Abstract PR05: ERK pathway activation is associated with acquired resistance to AZD9291, a third-generation irreversible inhibitor targeting EGFR sensitizing (EGFRm+) and resistance (T790M) mutations in NSCLC
Authors: Eberlein Cath , Al-Kadhimi Katie , Ross Sarah , Brown Henry , Fisher Paul , Stetson Daniel , Lai Zhongwu , Thress Kenneth , Dougherty Brian , Pao William , Cross Darren 
Published: May 2015

Abstract
First- and second-generation EGFR tyrosine kinase inhibitors (TKIs) are established first line therapies for patients with advanced NSCLC with activating/sensitising mutations in EGFR. Unfortunately, patients ultimately develop disease progression with acquisition of a second-site EGFR T790M mutation in more than half of cases. This has led to the development of third generation EGFR TKIs such as AZD9291 which inhibit both the EGFRm+ and T790M mutations in preclinical models, and are showing activity in patients with TKI-resistant tumors harbouring T790M in Phase I studies. Despite the potential improvements brought by third generation EGFR-TKIs, advanced EGFRm+ tumor cells will still remain highly adaptable, and the inevitability of further resistance will potentially limit the effectiveness of these drugs. As such, the identification of resistance mechanisms to these agents is essential to guide future therapeutic strategies and identify novel:novel combinations.
To interrogate resistance to AZD9291, we have generated panels of EGFRm+ cell populations resistant to gefitinib (first generation TKI), and EGFRm+ and EGFRm+/T790M cell populations resistant to afatinib (second generation TKI) and WZ4002 or AZD9291 (third generation TKIs). Subsequently, we have characterized the cell lines using a phenotypic screen to compare the sensitivity of small molecule inhibitors of canonical signaling pathways between the resistant and parental cell populations. In addition we have used a variety of molecular profiling techniques to determine the DNA mutation and copy number status and mRNA expression profile of a panel of cancer associated genes within the resistant cell populations.
The effects on cell survival across the range of resistant models by a panel of pathway inhibitors, in combination with the originating TKI, indicated that resistance to the EGFR inhibitors was frequently associated with increased sensitivity to selumetinib (AZD6244; ARRY-142886) (MEK1/2 inhibitor), suggesting that ERK signaling is commonly reactivated to circumvent inhibition of the EGFR pathway. Further, molecular analysis indicated the presence of mutations in NRAS, including a novel E63K mutation, or increased copy number of NRAS or KRAS within 12/25 and 3/9 resistant cell populations representing resistance in EGFRm+ and EGFRm+/T790M settings respectively. Analysis of the functional consequence of the observed RAS modifications confirmed their role in driving the survival of EGFR pathway addicted cells when EGFR signaling is inhibited.
Collectively, these data suggest that ERK pathway activation, in particular as a result of increased RAS activation, is frequently associated with acquired resistance to AZD9291 and other EGFR TKI inhibitors. In addition the data suggests that combining AZD9291 with selumetinib could prevent or delay resistance, and therefore potentially drive superior duration of benefit compared to TKI alone.
Citation Format: Cath Eberlein, Katie Al-Kadhimi, Sarah Ross, Henry Brown, Paul Fisher, Daniel Stetson, Zhongwu Lai, Kenneth Thress, Brian Dougherty, William Pao, Darren Cross. ERK pathway activation is associated with acquired resistance to AZD9291, a third-generation irreversible inhibitor targeting EGFR sensitizing (EGFRm+) and resistance (T790M) mutations in NSCLC. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr PR05.