Targeting PLK1 signaling for the treatment of fibrolamellar carcinoma

PROJECT SUMMARY Fibrolamellar carcinoma (FLC) is a childhood liver cancer with a high case mortality rate. Patients with FLC typically present with advanced disease, as there are no early warning signs. Thus, a cure by surgical resection is rarely attainable. In addition, FLCs are notoriously resistant to chemotherapies and other targeted therapies

currently approved for liver cancer, leading to a 5-year survival of just 30%. New therapeutic strategies that counteract the molecular signaling events that go awry in FLC are urgently needed. FLC is characterized by a fusion event resulting in a novel chimeric protein that joins the N-terminal domain of DNAJ with the catalytic

subunit of protein kinase A (PKAc) in hepatocytes. However, the underlying mechanism by which DNAJ-PKAc drives FLC tumor growth remains unknown. This project's overall goal is to apply an unbiased systems-based approach to identify and validate druggable signaling networks that regulate the growth of DNAJ-PKAc-

expressing FLC cells and uncover a mechanistic understanding of how DNAJ-PKAc chimeric protein drives FLC. The paucity of preclinical models such as immortalized primary human FLC cell lines has precluded many investigators. Our lab has established three new model systems to address this significant gap: patient-derived

cell lines bearing the FLC gene fusion, organotypic cultures, and patient-derived xenograft (PDX) mice. Utilizing these model systems, we carried out a systems-pharmacology-based functional kinase inhibitor screening in FLC cells and normal hepatocytes. We identified and confirmed the role of PLK1 kinases as essential for the

growth of FLC cells. Genetic depletion or pharmacological inhibition of PLK1 selectively reduces the growth of multiple patient-derived FLC cell lines and the viability of FLC organotypic tissue slices. Further, treatment of the FLC tumor with PLK1 inhibitor significantly reduced the tumor growth in the PDX model. PLK kinases are key

regulators of centrosome maturation and mitosis. Follow-up experiments suggest that DNAJ-PKAc chimera localizes to the centrosomes where it physically interacts with PLK1.Thus, we hypothesize that the heightened sensitivity of the FLC cells to PLK inhibition stems from the localization of the DNAJ-PKAc fusion protein to the

centrosome, its association with the PLK1 complex, thereby enhancing the activation of PLK1 and promoting mitotic progression. We propose to 1. uncover molecular mechanisms of how DNAJ-PKAc fusion alters PLK1 activation and function, and 2. evaluate the efficacy of clinical-grade PLK1 inhibitors alone and in combination

with chemotherapy in preclinical models.Functional analyses will highlight the mechanistic insights by which DNAJ-PKAc drives FLC tumor progression and the role of the PLK1 signaling complex in FLC survival, thus deepening our understanding of disease pathogenesis. Our cross-disciplinary team consisting of Drs. Gujral,

Scott, and Yeung represent a cohesive collaboration that brings systems biology, PKA biochemistry, and state- of-art human-derived FLC cancer models to address this deadly disease. Our findings have translational significance as they will provide a rationale for targeting critical signaling nodes that sustain FLC tumors' survival.