Lung cancer is associated with high prevalence and mortality, and despite significant successes with targeted drugs in genomically defined subsets of lung cancer and immunotherapy, the majority of patients currently does not benefit from these therapies. Through a targeted drug screen, we found the recently approved multi-kinase inhibitor midostaurin to have potent activity in several lung cancer cells independent of its intended target, PKC, or a specific genomic marker. To determine the underlying mechanism of action we applied a layered functional proteomics approach and a new data integration method. Using chemical proteomics, we identified multiple midostaurin kinase targets in these cells. Network-based integration of these targets with quantitative tyrosine and global phosphoproteomics data using protein-protein interactions from the STRING database suggested multiple targets are relevant for the mode of action of midostaurin. Subsequent functional validation using RNA interference and selective small molecule probes showed that simultaneous inhibition of TBK1, PDPK1 and AURKA was required to elicit midostaurin's cellular effects. Immunoblot analysis of downstream signaling nodes showed that combined inhibition of these targets altered PI3K/AKT and cell cycle signaling pathways that in part converged on PLK1. Furthermore, rational combination of midostaurin with the potent PLK1 inhibitor BI2536 elicited strong synergy. Our results demonstrate that combination of complementary functional proteomics approaches and subsequent network-based data integration can reveal novel insight into the complex mode of action of multi-kinase inhibitors, actionable targets for drug discovery and cancer vulnerabilities. Finally, we illustrate how this knowledge can be used for the rational design of synergistic drug combinations with high potential for clinical translation.

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Rearranged during transfection (RET), a receptor tyrosine kinase that is activated by the glial cell line-derived neurotrophic factor family ligands (GFLs), plays a crucial role in the development and function of the nervous system and additionally is required for kidney development and spermatogenesis. RET encodes a transmembrane receptor that is 20 exons long and produces two known protein isoforms differing in C-terminal amino acid composition, referred to as RET9 and RET51. Studies of human pheochromocytomas identified two additional novel transcripts involving the skipping of exon 3 or exons 3, 4, and 5 and are referred to as RET ⌬E3 and RET ⌬E345 , respec- higher baseline autophosphorylation, specifically on the catalytic tyrosine, Tyr 905 , and also on one of the most important signaling residues, Tyr 1062 . These data provide the first evidence for a physiologic role of these isoforms in RET pathway function.RET is a receptor tyrosine kinase that is critical for kidney morphogenesis, spermatogenesis, and development of the nervous system (1-4). RET is activated by a family of four growth factors known as the glial cell line-derived neurotrophic factor (GDNF) 2 family ligands (GFLs), which includes GDNF, neurturin, artemin, and persephin (1). Each GFL binds to one of four cognate glycosylphosphatidylinositol-anchored co-receptors known as the GDNF family receptor ␣s (GFR␣s) (2, 5). The GFL-GFR␣ complex binds to RET, inducing RET dimerization and subsequent autophosphorylation on multiple tyrosine residues within the intracellular tyrosine kinase domain. This enhances tyrosine kinase activity and initiates the association of adaptor proteins and enzymes that trigger multiple second messenger cascades (6). The presence of two major Ret isoforms, RET9 and RET51, has been extensively described in the literature, and a third isoform, RET43, has also been observed in humans (7-9). Ret is 20 exons long, and Ret9 and Ret51 transcripts differ in alternative splicing of intron 19. Intron 19 in the Ret51 transcript is excised properly, whereas, in the Ret9 transcript, the intron is retained, changing the reading frame and inserting a premature stop codon into the amino acid sequence. This creates a unique nine-amino acid C-terminal sequence for RET9 and a unique 51-amino acid C-terminal sequence for RET51. Interestingly, these two different isoforms display marked differences in their degradation and function (7, 10, 11).Three additional Ret transcripts have been reported in various tumor sources as well as adult human tissues (12). These novel transcripts are a product of exon skipping in the 5Ј region of RET, which encodes for the extracellular domain of the protein. Skipping of exons 3 (RET ⌬E3 ) or exons 3, 4 and 5 (RET ⌬E345) gives rise to transcripts that encode for full-length Ret proteins but with deletions in the extracellular domain, specifically cadherin-like domain 1 (CLD1) or CLD1-3, respectively. These deletions are hypothesized to change the extracellular domain structure as well as binding to GFL-GFR...