archives@tulane.edu / Triple negative breast cancers (TNBCs) are clinically and biologically aggressive, with higher recurrence and metastasis rates compared to other subtypes. Acquisition of a mesenchymal and migratory cell phenotype is consequential process that promotes metastasis. There are no clinically approved small molecule targeted therapies for TNBC; kinases are effective drug targets in cancer research. Although some kinases are known to regulate the mesenchymal phenotype, a large subset of the human kinome is understudied. There are many approaches to discovering novel kinase targets in cancer. Here, a phenotypic screen approach is described to identify understudied kinases using the Published Kinase Inhibitor Set (PKIS). Initial screens using TNBC cell lines (MDA-MB-231, BT549 and MDA-MB-157) identified 36 hits representative of twelve kinase inhibitor chemotypes based on reversal of the mesenchymal cell morphology. Our hits were further prioritized based on gene expression changes of the epithelial marker E-cadherin and migratory behavior. Active compounds were confirmed to reverse EMT on transcript and protein levels with qRT-PCR and Western blot. When pharmacologically similar compounds were more closely examined, different effects on cancer biology were observed (‘active’ versus ‘inactive’ compounds). Based on these observations, a kinase array was employed to compare both the active and inactive compounds to demonstrate how to identify candidate kinases responsible for the EMT reversal. Using this screening approach, small molecule inhibitors from the PKIS library (GSK346294A, GSK448459A, GSK237700A) were identified that were pharmacologically similar that reversed the mesenchymal phenotype in TNBC. These compounds have different biological effects in TNBC, despite having similar pharmacophores. Differential effects of the PKIS compounds on transwell migration, gene (qRT-PCR) and protein (Western blot) expressions, and mammosphere formation in TNBC cells was observed. In follow-up in vivo studies, our most active compound (GSK346294A) suppressed tumorigenesis and metastasis. RNA-sequencing confirmed downregulated pathways induced by GSK346294A treatment in TNBC cells included EMT, cytoskeletal rearrangement and cell cycle regulation. Because these compounds have different off-target activities, this approach can be used to identify candidate unique kinases responsible for the observed effects. NEK5 was one of these kinases candidates. NEK5 function remains understudied in cancer, and even more understudied in breast cancer. This study is the first, to our knowledge, to describe the function of NEK5 in breast cancer, specifically its roles in acquisition of a mesenchymal and migratory cell phenotype. Overexpression of NEK5 promotes a migratory and mesenchymal phenotype, and knockdown with a shRNA construct suppresses this migratory behavior. Data obtained using both qRT-PCR of the knockdown and overexpression cell lines, and follow-up RNA sequencing, revealed NEK5 regulates the PLAU/PAI-1/SRC axis. Furthermore, a role for NEK5 in resistance to SRC-targeting anticancer agents is demonstrated. The work described here demonstrates the utility of a novel approach to identify understudied kinases in cancer, and characterization of these kinases has potential impact in other metastatic diseases not limited to breast cancer. / 1 / Margarite Matossian