Abstract 3888: Systematic characterization of aberrant signaling induced by oncogenic fusions in non-small cell lung cancer

Abstract

Abstract Recent studies have greatly expanded the knowledge about genetic alterations in non-small cell lung cancer (NSCLC). Gene fusions involving the tyrosine kinases ALK, ROS and RET are found in 5 - 10% of NSCLC patients and are considered as oncogenic drivers. However, the cellular signaling activated downstream of oncogenic fusions has not been systematically investigated and it remains unclear if different fusions induce specific targetable signaling patterns. In this study we have employed quantitative mass spectrometry (MS)-based phosphoproteomics to systematically characterize the signaling induced by oncogenic fusions frequently found in NSCLC. To this end, cDNA sequences of oncogenic fusions containing the tyrosine kinases ALK, ROS and RET and different fusion partners were constructed and ectopically expressed in HEK 293 and lung cancer cell lines. Subsequently, tyrosine phosphorylated peptides were enriched and identified by LC-MS. Stable isotope labeling with amino acids in cell culture was employed to precisely quantify the abundance of phosphorylation sites in cells expressing different oncogenic fusions. Our data demonstrate that all investigated oncogenic fusions induce massive tyrosine phosphorylation of proteins and lead to increased phosphorylation of the majority of all quantified phospho-tyrosine sites. For instance, we quantified ∼1300 unique phosphotyrosine sites from HEK 293 cells expressing EML4-ALK. Of those, 952 increased more than 4-fold in abundance. Comparative analyses of the signaling patterns allowed defining kinase-specific phosphorylation signatures. In addition, comparison of signaling patterns induced by different fusions permitted us to investigate the contribution of the non-kinase fusion partner to the downstream signaling. We were able to identify several phosphorylation events that were associated with specific non-kinase fusion partners. For example, kinase fusions containing the kinesin-1 heavy chain (KIF5B) specifically led to phosphorylation of cytoskeletal proteins, including cytoskeleton-associated protein 5 (CKAP5). We are currently investigating the functional role of these phosphorylation events for the oncogenic transformation. In summary, we demonstrate that MS-based phosphoproteomics is a powerful tool to investigate aberrant signaling induced by oncogenic fusions in non-small cell lung cancer. Importantly, we find that the kinases involved in the oncogenic fusions largely define the downstream signaling and that the investigated fusion partners only exhibited a minor effect on the downstream signaling. Citation Format: Sebastian A. Wagner, Petra Beli, Hubert Serve. Systematic characterization of aberrant signaling induced by oncogenic fusions in non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3888.