Abstract A10: Establishment and characterization of a patient-derived non-small cell lung cancer mouse model of acquired resistance towards anti-EGFR treatment
Authors: Tschuch Cordula , Klingner Kerstin , Lehnhard Dorothee , Löhr Anne , Raeva Yana , Peille Anne-Lise , Oswald Eva , Schüler Julia B. 
Published: May 2016

Non-small cell lung cancer (NSCLC) is the largest subgroup of lung cancer, occurring at a frequency of over 80% of lung cancer cases. In up to 30% of NSCLC patients the oncogenic driver of tumor growth is a constitutively activated EGF receptor (EGFR), which plays a critical role in regulating multiple cellular processes, including proliferation, survival and apoptosis. Although these patients gain great benefit from treatment with EGFR tyrosine kinase inhibitors (TKI, e.g. erlotinib or gefitinib), development of resistance is inevitable.

To model the emergence of drug resistance, an EGFR driven, gefitinib sensitive, patient-derived xenograft (PDX) NSCLC model was treated continuously with gefitinib in immunocompromised mice. The dose of daily treatment was adjusted according to tumor growth over a period of up to 91 days. In a first phase, dosing was high (40-50 mg/kg) to eradicate EGFR TKI sensitive cells. At the time point of maximal antitumoral activity dosing was reduced to 20-30 mg/kg ( = low dose) to preserve selection pressure. Between 69 and 91 days after dosing was initiated, drug-resistant tumors emerged in 4 out of 10 mice under high dose treatment. Resistant tumor fragments, which were re-implanted into a new cohort of mice and continuously treated with gefitinib, kept resistance also under high dose treatment. A comprehensive analysis using Western blot (WB), qPCR and sequencing was performed to identify the reason for resistance.

In WB analysis we could show that signalling through EGFR was completely abrogated in all four resistant tumor sublines. Neither secondary mutations in EGFR (ex19-21) or KRAS (ex 1+2) could be detected, nor was the expression of cMET, AXL, HGF, PTEN or HER3 significantly increased in resistant tumors as shown by sequencing and qPCR respectively. However a more comprehensive WB analysis revealed several genes being activated in resistant compared to primary tumors. Depending on the subline, phospho-(p)-cMET, p-AKT, AXL & p-AXL, p-cRAF, p-MEK, p-ERK as well as HER3, IGF-R and ALK were up-regulated in resistant tumors. Based on these data we determined that signalling pathways such as the (RAS)-cRAF-MEK-ERK signalling cascade or enhanced cMET/AKT signalling were activated in drug-resistant tumors. These signalling pathways are known to be alternative pathways for EGFR signalling also in patients with acquired resistance indicating the clinical relevance of these models. To shed more light into the mechanism of resistance, whole exome sequencing analyses of the four resistant sublines as well as the original tumor model are underway.

In summary, we have developed four NSCLC tumor sublines each harbouring a different mechanism of resistance to EGFR TKI treatment, modelling the emergence of drug-resistant NSCLC in patients. Herewith a strong preclinical tool for the development of innovative compounds targeting acquired EGFR resistance is now available.

Citation Format: Cordula Tschuch, Kerstin Klingner, Dorothee Lehnhard, Anne Löhr, Yana Raeva, Anne-Lise Peille, Eva Oswald, Julia B. Schüler. Establishment and characterization of a patient-derived non-small cell lung cancer mouse model of acquired resistance towards anti-EGFR treatment. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A10.