Abstract 2314: Erlotinib response in lung- and glioma-derived EGFR alleles: Correlating kinase site occupancy with efficacy
Authors: Barkovich Krister J. , Nicolaides Theodore , Hariono Sujatmi , Garske Adam , Fan Qi Wen , Shokat Kevan , Weiss William A. 
Published: June 2012

Abstract
INTRODUCTION: Mutational activation of the Epidermal Growth Factor Receptor (EGFR) gene features prominently in malignant glioma (MG) and non-small-cell lung cancer (NSCLC). However, while NCSLC-derived mutants of EGFR exhibit high sensitivity to small molecule tyrosine kinase inhibitors, patients with MG-derived EGFR alleles respond poorly to TKIs. We tested MG- and NSCLC-derived alleles of EGFR to determine whether differences in occupancy within the mutant kinase domains determine response to therapy.
METHODS: Human malignant glioma-derived cell lines expressing wild-type and mutant alleles of EGFR (derived from MG (EGFRvIII) or NSCLC (EGFR L858R and EGFRdel746-750)) were generated by viral transduction. Cell cycle analyses and viability assays were performed to confirm the in vitro recapitulation of the range of erlotinib's clinical efficacy in cells expressing MG- or NSCLC-specific alleles of EGFR. A novel affinity probe selective for the kinase site of EGFR was used to quantify the level of erlotinib binding in this panel of EGFR-allele expressing cell lines. Cell-signaling was analyzed through western-blotting and quantified with densitometry.
RESULTS: Using the EGFR-specific affinity probe, erlotinib's kinase site occupancy was measured at clinically relevant doses. In cells expressing MG-derived EGFRvIII, significantly lower levels of kinase site occupancy were reached as compared to NSCLC-derived EGFR L858R and EGFRdel746-750. Surprisingly, these data did not correlate with levels of phospho-EGFR. Across all alleles, kinase site occupancy rather than levels of p-EGFR correlated with downstream signaling through Akt and mTOR, and with growth inhibition and cell cycle arrest.
CONCLUSION: Erlotinib, a small molecule tyrosine kinase inhibitor, achieves higher levels of kinase site occupancy in NSCLC-derived mutations of EGFR, as compared with MG-derived mutations. Kinase site occupancy correlated directly with efficacy. In contrast, levels of p-EGFR correlated poorly with efficacy for these agents. These data suggest kinase site occupancy as a potential biomarker for efficacy of EGFR inhibitors. This also raises the intriguing possibility that the reason for the differential efficacy of erlotinib in lung- and MG-derived activating mutations in EGFR is due to differences in ATP binding affinity: the lung-derived activating mutations in EGFR have lower ATP affinity as compared to the wild-type kinase, resulting in higher relative erlotinib affinity, while MG-derived activating mutations in EGFR have a higher affinity for ATP, which outcompetes erlotinib and results in poor drug affinity for the kinase site.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2314. doi:10.1158/1538-7445.AM2011-2314