Targeting understudied kinases in cancer cell plasticity and drug resistance

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Project Summary Metastasis is responsible for 90% of cancer-related deaths and novel drugs that target this disease stage are urgently needed. Metastatic cancer cells exploit developmental pathways to acquire a mesenchymal stem cell-like state. This epithelial-mesenchymal transition (EMT) enhances cancer cell motility, invasion and survival. Inhibition of EMT, in turn, can block metastasis and, strikingly, can sensitize cancer cells to chemotherapy. A critical goal of cancer drug discovery, therefore, is to identify druggable proteins that promote the EMT. In a detailed pharmacoproteomics study of hepatocellular carcinoma (HCC), we demonstrated that protein kinases are critical drivers of EMT and drug resistance. Remarkably, our study predicted 71 kinases to promote EMT, among them six understudied IDG targets. We will evaluate the translational potential of these six IDG kinases, i.e. NUAK2, CAMK1D, STK17A, STK17B, STK32B and CDK10, by scrutinizing their function in EMT and drug resistance. We will use targeted gene disruption with CRISPR/Cas9 to knock-out (KO) these kinases in mesenchymal HCC cell lines and ectopically express them in epithelial HCC lines. Impact on EMT state and drug resistance will be tested (1) by qPCR and western blot analysis of 15 reliable EMT markers, (2) in a robust trans-well invasion assay, and (3) a small-scale drug screen. Combining efficient KO with these orthogonal assays will ensure unambiguous specification of kinase roles in HCC cell EMT and drug response.