Development of cell-based models for fibrolamellar carcinoma

纤维板层癌细胞模型的开发

基本信息

批准号：
10656539
负责人：
KHASHAYAR VAKILI
金额：
$ 20.81万
依托单位：
BOSTON CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-01 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10656539
关键词：
Animals Biological Assay Biology CRISPR/Cas technology Catalytic Domain Cell Culture Techniques Cell Density Cell Line Cell fusion Cell model Cell-Matrix Junction Cells Cellular biology Chemistry Child Chimeric Proteins Chromosome 19 Collection Cyclic AMP-Dependent Protein Kinases Development Disease Disease-Free Survival Dose Engineering Event Excision Exons Fibrolamellar Hepatocellular Carcinoma Fluorescence-Activated Cell Sorting Gene Fusion Genes Genetic Goals Guide RNA Heat shock proteins Heterozygote Human Human Engineering In Vitro Incubated Kinetics Libraries Liver Luciferases Malignant Childhood Neoplasm Malignant Epithelial Cell Malignant Neoplasms Malignant neoplasm of liver Measurement Measures Methods Mitochondria Modeling Mus Oncogenic Operative Surgical Procedures Organoids Output PRKACA gene Patients Pharmaceutical Preparations Prognosis Proteins Proteomics Protocols documentation Reaction Recurrence Regimen Reporter Reproducibility Research Proposals Role Secondary to Small Interfering RNA Testing Therapeutic Therapeutic Agents Time Transcript Transfection Untranslated RNA chemotherapeutic agent cholangiocyte clinical efficacy fusion gene high risk high throughput screening high-throughput drug screening inhibitor interest knock-down leukemia luminescence novel overexpression pharmacologic promoter protein kinase A kinase response sarcoma screening single nucleus RNA-sequencing small hairpin RNA stem cells therapeutic target therapeutically effective tool transcriptome transcriptomics tumor young adult

项目摘要

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.

项目摘要纤维状癌（FLC）是一种肝恶性肿瘤，最常见于儿童或年轻人中鉴于对常规化学治疗剂的反应最少，预后的预后异常差。进行完整手术切除的患者复发的高风险，大多数会屈服于他们的疾病，5年的无事件生存率低于10％。 FLC中的遗传驱动器首先在2014年描述为染色体19中的缺失事件，导致DNAJB1-PRKACA（DP）基因融合。尽管目前尚不清楚这种融合蛋白导致癌症的确切机制，但研究已经证明，小鼠中这种融合基因的诱导足以发展FLC肿瘤，支持其作为癌症驱动力的角色。融合基因在白血病等其他各种癌症中的作用已经描述了肉瘤，并靶向负责任的融合蛋白的方法已证明临床功效。因此，我们建议的总体目标是开发基于细胞的治疗工具由于目前缺乏此类模型，因此靶向DP蛋白。我们的第一个目标是发展高通过在我们的实验室。 HEK-DP是通过CRISPR/CAS9编辑创建的细胞系，用于设计HEK293T中的DP基因融合细胞。我们以前已经进行了广泛的转录组，蛋白质组学和线粒体动力学研究在HEK-DP细胞上。我们计划将HEK-DP细胞改成发光报告基因的DP活性基于linc00473启动子的蛋白质。 linc00473是一个长的非编码RNA，在FLC中上调肿瘤以及HEK-DP细胞。我们的研究表明，DP活性直接调节Linc00473 因此，在HEK-DP细胞中的表达，因此，我们预计开发此报告基细胞系将提供用于高通量筛查的新工具，可以针对DP活性。我们的第二个目标将重点放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-Org-DP）进行HEK-DP工程。由于FLC细胞的起源与肝脏祖细胞相似，因此Chol-Org-DP细胞将为我们提供强大的新型细胞模型，以研究DP的致癌机制治疗筛查。