Integrated Functional Proteomics Of Met/Vegfr Inhibitors Reveals Complex Mechanism Of Action Of Foretinib In Nsclc

BACKGROUND: Foretinib (FORE) and cabozantinib (CABO) are two MET/VEGFR inhibitors with similar chemical structures. CABO is FDA-approved for medullary thyroid and renal cancer; in addition, it is in clinical trials for treatment of non-small cell lung cancer (NSCLC). Through an unbiased viability screen we have observed potent cellular activity of FORE, but not CABO, in several NSCLC cell lines. We have previously shown that most NSCLC cell lines are insensitive to MET or VEGFR inhibition, suggesting off-target activity of FORE in these cells. The aim of this project is to identify the mechanism of action of FORE in NSCLC and design an optimized combination therapy. METHODS: Cellular viability assays were done using CellTiter-Glo, cell cycle analysis by flow cytometry. Western blotting was performed to evaluate the induction of apoptosis through PARP1 and caspase cleavage, as well changes in signaling. We synthesized FORE and CABO analogues and performed differential quantitative chemical and phosphoproteomics to determine the target kinase profile and pathway effects in NSCLC cells. Changes in gene expression upon drug treatment were measured by RNA-seq. RNAi in combination with pharmacological inhibitors was performed to interrogate targets and pathways. RESULTS: FORE showed greater potency in NSCLC cell lines than CABO with regard to inhibition of viability and induction of apoptosis. FORE decreased phosphorylation of AKT and ERK. Chemical and phosphoproteomics revealed several kinases, such as MEK and MAP4K5, to bind preferentially by FORE over CABO that differentially affect the adherens junction and MAPK signaling pathways. Target validation showed differential inhibition of MEK1/2 and MAP4K5. Cellular validation with RNAi in combination with pharmacological inhibitors suggested that MEK1/2, MAP4K5 and IGF1R are involved in the mechanism of action of foretinib in NSCLC cells. RNA-seq pathway analysis furthermore suggested regulation of chromatin organization and Wnt pathway signaling by foretinib. CONCLUSION: Our results suggest that the difference in the efficacy between FORE and CABO is related to polypharmacology of FORE, which simultaneously targets IGF1R, MEK1/2 and MAP4K5. This difference results in divergence in signaling pathway inhibition and induces distinct effects in NSCLC. The establishment of FORE targets and signaling pathways can lead to optimized combination therapy for NSCLC and identification of new actionable kinases in lung cancer cells. Citation Format: Natalia J. Sumi, Bin Fang, Lily L. Rix, Muhammad Ayaz, Fumi Kinose, Eric A. Welsh, Steven A. Eschrich, Harshani R. Lawrence, John M. Koomen, Eric B. Haura, Uwe Rix. Integrated functional proteomics of MET/VEGFR inhibitors reveals complex mechanism of action of foretinib in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 221. doi:10.1158/1538-7445.AM2017-221