Using CRISPR/Cas9 to dissect the role of FUS-CHOP in tumor response to radiation therapy

使用 CRISPR/Cas9 剖析 FUS-CHOP 在肿瘤放射治疗反应中的作用

• 批准号: 9270422
• 负责人: Mark Shuo Chen
• 金额: $ 3.76万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9270422
• 关键词: Adenoviruses; Adult; Apoptosis; Biological Assay; CRISPR/Cas technology; Cancer Burden; Cancer Patient; Caring; Cell Line; Chimeric Proteins; Chromosomal Rearrangement; Clinical; Connective Tissue; DNA; DNA Damage; Engineering; Fatty acid glycerol esters; Fusion Oncogene Proteins; Generations; Genes; Goals; Guide RNA; Histology; Human; In Vitro; Ionizing radiation; Knowledge; Liver; Lung; Malignant - descriptor; Malignant Neoplasms; Mediating; Mission; Modeling; Molecular; Mouse Cell Line; Mus; Myxoid Liposarcoma; Neoplasm Metastasis; Orthologous Gene; Pathway interactions; Patients; Pharmacology; Phenotype; Pre-Clinical Model; Primary Neoplasm; Process; Property; Public Health; RNA-Binding Proteins; Radiation; Radiation Oncology; Radiation Tolerance; Radiation therapy; Radiation-Sensitizing Agents; Radiosensitization; Role; Site; Soft tissue sarcoma; Specificity; Stress; System; Technology; Testing; biological adaptation to stress; bone; genome editing; human disease; improved; in vivo; irradiation; liposarcoma; mouse model; neoplastic cell; novel; overexpression; promoter; public health relevance; radiation response; radioresistant; radiosensitive; response; sarcoma; soft tissue; transcription factor; transcription factor CHOP; tumor; tumor growth; tumorigenesis

 DESCRIPTION (provided by applicant): Soft tissue sarcomas are tumors of the connective tissue that can arise anywhere in the body and is fatal in nearly 1/3 of patients. Myxoid liposarcoma (MLPS) is a type of malignant sarcoma that accounts for 1/3 of all liposarcomas, the most common soft tissue sarcoma. Clinically, MLPS has two unique properties. First, they have been observed to be more radiosensitive compared to other sarcomas, which are mostly radioresistant. Second, these tumors metastasize to bone, liver, and other soft tissue sites rather than to the lung, the most common site of metastasis for sarcomas. Genetically, MLPS is characterized by a t (12;16)(q13;p11) translocation in humans. Previous studies have shown that the translocation produces a fusion oncoprotein, FUS- CHOP, which is sufficient to induce MLPS in mice. How FUS-CHOP drives tumorigenesis remains unclear, and whether FUS-CHOP contributes to the distinctive radiosensitivity of MLPS is unknown. To most accurately model human disease, here I apply CRISPR-Cas9 technology for genome editing to generate a rapid, tempo- rally and spatially restricted mouse model of MLPS with the Fus-Chop translocation at the endogenous locus. The long-term goal of this project is to elucidate and harness mechanisms of radiosensitivity to radiosensitize cancers. The overall goal of this proposal is to dissect the mechanisms by which Fus-Chop mediates tumor- igenesis and radiosensitivity in MLPS. These processes will be studied by using CRISPR/Cas9 technology in vitro and in vivo to generate the Fus-Chop translocation. The central hypothesis of this study is that Fus-Chop creates a radiosensitive phenotype by modifying the ER stress and DNA damage responses. Using in vitro and in vivo systems generated with CRISPR/Cas9, I will test this hypothesis in the following three specific aims: Aim 1: Elucidate the mechanisms of Fus-Chop in tumorigenesis of MLPS via CRISPR/Cas9-mediated chromosomal rearrangements in vitro. Aim 2: Determine the role of Fus-Chop in modifying cellular response to ER stress and ionizing radiation. Aim 3: Determine the role of Fus-Chop in mediating response to radiotherapy in a primary mouse model of MLPS using CRISPR/Cas9 technology. I will define the role of Fus-Chop in the radiation response of MLPS and also generate a valuable radiosensitive pre-clinical model for radiation oncology.

 描述（由申请人提供）：软组织肉瘤是结缔组织肿瘤，可发生在身体的任何地方，并且在近1/3的患者中是致命的。粘液样脂肪肉瘤（MLPS）是一种恶性肉瘤，占所有脂肪肉瘤的1/3，是最常见的软组织肉瘤。临床上，MLPS具有两个独特的特性。首先，与其他肉瘤相比，它们被观察到对辐射更敏感，而其他肉瘤大多具有辐射抗性。其次，这些肿瘤转移到骨、肝和其他软组织部位，而不是转移到肺，肺是肉瘤最常见的转移部位。在遗传学上，MLPS的特征在于人类的t（12;16）（q13;p11）易位。先前的研究表明，易位产生融合癌蛋白FUS-CHOP，其足以在小鼠中诱导MLPS。FUS-CHOP如何驱动肿瘤发生尚不清楚，FUS-CHOP是否有助于MLPS的独特放射敏感性尚不清楚。为了最准确地模拟人类疾病，在此，我应用CRISPR-Cas9技术进行基因组编辑，以生成在内源基因座处具有Fus-Chop易位的MLPS的快速、克里思和空间限制的小鼠模型。该项目的长期目标是阐明和利用放射敏感性的机制来使癌症放射敏感。该提案的总体目标是剖析Fus-Chop介导MLPS中肿瘤发生和放射敏感性的机制。这些过程将通过使用CRISPR/Cas9技术在体外和体内进行研究，以产生Fus-Chop易位。本研究的中心假设是，Fus-Chop通过改变ER应激和DNA损伤反应来产生辐射敏感表型。使用CRISPR/Cas9产生的体外和体内系统，我将在以下三个具体目标中检验这一假设：目标1：阐明Fus-Chop通过CRISPR/Cas9介导的体外染色体重排在MLPS肿瘤发生中的机制。目的2：确定Fus-Chop在改变细胞对ER应激和电离辐射的反应中的作用。目的3：使用CRISPR/Cas9技术确定Fus-Chop在MLPS的原代小鼠模型中介导对放射治疗的反应中的作用。我将定义Fus-Chop在MLPS辐射反应中的作用，并为放射肿瘤学产生有价值的放射敏感性临床前模型。