Proteome-Wide Analysis Of Echinoderm Microtubule Associated Protein Like 4-Anaplastic Lymphoma Kinase (Eml4-Alk) Network In Lung Cancer

The translocation of echinoderm microtubule associated protein like 4 – anaplastic lymphoma kinase (EML4-ALK) defines a unique, new subset of patients with non small cell lung cancer (NSCLC). It represents a novel diagnostic biomarker and therapeutic target for NSCLC. PF 02341066 is a ALK small tyrosine kinase inhibitor that has demonstrated high clinical activity in EML4-ALK- positive NSCLC patients. We sought to characterize the signaling network used by EML4-ALK and reveal potential new therapeutic targets. We used tandem affinity purification (TAP) and liquid chromatography-mass spectrometry (LC-MS/MS) to map protein-protein interactions and anti-phosphotyrosine immunoprecipitation coupled with LC-MS/MS to map tyrosine phosphorylation. We selected the human NSCLC H3122 cell line for our studies as it is sensitive to PF 02341066 and harbors ELM4-ALK. Quantitative western blotting results show that activities of MET, ALK, AKT, ERK and STAT3 were inhibited by treatment with PF 02341066. Proteome-wide analysis of tyrosine phosphorylation of the ALK signaling pathway were examined. In total, 311 tyrosine phosphorylated proteins (487 unique phosphorylated sites) were identified. Of those, 73 pTyr sites (62proteins) were upregulated and 78 pTyr sites (58 proteins) were downregulated by 1uM PF02341066. Using TAP, we identified 13 proteints interacting with EML4-ALK including GRB2 and SHC1. Core interactions were verified by coupling of immunoprecipitation to Western blot and mass spectrometry. A comprehensive physical ALK network was generated by integrating both phosphoproteomic data and interactional partners using pathway analysis algorithms. Two adaptors, SHC1and GAB2, are suggested as the first outlets of signaling propagation from ALK. Other sublevels directly connected with ALK may allow further understanding the mechanism of ‘ALK addiction’. A sub-network composed of 35 cell death associated proteins was created. An ongoing functional analysis including siRNA and small inhibitor screen on this small network should validate druggable targets. This network approach has potential translational applications to identify novel approaches to treatment of ALK addicted lung cancers and could be useful in patients with acquired resistance to ALK inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1627. doi:10.1158/1538-7445.AM2011-1627