Modeling p53 Mutant-Associated Cancer With LFS Patient-Derived iPSCs

描述（由申请人提供）：胚胎干细胞（ESCs）和诱导多能干细胞（iPSCs）作为一种无限的细胞来源，在生物医学领域具有巨大的前景，可用于产生以移植为基础的分化后代，研究各种疾病的病因，以及开发新的药物治疗。在我之前发表在《细胞干细胞和自然协议》上的工作中，我发现了Aurka-p53信号通路在调节ESC/iPSC身份和体细胞编程中的新作用。一个由Aurka介导的p53磷酸化事件将ESCs从分化转变为自我更新状态。通过对ESCs中p53直接结合靶基因的全基因组分析，我揭示了p53通过调节中胚层和外胚层谱系基因表达和作为分化激活因子，负向控制ESC自我更新的独特功能。最近的研究，包括我自己的研究，揭示了p53在调节细胞分化而不是细胞凋亡和细胞周期中的作用。为了在我之前发现p53是分化的守护者的基础上，扩大我们对p53作为肿瘤抑制因子和分化激活因子的关键作用的认识，我建议建立第一个癌症相关的Li-Fraumeni综合征（LFS） iPSC模型来研究这种p53突变相关疾病。LFS是一种遗传异质性的遗传性癌症综合征，其特征是种系p53突变的孟德尔常染色体显性遗传，可导致个体患者多发肿瘤的早期发病。与其他主要是组织特异性的遗传性癌症不同，LFS患者表现为多种肿瘤。p53蛋白在调节正常生理稳态中起着至关重要的作用，p53的突变不仅破坏了其正常的抑瘤活性，而且将其功能转化为致癌潜能。本提案的目标是首先通过使用来自患者特异性iPSCs的成骨细胞来模拟人类lfs相关的骨肉瘤，并创建一个“疾病培养皿”平台来阐明这些p53突变引起的潜在发病机制。通过转录激活样效应核酸酶（TALEN）介导的精确基因靶向，纠正LFS患者来源的iPSCs中LFS相关突变，有望逆转疾病相关的去分化表型。下面的建议将通过系统的分析来确定p53突变相关骨肉瘤的分子机制，并进一步为治疗未来的骨肉瘤或其他p53突变的癌症提供治疗靶点。该实验的成功完成将极大地促进我们对突变型p53在骨肉瘤发展中的作用的理解。此外，这些拟议的研究将有可能导致确定新的治疗靶点，以改善骨肉瘤患者的临床结果。

DESCRIPTION (provided by applicant): Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) hold great promise for biomedicine as an unlimited source of cells for generating differentiated progenies for transplantation-based therapies, studying the etiology of various diseases, and developing new drug treatments. In my previous work published in Cell Stem Cell and Nature Protocols, I discovered a novel role for the Aurka-p53 signaling pathway in regulating ESC/iPSC identity and somatic cell programming. A single phosphorylation event mediated by Aurka on p53 shifts ESCs from differentiating to a self-renewal state. Through genome-wide analysis of direct p53 binding target genes in ESCs, I revealed a unique p53 function in negatively controlling ESC self-renewal by regulating mesodermal and ectodermal lineage gene expression and functioning as a differentiation activator. Collectively recent studies, including my own, revealed the unappreciated role of p53 in regulating cell differentiation instead of cell apoptosis and cell cycle. To build on my previous finding that p53 serves as a guardian of differentiation and expand our knowledge into the critical role of p53 as a both tumor suppressor and differentiation activator, I propose to establish the first cancer-related Li-Fraumeni Syndrome (LFS) iPSC model to study this p53 mutation-associated disorder. LFS is a genetically heterogeneous inherited cancer syndrome characterized by Mendelian autosomal dominant inheritance of germline p53 mutations that cause early onset of multiple tumors in individual patients. In contrast to other inherited cancers that are predominantly tissue-specific, LFS patients present with a variety of tumors. The p53 protein plays critical roles in regulating normal physiological homeostasis and mutations in p53 not only abolish its normal tumor suppressor activity but convert this function to oncogenic potential. The objectives of this proposal are first to model human LFS-associated osteosarcomas by using osteoblasts derived from patient-specific iPSCs and creating a "disease in a dish" platform to elucidate the underlying pathogenesis caused by these p53 mutations. Correction of the LFS-linked mutation in LFS patient-derived iPSCs by Transcription Activation-Like Effector Nuclease (TALEN)-mediated precise gene targeting is expected to reverse the disease-associated dedifferentiation phenotype. The following proposal will use systematical analyses to identify the molecular mechanisms involved in p53 mutant-associated osteosarcoma and further provide therapeutic targets for treating future osteosarcomas or other cancers with p53 mutations. Successful completion of the proposed experiment will significantly advance our understanding of the role of mutant p53 in osteosarcoma development. In addition, these proposed studies will potentially lead to identifying novel therapeutic targets to improve clinical outcomes for osteosarcoma patients.