SRP477727 PRJNA1051952 GSE250059 Centrosomal Localization of DNAJ-PKAc Fusion Heightens PLK1 Inhibitor Sensitivity in Fibrolamellar Cancer Fibrolamellar carcinoma (FLC), a rare and fatal liver cancer lacking effective drug therapy, is driven by the DNAJ-PKAc fusion oncoprotein. However, the underlying mechanism of DNAJ-PKAc's role in FLC tumor growth remains enigmatic. Employing an unbiased systems-based approach, we uncovered a new role of DNAJ-PKAc oncoprotein in FLC and identified downstream kinases involved in this process. Functional screening, coupled with computational analysis, highlighted Polo-like kinase 1 (PLK1) as vital for FLC cell viability. Genetic and pharmacological PLK1 inhibition significantly reduced FLC cell growth, inducing apoptosis. Further studies showed DNAJ-PKAc's centrosomal presence and direct interaction with PLK1, revealing a novel mechanism that promotes PLK1 activation and mitotic progression. Clinical-grade PLK1 inhibitors effectively suppressed FLC tumor growth across multiple preclinical models, including patient-derived xenograft and an orthotopic model of FLC, suggesting promising therapeutic avenues. Our findings underscore the role of DNAJ-PKAc in rewiring signaling networks and highlight valuable clinical implications for PLK1-targeted therapies for FLC. Overall design: Bulk-RNA seq of Fibrolamellar tumors, non-tumor livers, patient-derived xenograft tumors, and cancer cells. *************************************************************** Submitter states that missing raw files are due to file loss. Sample "FLC1, rep2". *************************************************************** GSE250059