System analysis of p53 mutant associated cancer development with LFS patient- specific iPSCs

项目摘要/摘要 人诱导多能干细胞(IPSCs)不仅是一种很有前途的再生资源 医学，但它们作为研究人类疾病的工具的效用也在上升。具体地说，建立癌症模型 使用患者特异的IPSCs为研究人类癌症的发病机制提供了机会。在我们之前的 在《细胞2015》的《用诱导多能干细胞建立家族性癌症模型》中，我们证明了 家族性Li-Fraumeni综合征(LFS)患者来源的IPSCs类似肿瘤 培养皿中的致病机理。此外，对LFS患者骨肉瘤发展的系统分析显示， 基因H19及其印迹基因网络是正常成骨细胞分化所必需的， LFS患者成骨细胞分化过程中的调控失调。 尽管我们报道了LFS iPSC来源的H19及其印记基因网络的异常调节 成骨细胞，没有已知的临床可行的策略来靶向异常的H19及其非编码RNA 监管。为了发现临床相关和治疗靶向的分子，我继续说 研究我们的LFS iPSC来源的骨肉瘤模型并鉴定分泌型卷曲相关蛋白2 (SFRP2)作为骨肉瘤发生发展的重要致癌因子。我进一步证明了SFRP2 增加成骨细胞中Axl受体的磷酸化，这是一种已知的与结肠相关的癌基因 癌症、黑色素瘤和慢性粒细胞白血病。此外，使用CRISPR/Cas9基因编辑 技术I纠正了我们的LFS iPSC系中的p53突变。 在这项建议中，我将使用功能和比较分析来确定LFS的分子差异- 野生型和原始LFS iPSCs的等基因对照。从这一分析中，我预计将表明 用CRISPR/Cas9纠正骨肉瘤中致病的P53突变可以逆转骨肉瘤的发病机制 IPSC平台上的骨肉瘤。此外，我还将演示针对SFRP2和AXL的有效性 受体作为LFS相关骨肉瘤的潜在治疗策略。最后，检查临床 与我的方法相关，我将使用以下方法分析人骨肉瘤样本中SFRP2的表达水平 骨肉瘤组织芯片。 总体而言，拟议的研究将提供纠正的IPSC克隆，作为完美的等位基因对照 研究P53突变的作用以及由此导致的SFRP2表达和Axl的下游变化 受体磷酸化，在LFS相关骨肉瘤的发展中。这项工作不仅将扩大我们的 关于P53突变介导骨肉瘤进展的知识，但也描述了多重潜力 提高p53基因突变骨肉瘤患者临床疗效的治疗方法。

Project Summary/Abstract Human induced pluripotent stem cells (iPSCs) not only represent a promising resource for regenerative medicine, but are also rising in their utility as a tool for studying human disease. Specifically, modeling cancer using patient specific iPSCs provides an opportunity to study human cancer pathogenesis. In our previous work in Cell 2015, `Modeling Familial Cancer with Induced Pluripotent Stem Cells', we demonstrated that iPSCs derived from human familial cancer patients of Li-Fraumeni Syndrome (LFS) mimic cancer pathogenesis in a dish. Additionally, systems analysis of LFS patient osteosarcoma development revealed that the gene H19 and its imprinted gene network, which is required for normal osteoblast differentiation, become dysregulated during LFS patient osteoblast differentiation. Although we reported the abnormal regulation of H19 and its imprinted gene network in LFS iPSC-derived osteoblasts, there is no known clinically feasible strategy to target abnormal H19 and its non-coding RNA regulation. In order to uncover clinically relevant and therapeutically targetable molecules, I continued researching our LFS iPSC-derived osteosarcoma model and identified secreted frizzled-related protein 2 (sFRP2) as an important oncogenic factor of osteosarcoma development. I further demonstrated that sFRP2 increases the phosphorylation of AXL receptor in osteoblasts, a known oncogene associated with colon cancer, melanoma, and chronic myelogenous leukemia. Additionally, using CRISPR/Cas9 gene editing technology I corrected the p53 mutation in our LFS iPSC lines. In this proposal, I will use functional and comparative analysis to identify the molecular differences of LFS- isogenic controls to wild type and the original LFS iPSCs. From this analysis, I anticipate showing that correcting the causative p53 mutation in osteosarcoma using CRISPR/Cas9 can reverse pathogenesis of osteosarcoma in an iPSC platform. Additionally, I will demonstrate the efficacy of targeting sFRP2 and the AXL receptor as potential therapeutic strategies for LFS-associated osteosarcoma. Finally, to check the clinical relevance of my approach, I will analyze the expression level of sFRP2 in human osteosarcoma samples using osteosarcoma tissue microarrays. Overall, the proposed studies will provide corrected iPSC clones that will serve as perfect isogenic controls to study the role of p53 mutation, as well as the resulting downstream changes in sFRP2 expression and AXL receptor phosphorylation, in LFS-associated osteosarcoma development. This work will not only expand our knowledge of p53 mutation mediated osteosarcoma progression, but also describe multiple potential therapeutic methods to improve clinical outcomes for p53 mutation osteosarcoma patients.