Modeling Hepatoblastoma using pluripotent stem cell derived hepatocyte-like cells

使用多能干细胞衍生的肝细胞样细胞模拟肝母细胞瘤

• 批准号: 10222621
• 负责人: Robert E Schwartz
• 金额: $ 38.27万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-19 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10222621
• 关键词: Address; Adopted; Automobile Driving; CRISPR/Cas technology; CTNNB1 gene; Cause of Death; Cell Line; Cell Proliferation; Cells; Cessation of life; Child; Classification; Clinical; Communities; DNA Sequence Alteration; Data; Defect; Development; Disease; Dissection; Endoderm; Event; Generations; Genes; Genetic; Goals; Growth and Development function; Hepatic; Hepatoblastoma; Hepatocyte; Histology; Human; Impairment; Incidence; Knowledge; Lead; Liver neoplasms; Malignant Childhood Neoplasm; Malignant Neoplasms; Malignant neoplasm of liver; Modeling; Molecular; Mutate; Mutation; Operative Surgical Procedures; Pathway interactions; Phenotype; Play; Pluripotent Stem Cells; Primary carcinoma of the liver cells; Proliferating; Research; Role; Signal Transduction; Specific qualifier value; Stains; Survival Rate; Testing; WNT Signaling Pathway; Work; anticancer research; beta catenin; cell type; cellular engineering; clinically relevant; curative treatments; fetal; human pluripotent stem cell; human stem cells; improved; in vivo; induced pluripotent stem cell; insight; interest; metabolomics; mutant; nonsynonymous mutation; novel; novel strategies; overexpression; stem cell model; targeted treatment; tool; transcriptomics; tumor; tumorigenesis

Project Summary/Abstract Hepatoblastoma is the most common liver neoplasm in children and accounts for approximately 1% of all childhood malignancies. Five-year survival for hepatoblastoma is 59-74%; one of the lowest survival rates for childhood cancer and is dictated largely by surgical control. Therefore, an improved understanding of the mechanisms underlying this cancer will enable the development of improved and more targeted clinical therapies. Hepatoblastoma is an embryonal tumor with distinct phenotypes and whose classification and treatment is guided largely by both histology and immunohistochemical staining. The molecular defects present in hepatoblastoma have been identified but their testing and inclusion of targeted therapies into clinical therapy have not been adopted and consequently curative treatment largely consists of surgery alone. Our central hypothesis is that the most common mutation present in hepatoblastoma tumors, CTNNB1 cause differentiation defects, increased cellular proliferation, and tumorigenesis in a cell-specific manner. Therefore, the lack of robust models of hepatoblastoma in a relevant and appropriate cell type has significantly impaired our ability to better understand the molecular underpinnings underlying this cancer and to develop improved and effective clinical therapies. These tumors have relative genetic simplicity, consequently we can readily generate cell lines containing genetically similar and relevant mutations. We propose to use induced pluripotent stem cells (PSC) and PSC derived hepatoblasts and hepatocyte-like cells engineered using CrispR technology to generate mutations in β-catenin, the most common gene that is mutated in hepatoblastoma. In our preliminary data, we have used CrispR/Cas9 technologies to generate human PSC lines with similar mutations in CTNNB1. These PSC lines have increased activation of WNT signaling but differentiate normally into hepatoblast-like cells, at which point, these cells proliferate and have differentiation defects. Mutant CTNNB1 containing hepatoblasts and hepatocyte-like cells form tumors in vivo. The proposed work will determine the role that mutant CTNNB1 plays in driving hepatoblastoma formation. We will study CTNNB1- mutant containing PSC derived hepatoblasts and hepatocyte-like cells and determine the effect of mutant CTNNB1 on cellular differentiation, proliferation, the transcriptomic and metabolomic signature, and tumorigenesis in vivo. We will then use inducible Cas9 platforms to dynamically generate mutations to determine whether the developmental stage at which CTNNB1 mutations form leads to further downstream alterations in cellular differentiation, phenotype, and tumorigenesis. Finally we will determine whether the timing and order of coincident mutations in addition to the specific types of cells in which the mutations are generated impacts hepatoblastoma formation. The dissection of key regulators of hepatoblastoma formation will enable us to identify and target pathways regulating hepatoblastoma development and growth and will lead to the identification of downstream regulators and clinical targets for hepatoblastoma treatment.

项目总结/摘要 肝母细胞瘤是儿童最常见的肝脏肿瘤，约占所有肿瘤的1%。 儿童恶性肿瘤肝母细胞瘤的五年生存率为59-74%;肝母细胞瘤的最低生存率之一。 儿童癌症，主要取决于手术控制。因此，更好地了解 这种癌症的潜在机制将使开发改进的和更有针对性的临床治疗成为可能。 治疗肝母细胞瘤是一种具有不同表型的胚胎性肿瘤，其分类和 治疗主要由组织学和免疫组织化学染色指导。存在的分子缺陷 但他们的测试和纳入靶向治疗的临床治疗 没有被采用，因此治愈性治疗主要包括单独的手术。我们的中央 一种假说认为，肝母细胞瘤中最常见的突变CTNNB 1导致了 分化缺陷、增加的细胞增殖和以细胞特异性方式的肿瘤发生。因此，我们认为， 在相关和适当的细胞类型中缺乏强有力的肝母细胞瘤模型， 我们能够更好地了解这种癌症的分子基础， 有效的临床治疗。这些肿瘤具有相对的遗传简单性，因此我们可以很容易地 产生含有遗传相似和相关突变的细胞系。我们建议使用诱导 使用CrispR工程化的多能干细胞（PSC）和PSC衍生的成肝细胞和肝细胞样细胞 技术来产生β-连环蛋白的突变，β-连环蛋白是肝母细胞瘤中最常见的突变基因。在 根据我们的初步数据，我们已经使用CrispR/Cas9技术来产生具有类似于 CTNNB 1突变。这些PSC系具有增加的WNT信号传导激活，但正常分化 成肝细胞样细胞，此时，这些细胞增殖并具有分化缺陷。突变体 含有CTNNB 1的成肝细胞和肝细胞样细胞在体内形成肿瘤。拟议的工作将 确定突变CTNNB 1在驱动肝母细胞瘤形成中的作用。我们将研究CTNNB 1- 含有PSC衍生的成肝细胞和肝细胞样细胞的突变体，并确定突变体的作用 CTNNB 1对细胞分化、增殖、转录组学和代谢组学特征的影响，以及 体内肿瘤发生。然后，我们将使用诱导型Cas9平台动态生成突变， 确定CTNNB 1突变形成的发育阶段是否导致进一步的下游突变， 细胞分化、表型和肿瘤发生的改变。最后，我们将确定 除了发生突变的特定类型的细胞之外，同时发生突变的时间和顺序 产生的影响肝母细胞瘤的形成。肝母细胞瘤形成关键调控因子的研究 将使我们能够识别和靶向调节肝母细胞瘤发育和生长的途径， 涉及肝母细胞瘤治疗的下游调节剂和临床靶点的鉴定。