Investigating the role of the Hippo pathway in epithelial-mesenchymal transition and drug resistance

Abstract

The Hippo pathway is an evolutionarily conserved signaling cascade and comprises the core Hippo kinases MST1/2 and LATS1/2, which phosphorylate the transcriptional YES associated protein (YAP), promoting its nuclear exclusion and degradation. Excess YAP activity is oncogenic and is commonly observed in various human malignancies. Despite increased understanding of phenotypes associated with Hippo aberration and excess YAP activity, specific mediators of YAP-driven phenotypic changes remain elusive. In this thesis, I present two projects investigating the kinome signaling downstream of YAP and a survey of YAP-activity mediated changes to drug response. In the first project, I describe the first comprehensive survey of kinome signaling downstream of YAP. YAP hyper-activity results in widespread changes to the activity state of the kinome. I identified the ZAK kinase as highly phosphorylated in YAP-driven cells and identified a strong correlation with this kinase in mesenchymal-like cells. Inhibition of ZAK reversed the mesenchymal phenotype, and overexpression of ZAK resulted in epithelial-mesenchymal transition (EMT). Analysis of The Cancer Genome Atlas (TCGA) data shows a strong correlation between ZAK expression and mesenchymal-characterized tumors and Hippo pathway output. These data implicate the ZAK kinase as a critical mediator of YAP-driven EMT and provide an in-depth look at kinome signaling changes resulting from YAP activity. In the second project, I investigated YAP-mediated changes to the cellular response to a collection of >2000 compounds. To uncover therapeutic vulnerabilities caused by increased YAP activity, I analyzed results from a quantitative, high-throughput chemical screen in Panc02.13 cells, a pancreatic ductal adenocarcinoma (PDAC) cell line. I confirmed previously annotated resistances and sensitivities conferred by YAP-activity and identified a significant resistance to MEK inhibition (MEKi) in YAP-driven cells. This resistance was robust; YAP-driven cells survived MEKi treatment even with a combined inhibition of common compensatory signaling through pro-survival kinase, AKT and receptor tyrosine kinase, AXL. Follow-up studies identified alterations in the expression of at least four apoptotic regulators and EMT transcription factors. Thus, I posit that YAP-induced EMT enables resistance to MEK inhibitors through kinome re-wiring and apoptotic re-programming changes. These results may help explain the failure of MEKi targeted therapy in clinical trials treating PDAC. Overall, these studies provide a comprehensive overview of the signaling landscape downstream of aberrant YAP activity and may be useful in understanding how aberrant Hippo signaling results in resistance to targeted therapy.