocarcinoma who acquired resistance to crizotinib were stained with IHC, and the epithelial regions (ALK+, vimentin+, and E-cadherin－) and the mesenchymal regions (ALK+, vimentin－, and E-cadherin+) were collected by microdissection. The DNA from the each regions were isolated and the ALK L1196M mutation was detected by degital PCR analysis. Crizotinib-resistant cell line was developed by continuous treatment with crizotinib in the pleural carcinomatosis mouse model inoculated with the crizotinib-sensitive human lung cancer cell line, A925LPE3, which harbors the EML4-ALK gene fusion. The clones were estabilish by limiting dilution and the mechanism of crizotinib resistance was examined by microarray analysis, miRNA array analysis, western blot, and MTT assay. Compounds that overcame crizotinib resistance were screened from a library of 200 kinase inhibitors. Digital PCR analyses combined with microdissection after IHC staining for EMT markers revealed that ALK L1196M was predominantly detected in epithelial-type tumor cells, indicating that mesenchymal phenotype and ALK mutation can coexist as independent mechanisms underlying ALK inhibitor–resistant cancers. Preclinical experiments with crizotinib-resistant lung cancer cells showed that EMT associated with decreased expression of miR-200c and increased expression of ZEB1 caused cross-resistance to new-generation ALK inhibitors alectinib, ceritinib, and lorlatinib. Moreover, pretreatment with the histone deacetylase (HDAC) inhibitor quisinostat overcame this resistance by reverting EMT in vitro and in vivo. These findings indicate that HDAC inhibitor pretreatment followed by a new ALK inhibitor may be useful to circumvent resistance constituted by coexistence of resistance mutations and EMT in the heterogeneous tumor.