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

项目总结/摘要 人类诱导多能干细胞（iPSC）不仅代表了再生的有前途的资源， 医学，而且作为研究人类疾病的工具，它们的效用也在上升。具体来说， 使用患者特异性iPSC提供了研究人类癌症发病机制的机会。在我们以前 在细胞2015年的工作中，“用诱导多能干细胞建模家族性癌症”，我们证明了， 来源于Li-Fraumeni综合征（LFS）模拟癌症的人类家族性癌症患者的iPSC 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， 正常成骨细胞分化所需的基因H19及其印记基因网络， 在LFS患者成骨细胞分化期间失调。 虽然我们报道了H19及其印迹基因网络在LFS诱导的iPSC中的异常调节， 在成骨细胞中，没有已知的临床可行的策略来靶向异常H19及其非编码RNA 调控为了发现临床相关和治疗靶向分子，我继续 研究我们的LFS iPSC衍生的骨肉瘤模型，并鉴定出分泌的卷曲相关蛋白2 （sFRP 2）是骨肉瘤发生发展的重要致癌因子。我进一步证明了sFRP 2 增加成骨细胞中AXL受体的磷酸化，AXL受体是一种已知的与结肠相关的癌基因 癌症、黑色素瘤和慢性骨髓性白血病。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。 在这个建议中，我将使用功能和比较分析，以确定LFS的分子差异- 与野生型和原始LFS iPSC的同基因对照。从这一分析中，我预计表明， 使用CRISPR/Cas9纠正骨肉瘤中的致病性p53突变可以逆转骨肉瘤的发病机制。 在iPSC平台上的骨肉瘤。此外，我将证明靶向sFRP 2和AXL的疗效。 受体作为LFS相关骨肉瘤的潜在治疗策略。最后，检查临床 我的方法的相关性，我将分析sFRP 2在人类骨肉瘤样本的表达水平，使用 骨肉瘤组织芯片 总的来说，拟议的研究将提供校正的iPSC克隆，其将作为完美的等基因对照， 研究p53突变的作用，以及sFRP 2表达和AXL的下游变化。 受体磷酸化，在LFS相关的骨肉瘤的发展。这项工作不仅将扩大我们的 了解p53突变介导骨肉瘤进展，但也描述了多种可能性 探讨改善p53突变骨肉瘤患者临床预后的治疗方法。

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.