Development of cell-based models for fibrolamellar carcinoma

项目摘要 纤维板层癌（FLC）是一种肝脏恶性肿瘤，最常见于儿童或年轻人， 由于对常规化疗剂的反应最小，因此预后极差。 接受完全手术切除的患者复发的风险很高，大多数患者将死于他们的癌症。 疾病，如5年无事件生存率低于10%所示。FLC中的遗传驱动因素首先是 在2014年被描述为19号染色体中的缺失事件，导致DNAJB 1-PRKACA（DP）基因融合。 虽然这种融合蛋白导致癌症的确切机制目前还不清楚，但研究表明， 已经证明在小鼠中诱导这种融合基因足以发展FLC肿瘤， 支持其作为癌症驱动因素的作用。融合基因在各种其他癌症中的作用，如白血病和 已经描述了肉瘤，并且已经证明了靶向负责融合蛋白的方法 临床疗效因此，我们建议的首要目标是开发基于细胞的治疗工具， 靶向DP蛋白，因为目前缺乏这样的模型。我们的第一个目标将是发展一个高 通过进一步修饰我们开发的新型细胞系（HEK-DP）， 实验室HEK-DP是通过CRISPR/Cas9编辑创建的细胞系，以在HEK 293 T中工程化DP基因融合 细胞我们以前进行了广泛的转录组学，蛋白质组学和线粒体动态研究 在HEK-DP细胞上。我们计划将HEK-DP细胞修饰成DP活性的发光报告基因 基于LINC 00473启动子的蛋白。LINC 00473是在FLC中上调的长非编码RNA 肿瘤以及HEK-DP细胞。我们的研究表明DP活性直接调节LINC 00473 因此，我们预计开发这种报告细胞系将提供一种在HEK-DP细胞中表达的新方法。 用于高通量筛选可靶向DP活性的药物的新工具。我们的第二个目标将集中在 使用类似的策略编辑人源性肝祖细胞以表达DP融合基因（Chol-DP-DP 到HEK-DP工程。由于FLC细胞来源类似于肝祖细胞，因此Chol-DP-DP细胞 将为我们提供一个强大的新的细胞模型，以研究DP的致癌机制，以及 治疗筛选

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.