Abstract 2882: Elucidation of the mechanism of action of the multi-kinase inhibitor midostaurin in NSCLC

Abstract

Abstract Background: Non-small cell lung cancer (NSCLC) constitutes a strong unmet medical need in spite of recent successes with targeted drugs and immunotherapy in some subsets. We had observed the recently FDA-approved drug midostaurin to exhibit potent anticancer activity in a subset of NSCLC cell lines that was unrelated to its known targets FLT3 or PKC. We here explored a new repurposing opportunity and investigated the mechanism of action (MoA) of midostaurin in NSCLC utilizing a multilayered functional proteomics approach. Methods: Chemical proteomics identified kinase targets and global and tyrosine phosphoproteomics generated a network view of intracellular signaling upon midostaurin treatment. Functional validation was performed using RNA interference and selective small-molecule probes. Cell viability assays were carried out using CellTiterGlo and colony formation assays. Induction of apoptosis was determined by immunoblotting and fluorescent microscopy. Flow cytometry assessed cell cycle stages. Signaling changes were determined by immunoblotting. Results: Viability screening confirmed anticancer activity in several midostaurin-sensitive NSCLC cell lines at clinically relevant concentrations independent of its canonical targets. Using chemical proteomics, we identified multiple midostaurin kinase targets. Network-based integration of the chemical proteomics and quantitative tyrosine and global phosphoproteomics data using STRING for protein-protein interactions and NetworKIN for substrate-kinase predictions revealed several nodes to be relevant for the MoA of midostaurin. Subsequent functional validation showed that simultaneous inhibition of TBK1, PDK1 and AURKA was required to mimic midostaurin's cellular effects in these cells. Immunoblotting for downstream targets of these kinases revealed PLK1 as a critical signaling effector. Rational combination of midostaurin with the more potent PLK1 inhibitor BI2536 caused strong synergy. Conclusion: Midostaurin showed activity in a subset of NSCLC cells. Combination of complementary functional proteomics approaches and subsequent network-based integration revealed novel insight into the MoA of midostaurin and NSCLC vulnerabilities. We utilized this mechanistic knowledge for the rational combination of midostaurin with the clinical PLK1 inhibitor BI2536, which is highly synergistic and has strong potential for clinical translation. Citation Format: Claudia Ctortecka, Brent Kuenzi, Bin Fang, Natalia Sumi, Victoria Izumi, Fumi Kinose, Eric Haura, John Koomen, Uwe Rix. Elucidation of the mechanism of action of the multi-kinase inhibitor midostaurin in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2882.