Abstract 4444: Switching oncogene signaling in a highly selective ALK tyrosine kinase inhibitor CH5424802 resistant cells.
Authors: Isozaki Hideko , Ichihara Eiki , Yasugi Masayuki , Ninomiya Takashi , Honda Yoshihiro , Murakami Toshi , Minami Daisuke , Kato Yuka , Kudo Kenitiro , Sato Akiko , Hotta Katsuyuki , Takigawa Nagio , Sendo Toshiaki , Tanimoto Mitsune , Kiura Katsuyuki 
Published: August 2014

Abstract
We experienced dramatic responses to crizotinib, a multitargeted tyrosine kinase inhibitor including anaplastic lymphoma kinase (ALK), MET, and ROS1 in patients with non-small cell lung cancer (NSCLC) harboring ALK fusion gene; however, majority of the patients relapsed until around 10 months. The emergence of resistance to crizotinib is reportedly conferred by genetic alterations, such as ALK kinase domain mutation, ALK copy number gain or activation of the bypass pathways. CH5424802 (Chugai Pharmaceutical Co., Ltd.) is a new, highly selective ALK tyrosine kinase inhibitor that partially overcome acquired resistance to crizotinib, thereby inhibiting crizotinib resistant mutations of ALK in a preclinical experiment (Cancer Cell 19, 2011: 679-90) A phase II study of CH5424802 demonstrated 93.5% response rate (JLCS 2012); however, we also encountered acquired resistance phenomenon.
To elucidate the acquired resistant mechanisms, we established CH5424802-resistant H2228 cells harboring EML4-ALK fusion gene under the step-wise continuous exposures of CH5424802. Six sublines that could grow in the presence of 1 μmol/L of CH5424802 (designated H2228CHR clones -1, -2, -3, -5, -6, and -9) were obtained. All sublines revealed more than 30-fold resistance to CH5424802 (50% inhibitory concentration [IC50]: 1.0-4.8 μmol/L) compared with the parental H2228 cells (IC50: 0.03 μmol/L). In addition, the sublines similarly indicated resistance to Crizotinib. H2228CHR clones showed the constitutive phosphorylation of Akt and Erk1/2 even in the presence of CH5424802, whereas phosphorylation of Akt and Erk1/2 was markedly decreased in the parental H2228 cells. Surprisingly, ALK fusion gene disappeared in all H2228 CHR clones, which were confirmed immunostaining, reverse transcription PCR, FISH and Western blotting. H2228 CHR clones -1 and -2 presented higher phosphorylated levels of epidermal growth factor receptor (EGFR) than the parental H2228 cells and showed sensitivity to EGFR tyrosine kinase inhibitors, gefitinib and erlotinib.
Furthermore, we also established CH5424802-resistant ABC-11CHR cells derived from ABC-11 cells from pleural effusion of an ALK inhibitor naïve patient. In ABC-11CHR cells, phosphorylated ALK was markedly decreased and phosphorylated MET was increased.
In conclusion, signal switching from ALK to other tyrosine kinase receptors induced resistance to a highly selective ALK tyrosine kinase inhibitor CH5424802 in NSCLC cell lines harboring ALK fusion gene.
Citation Format: Hideko Isozaki, Eiki Ichihara, Masayuki Yasugi, Takashi Ninomiya, Yoshihiro Honda, Toshi Murakami, Daisuke Minami, Yuka Kato, Kenitiro Kudo, Akiko Sato, Katsuyuki Hotta, Nagio Takigawa, Toshiaki Sendo, Mitsune Tanimoto, Katsuyuki Kiura. Switching oncogene signaling in a highly selective ALK tyrosine kinase inhibitor CH5424802 resistant cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4444. doi:10.1158/1538-7445.AM2013-4444