Modeling Hepatoblastoma using pluripotent stem cell derived hepatocyte-like cells

项目摘要/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%。 儿童时期的恶性肿瘤。肝母细胞瘤的五年存活率为59%-74%； 儿童癌症，主要由手术控制决定。因此，更好地理解 这种癌症的潜在机制将使改善和更有针对性的临床研究成为可能。 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 治疗主要通过组织学和免疫组织化学染色来指导。目前存在的分子缺陷 在肝母细胞瘤中已被发现，但他们的测试和将靶向治疗纳入临床治疗 尚未被采用，因此根治性治疗很大程度上只包括手术。我们的中央 假设是肝母细胞瘤中存在的最常见的突变，CTNNB1引起 分化缺陷，促进细胞增殖，并以细胞特有的方式发生肿瘤。因此， 在相关和适当的细胞类型中缺乏强大的肝母细胞瘤模型已显著受损 我们能够更好地理解这种癌症的分子基础，并发展出更好的 和有效的临床治疗。这些肿瘤具有相对的遗传简单性，因此我们很容易 产生包含遗传相似和相关突变的细胞系。我们建议使用诱导式 利用CRISPR构建多能干细胞(PSC)、PSC来源的肝母细胞和肝细胞样细胞 产生β-连环蛋白突变的技术，这是肝母细胞瘤中最常见的突变基因。在……里面 我们的初步数据，我们已经使用CRISPR/Cas9技术生成了类似于 CTNNB1基因突变。这些PSC株系WNT信号的激活增加，但分化正常 转化为肝母细胞样细胞，在这一点上，这些细胞增殖并有分化缺陷。突变型 含CTNNB1的肝母细胞和肝细胞样细胞在体内形成肿瘤。拟议的工作将 确定突变型CTNNB1在肝母细胞瘤形成中的作用。我们将研究CTNNB1- 含PSC来源的肝母细胞和肝细胞样细胞的突变体及其效应的测定 CTNNB1对细胞分化、增殖、转录和代谢特征的影响，以及 体内的肿瘤发生。然后，我们将使用可诱导的Cas9平台动态生成突变 确定CTNNB1突变形成的发育阶段是否会导致进一步的下游 细胞分化、表型和肿瘤发生的改变。最后，我们将确定是否 除了突变所在的特定细胞类型之外，重合突变的时间和顺序 会影响肝母细胞瘤的形成。肝母细胞瘤形成的关键调控因子剖析 将使我们能够识别和靶向调节肝母细胞瘤发展和生长的途径，并将导致 以确定肝母细胞瘤治疗的下游调节因子和临床靶点。

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.