Development of cell-based models for fibrolamellar carcinoma

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PROJECT SUMMARY Fibrolamellar carcinoma (FLC) is a liver malignancy, most commonly occurring in children or young adults that carries an exceptionally poor prognosis given minimal response to conventional chemotherapeutic agents. Patients who undergo complete surgical resection are at high risk of recurrence and most will succumb to their disease, as demonstrated by a 5-year event-free survival of less than 10%. The genetic driver in FLC was first described in 2014 as a deletion event in chromosome 19 resulting in the DNAJB1-PRKACA (DP) gene fusion. Although the exact mechanisms by which this fusion protein results in cancer are unclear at this time, studies have demonstrated that induction of this fusion gene in mice is sufficient for the development of FLC tumors, supporting its role as a cancer driver. The role of fusion genes in various other cancers such as leukemia and sarcoma have been described and approaches to target the responsible fusion proteins have demonstrated clinical efficacy. Therefore, the overarching goal of our proposal is to develop cell-based tools for therapeutic targeting of DP protein since such models are currently lacking. Our first aim will be to develop a high throughput drug screening cell-based model by further modifying a novel cell line (HEK-DP) developed in our lab. HEK-DP is a cell line created through CRISPR/Cas9 editing to engineer the DP gene fusion in HEK293T cells. We have previously performed extensive transcriptomic, proteomic, and mitochondrial dynamic studies on the HEK-DP cells. We plan to modify the HEK-DP cells into a luminescent reporter for the activity of DP protein based on LINC00473 promoter. LINC00473 is a long non-coding RNA that is upregulated in FLC tumors as well as the HEK-DP cells. Our studies demonstrate that DP activity directly regulates LINC00473 expression in the HEK-DP cells and therefore, we anticipate that developing this reporter cell line will provide a novel tool for high throughput screening of drugs that can target DP activity. Our second aim will focus on editing human-derived liver progenitor cells to express DP fusion gene (Chol-Org-DP) using a similar strategy to HEK-DP engineering. Since the FLC cell origin is similar to the liver progenitor cells, the Chol-Org-DP cells will provide us with a powerful novel cell model to study the oncogenic mechanisms of DP as well as therapeutic screening.