Abstract C95: Reversibility of acquired resistance and “addiction” to MET inhibitors.
Authors: Funakoshi Yohei , Mukohara Toru , Tomioka Hideo , Ekyalongo Roudy Chiminch , Kataoka Yu , Shimono Yohei , Chayahara Naoko , Toyoda Masanori , Kiyota Naomi , Fujiwara Yutaka , Minami Hironobu 
Published: August 2014

Abstract
Background: Acquired resitance is one of the biggest unsolved issues around tyrosine kinase-targeted anticancer therapies. Previously, we established resistant models by continuously exposing the MET-amplified gastric cancer cell line MKN45 to two different MET-tyrosine kinase inhibitors (TKIs), PHA665752 (MKN45-PR) or GSK1368089 (MKN45-GR). We found resistant mechanisms caused by increased copy number of MET in both MKN45-PR and MKN45-GR, and Y1230H gain-of-function mutation in MKN45-PR (Funakoshi Y et al, Invest New Drugs, in press). In addition, we found that excessive MET signaling resulting from the above mentioned MET gene alterations induced replication stress leading to intra-S phase arrest in the absent of MET-TKIs. Thereby, MKN45-PR and MKN45-GR showed the highest growth rate in the presence of MET-TKIs enough to suppress the excessive MET signaling. As a result, Y1230H mutation and/or copy number increase in MET gene were found to cause both acquired resitance and “addiction” to MET-TKIs. In this study, we investigated reversibility of the resistance and “addiction” to MET-TKIs and their causative MET gene alterations, so as to explore treatment strategies for overcoming acquired resistance to MET-TKIs.
Material and Methods: We sequentially cultured MKN45-PR and MKN45-GR in reducing concentrations of MET-TKIs until cells became able to grow in drugs-free condition. We designated these “revertant” cell lines as MKN45-PR-RE and MKN45-GR-RE. We comparatively analized these cell lines for cell growth, MET gene copy number and exon 19 sequence, and cell signaling, using MTS assay, RT-PCR and direct sequence, and Western blotting, respectively.
Results: Growth assay showed that both MKN45-PR-RE and MKN45-GR-RE partially lost the property of “addiction” to MET-TKIs, which we found in MKN45-PR and MKN45-GR. On the other hand, while MKN45-GR-RE lost the property of resistance to GSK1368089, MKN45-PR-RE retained the resistance to PHA665752. Correspondingly, while MET copy number was reduced in both MKN45-PR-RE and MKN45-GR-RE compared to MKN45-PR and MKN45-GR, respectively, Y1230H mutation remained in MKN45-PR-RE. Consistent with these findings, protein expression and phosphorylation of MET were higher in MKN45-PR and MKN45-GR than parental MKN45, and they were partially reduced in MKN45-PR-RE and MKN45-GR-RE compared to corresponding resistant cell lines. Further, biochemical resistance to MET-TKIs was retained only in MKN45-PR-RE, but not in MKN45-GR-RE.
Conclusions: The “addiction” to MET-TKIs appeared attributable to increased MET copy number, and the property and the MET alteration were reversible. Y1230H MET mutation appeared enough to keep cells resistant to MET-TKIs by itself, and the MET mutation was irreversible. Our data may justify re-introduction of MET-TKIs after transient withdrawal of the drug only when emergence of Y1230H mutation is not observed.
Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C95.
Citation Format: Yohei Funakoshi, Toru Mukohara, Hideo Tomioka, Roudy Chiminch Ekyalongo, Yu Kataoka, Yohei Shimono, Naoko Chayahara, Masanori Toyoda, Naomi Kiyota, Yutaka Fujiwara, Hironobu Minami. Reversibility of acquired resistance and “addiction” to MET inhibitors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C95.