Abstract A45: In depth molecular characterization of novel PI3K-mTOR inhibitor resistant NSCLC cell lines
Authors: Heavey Susan , Dowling Paul , Toomey Sinéad , Carr Aoife , Hennessy Bryan , O'Neill Michael , Barr Martin P , Finn Stephen , Cuffe Sinéad , O'Byrne Kenneth J. , Gately Kathy 
Published: October 2015

Abstract
Introduction: Non-small cell lung cancer (NSCLC) is the leading cause of cancer morbidity and mortality globally in males, with rates increasing in females in recent years. New therapeutic intervention strategies will be crucial in improving the current survival rates for patients with this disease, which currently stand at less than 15% at 5 years. The PI3K-Akt- mTOR pathway regulates cell growth and proliferation and is often dysregulated in cancers including NSCLC, making it an attractive new therapeutic target in this setting. GDC-0980 is a selective dual inhibitor of PI3K and mTOR, which is currently in Phase II clinical trials for solid tumours. As with all targeted therapies, acquired resistance to GDC-0980 is anticipated to be a major hurdle in the success of this drug. The aim of this project is to develop and characterise four cell line models of resistance to GDC-0980, each representing a different molecular subtype of NSCLC, in order to identify biomarkers of response/resistance to the drug that may dictate beneficial treatment strategies.
Methods: H460, A549, H1975 and SKMES-1 cells were cultured in GDC-0980 at IC50 concentrations over a period of several months, along with matched ‘parent’ cell lines. Development of resistance was assessed by monthly BrdU proliferation assays. Cell growth patterns were compared across the sensitive and resistant cell lines in real time using the xCELLigence platform. Cell lines were then interrogated for alterations in DNA (Sequenom sequencing of 547 mutations in 46 genes), mRNA (SABiosciences arrays profiling expression of &gt;150 genes), miRNA (Exiqon expression profiling of 2100 miRNAs) and protein (R&D Phospho Kinase array expression profiling of 43 kinases and 2 associated total proteins, bottoms-up label-free mass spectrometry and Western blot analysis).
Results: H1975 cells (PIK3CA mutant and activated pAkt (Ser473/Thr308), pmTOR, pS6R) were most sensitive to GDC-0980, however they were the first to develop resistance to the drug. Results obtained from xCELLigence studies identified H1975 resistant cells as having the highest cell index out of all parent and resistant cell lines after 100 hours of cell growth, suggesting that these are the most aggressive cells. A 33 miRNA signature was identified contrasting H1975 parent and resistant cells. 1,200 proteins were found to be differentially expressed between H1975 parent and resistant cells (p &lt; 0.05, fold change &gt;2) by bottoms-up label-free mass spectrometry. Preliminary results based on these screens have identified dysregulated metabolism and IGF-1 pathway signalling as potential mechanisms of resistance to GDC-0980 in H1975 cells. Comparison of Nanog, Sox2, Klf4, Oct4 and c-Myc expression highlighted a putative stem-like signature in all resistant cell lines. Several dysregulated genes and proteins will be presented including a kinase which plays a key role in cell proliferation and cancer progression and is targetable by dual and triple kinase inhibitors.
Discussion and Conclusion: While the panel of four NSCLC cell lines all responded well to GDC-0980 treatment initially, resistance to the drug developed rapidly in 3 of the cell lines, within 4-6 months. Initial data highlights IGF-1 signalling pathway proteins and the emergence of a stem-like signature as potential mediators of resistance to the drug. Of particular interest is the identification of a specific kinase which is targetable by third generation small molecule inhibitors. Further elucidation of these bypass strategies are under investigation and are crucial to the design of optimal treatment protocols for patients.
Citation Format: Susan Heavey, Paul Dowling, Sinéad Toomey, Aoife Carr, Bryan Hennessy, Michael O'Neill, Martin P Barr, Stephen Finn, Sinéad Cuffe, Kenneth J. O'Byrne, Kathy Gately. In depth molecular characterization of novel PI3K-mTOR inhibitor resistant NSCLC cell lines. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A45.