Abstract 1973: Targeting activated alleles of EGFR derived from glioma and lung cancer: Correlating kinase active-site occupancy with efficacy

Abstract

Abstract Introduction: The Epidermal Growth Factor Receptor (EGFR) features prominently in malignant glioma (MG), yet attempts to exploit this target in patients with MG have been somewhat disappointing. Unlike the highly sensitizing kinase site alleles of EGFR derived from patients with non-small cell lung cancer (NSCLCA), mutationally activated alleles of EGFR derived from patients with MG respond poorly to the currently available reversible clinical EGFR inhibitors. We hypothesize that the anti-tumor effects of EGFR inhibitors in MG-associated alleles of EGFR require more efficient blockade to achieve anti-tumor responses, in comparison with NSCLCA-associated alleles of EGFR, and that thisincreased efficiency can be achieved using irreversible inhibitors. Methods: We generated glioma and fibroblast cell lines that expressed wild-type or tumor-associated alleles of EGFR derived either from MG (EGFRvIII) or NSCLCA (L858R or del 747-753). Using an EGFR affinity probe (an irreversible EGFR inhibitor tethered to a flourophore), we quantified occupancy of the kinase active site in response to erotinib, a clinical reversible inhibitor of EGFR, correlating occupancy with proliferation and apoptosis. Next, we repeated these measurements using the tool compound and irreversible EGFR inhibitor PD-168394. Results: Using the affinity probe in pulse-chase experiments, we quantified active site occupancy of EGFR int response to increasing doses of inhibitors. In cells expressing MG-derived alleles, irreversible EGFR inhibitors were significantly more potent than reversible agents in abrogating signaling downstream of EGFR and in decreasing viability. Using the affinity probe, we found that irreversible inhibitors showed nearly complete occupancy of the active site, whereas treatment with reversible EGFR inhibitors resulted in only partial occupancy. Cells containing NSCLCA-derived EGFR alleles showed similar signaling and growth responses to both reversible and EGFR inhibitors, despite a large difference in EGFR kinase site occupancy with the two types of inhibitors. Flow cytometry studies correlating kinase site occupancy with downstream signaling in individual cells will also be presented. Conclusions: Activated alleles of EGFR derived from MG require significantly higher levels of kinase site blockade to achieve an anti-proliferative threshold, as compared with activated alleles derived from NSCLCA. This higher degree of kinase site blockade can be achieved using irreversible inhibitors of EGFR. These studies support the initiation of clinical trials using irreversible inhibitors of EGFR in MG. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1973.