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Structural basis for the altered drug sensitivities of non-small cell lung cancer-associated mutants of human epidermal growth factor receptor

Abstract

The epidermal growth factor receptor (EGFR) has an essential role in multiple signaling pathways, including cell proliferation and migration, through extracellular ligand binding and subsequent activation of its intracellular tyrosine kinase (TK) domain. The non-small cell lung cancer (NSCLC)-associated EGFR mutants, L858R and G719S, are constitutively active and oncogenic. They display sensitivity to TK inhibitors, including gefitinib and erlotinib. In contrast, the secondary mutation of the gatekeeper residue, T790M, reportedly confers inhibitor resistance on the oncogenic EGFR mutants. In this study, our biochemical analyses revealed that the introduction of the T790M mutation confers gefitinib resistance on the G719S mutant. The G719S/T790M double mutant has enhanced activity and retains high gefitinib-binding affinity. The T790M mutation increases the ATP affinity of the G719S mutant, explaining the acquired drug resistance of the double mutant. Structural analyses of the G719S/T790M double mutant, as well as the wild type and the G719S and L858R mutants, revealed that the T790M mutation stabilizes the hydrophobic spine of the active EGFR-TK conformation. The Met790 side chain of the G719S/T790M double mutant, in the apo form and gefitinib- and AMPPNP-bound forms, adopts different conformations that explain the accommodation of these ligands. In the L858R mutant structure, the active-site cleft is expanded by the repositioning of Phe723 within the P-loop. Notably, the introduction of the F723A mutation greatly enhanced the gefitinib sensitivity of the wild-type EGFR in vivo, supporting our hypothesis that the expansion of the active-site cleft results in enhanced gefitinib sensitivity. Taken together, our results provide a structural basis for the altered drug sensitivities caused by distinct NSCLC-associated EGFR mutations.

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Acknowledgements

We thank C Takemoto, T Kaminishi, M Kawazoe, Y Fujii and S Kishishita for assisting with the data collection; Y Ishizuka-Katsura, R Akasaka, M Yamaguchi-Hirafuji, T Uchikubo-Kamo, A Urushibata and N Maoka for technical assistance; S Kusano for helpful assistance; and K Murayama for helpful advice. We also thank the beamline staffs of the X06SA (SLS), BL-5A (PF), BL41XU (SPring-8) and BL26B2 (SPring-8) beamlines. Portions of the data were collected at the Southeast Regional Collaborative Access Team (SER-CAT) 22-ID beamline at the Advanced Photon Source, Argonne National Laboratory. Supporting institutions may be found at http://www.ser.anl.gov/. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W-31-109-Eng-38. This study was supported in part by the ‘High-quality Protein Crystal Growth Experiment Project in JEM’ promoted by the Japan Aerospace Exploration Agency. The Russian Spacecraft ‘Progress’ and ‘Soyuz’, provided by the Russian Federal Space Agency, were used for space transportation. A portion of the crystallization technology for the counter-diffusion method was developed by European Space Agency and University of Granada. This work was supported by the RIKEN Structural Genomics/Proteomics Initiative, the National Project on Protein Structural and Functional Analyses, the Targeted Proteins Research Program (TPRP), the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by a Japanese Society for the Promotion of Science Fellowship (LJP).

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Correspondence to T Yamamoto or S Yokoyama.

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Yoshikawa, S., Kukimoto-Niino, M., Parker, L. et al. Structural basis for the altered drug sensitivities of non-small cell lung cancer-associated mutants of human epidermal growth factor receptor. Oncogene 32, 27–38 (2013). https://doi.org/10.1038/onc.2012.21

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