• Kuo-Yen Huang, Shih-Han Kao, Wen-Lung Wang, Chi-Yuan Chen, Tzu-Hung Hsiao, Santosh B Salunke, Jeremy J W Chen, Kang-Yi Su, Shuenn-Chen Yang, Tse-Ming Hong, Ching-Shih Chen, and Pan-Chyr Yang.
• 1 Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan.
• Am. J. Respir. Crit. Care Med. 2016 Apr 1; 193 (7): 753-66.

RationaleDespite the fact that tyrosine kinase inhibitors (TKIs) have been found effective in treating patients harboring activating mutations of epidermal growth factor receptor (EGFR), an acquired secondary mutation, T790M, which lowers the affinity to TKIs, can lead to EGFR TKI resistance after this standard treatment.ObjectivesTo evaluate the effect of small molecule T315 on EGFR degradation and its therapeutic efficacy in vitro and in vivo.MethodsLung adenocarcinoma cells were treated with T315, and cell proliferation and apoptotic proportion were determined by the CellTiter 96 AQueous MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt) assay and flow cytometry. The effects of T315 on EGFR mRNA and protein levels, autophosphorylation, ubiquitination, and degradation were evaluated by real-time polymerase chain reaction and Western blot, respectively. Direct targeting of T315 to EGFR was confirmed by the in vitro kinase assay and mass spectrometry. Finally, the preclinical effect of T315 was validated in the murine xenograft model in combination with a second-generation TKI, afatinib.Measurements And Main ResultsWe identified T315 as a novel, potent small molecule for suppressing cancer cell proliferation in vitro and in vivo. The therapeutic effect was verified after T315 was combined with a second-generation TKI, afatinib, compared with a single drug administration. We found a new mechanism of action, in that T315 appears to directly bind EGFR and triggers EGFR-Y1045 autophosphorylation, whereby its degradation is triggered through the ubiquitin-proteasome pathway.ConclusionsOur evidence suggests that T315 is a novel class of anticancer drug that is able to inhibit the growth of EGFR-TKI-resistant lung adenocarcinoma cells by inducing the degradation of EGFR.

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