Abstract B02: Heterogeneity of epithelial-to-mesenchymal transition and resistance mutation in ALK inhibitor-resistant lung cancer and its circumvention
Authors: Fukuda Koji , Takeuchi Shinji , Arai Sachiko , Katayama Ryohei , Nanjo Shigeki , Tanimoto Azusa , Nisiyama Akihiro , Suzuki Takeshi , Takeuchi Kengo , Nishio Makoto , Yano Seiji 
Published: February 2019

ALK rearrangement, most commonly EML4-ALK, is detected in approximately 3–5% of non-small cell lung cancer (NSCLC). While ALK tyrosine kinase inhibitor (TKI) shows dramatic clinical efficacy in ALK-rearranged NSCLC patients, almost all patients acquire resistance over time. ALK-secondary mutations, including ALK L1196M, are detected in ~40% of ALK-rearranged lung cancers resistant to ALK inhibitors. Epithelial–mesenchymal transition (EMT) was also reported to be associated with various targeted drugs; however, its involvement in ALK-inhibitor resistance is largely unknown.

In this study, we continuously gave crizotinib treatment to SCID mice inoculated with EML4-ALK lung cancer cell line A925LPE into thoracic cavity and established crizotinib-resistant cells. We also obtained several single-cell clones with acquired EMT phenotypes (low E-cadherin, high vimentin and ZEB1). MicroRNA profile analysis revealed that EMT was induced by reducing the expression of miR-200 family members, including miR-200c and miR-141, which resulted in increasing ZEB1 and decreasing E-cadherin expression in the clone cells. A reporter assay on a 200-kinase inhibitor library indicated that the histone deacetylase (HDAC) inhibitor, quisinostat, had the highest potential to increase miR-200c-141 promoter activity. Interestingly, pretreatment of the cells with quisinostat reduced ZEB1 expression, increased E-cadherin expression, and thus restored sensitivity to crizotinib and alectinib, mediated by enhanced expression of miR-200c in vitro and in vivo. These results indicate that quisinostat induces mesenchymal-epithelial transition (MET) by upregulating miR-200c expression that targets ZEB1 and thereby resensitizes to ALK-TKI.

Furthermore, we analyzed tumor tissue obtained at autopsy from an ALK-rearranged NSCLC patient who acquired resistance to crizotinib. In specimens from the primary lung tumor, as well as from the brain and subcutaneous metastases, both ALK L1196M mutation and EMT were concomitantly detected in all crizotinib-resistant lesions. Therefore, we performed laser capture microdissection and measured the copy number of ALK L1196M in epithelial and mesenchymal type tumor lesions separately. Very interestingly, ALK L1196M mutation was predominantly detected in epithelial type tumor cell lesion; by sharp contrast, it was hardly detected in the mesenchymal type tumor cell lesion. These results clearly demonstrate that EMT is a clinically relevant independent mechanism for crizotinib resistance underlying ALK inhibitor-resistant cancers.

Together, our study demonstrates the intratumor heterogeneity constituted by coexistence of resistance mutations and EMT in crizotinib-resistant tumors. HDAC inhibitor pretreatment, which reverts EMT, followed by a new-generation ALK inhibitor may be useful to circumvent resistance due to such intratumor heterogeneity.

Citation Format: Koji Fukuda, Shinji Takeuchi, Sachiko Arai, Ryohei Katayama, Shigeki Nanjo, Azusa Tanimoto, Akihiro Nisiyama, Takeshi Suzuki, Kengo Takeuchi, Makoto Nishio, Seiji Yano. Heterogeneity of epithelial-to-mesenchymal transition and resistance mutation in ALK inhibitor-resistant lung cancer and its circumvention [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr B02.