Epigenetic Mechanisms Driving Synovial Sarcomagenesis

项目总结/摘要 这项申请加入了两个共同主要研究者的努力，一个是新的和早期阶段的研究者，一个是 整形外科医生兼科学家，最近完成了国家癌症研究所赞助的K 08职业生涯 发展奖侧重于小鼠模型的肉瘤，另一个经验丰富的生物化学家与 染色质重塑复合物和基因组学方面的专业知识。这些调查人员已经组建了一个小组， 将他们不同的经验应用于滑膜肉瘤形成的核心表观遗传机制。 滑膜肉瘤是最常见的软组织肉瘤的青春期和年轻的成年。其被驱动 由一个单一的遗传畸变：创造一个融合癌基因从平衡染色体易位 t（X;18）。在研究人员及其同事开发的模型中，这些SS 18-SSX融合蛋白的表达 小鼠中的癌基因驱动人类滑膜肉瘤的忠实再现。SS 18-SSX融合 在人滑膜肉瘤细胞系中已经显示癌蛋白利用两种表观遗传机制， 影响靶基因的转录。在一种机制中，SS 18-SSX形成将TLE 1-SSX重新定位的桥。 聚集的阻遏物与ATF 2结合位点的结合。第二种机制涉及染色质的调节 通过用融合物替换天然SS 18并排出另一个成员SNF 5来重塑BAF复合物。每个 已经在细胞系中的特定位点证明了这种机制，但不是全基因组或肿瘤中。 从SS 18-SSX表达错误调节细胞的募集和/或活性的假设出发， 染色质重塑和修饰复合物，以创建致癌转录谱，建议 实验将具体地（1）确定所述两种机制对滑膜的必要性， 肉瘤发生，（2）确定SS 18-SSX是否改变BAF功能，位置，或两者，超过SNF 5 排出，和（3）通过定义SS 18-SSX的基因组位置来确定SS 18-SSX肿瘤发生的驱动因素。 SS 18-SSX融合癌蛋白（和伴侣）通过ChIP-seq，以及它们对通过RNA-seq占据的基因座的影响。 在小鼠滑膜肉瘤形成的完全渗透模型中的反向遗传学实验 将测试每个机制的核心成员的必要性。有条件的和时间诱导的 SS 18-SSX的表达将测试其添加对小鼠中Snf 5缺失诱导的肿瘤发生的影响。 滑膜肉瘤将通过表达新的V5标记的SS 18-SSX融合体在小鼠中特异性诱导， 校正了先前用非融合特异性抗体进行的实验的困难解释，因此 能够在基因组范围内定位沉默的伴侣蛋白的融合和共定位， 激活位点因此，这些基因组定位评估将在理想化的环境中进行。 实验性肿瘤发生设置，在最富集的组织中进行比较对照评估 转化前的原始细胞。然后，来自模型中这个理想化实验范例的数据将 优先考虑和验证与比较基因组学从人类滑膜肉瘤样本。

Project Summary/Abstract This application joins the efforts of two co-principal investigators, one a new and early stage investigator, an orthopaedic surgeon-scientist who recently completed a National Cancer Institute sponsored K08 career development award focused on mouse modeling of sarcomagenesis, the other an experienced biochemist with expertise in chromatin remodeling complexes and genomics. These investigators have assembled a team to bring their varied experience to bear on the core epigenetic mechanisms of synovial sarcomagenesis. Synovial sarcoma is the most common soft-tissue sarcoma of adolescence and young adulthood. It is driven by a single genetic aberration: the creation of a fusion oncogene from a balanced chromosomal translocation t(X;18). In models developed by the investigators and their colleagues, expression of these SS18-SSX fusion oncogenes in the mouse drives faithful recapitulations of human synovial sarcoma. The SS18-SSX fusion oncoproteins have been shown in human synovial sarcoma cell lines to utilize two epigenetic mechanisms to impact the transcription of target genes. In one mechanism, SS18-SSX forms a bridge that relocates TLE1- aggregated repressors to ATF2-bound loci. The second mechanism involves the modulation of the chromatin remodeling BAF complex by replacing native SS18 with the fusion and ejecting another member, SNF5. Each mechanism has been demonstrated at specific loci in cell lines, but not genome-wide or in tumors. Working from the hypothesis that SS18-SSX expression misregulates the recruitment and/or activity of chromatin remodeling and modifying complexes to create an oncogenic transcriptional profile, the proposed experiments will specifically (1) Determine the necessity of the two described mechanisms to synovial sarcomagenesis, (2) Determine whether SS18-SSX alters BAF function, location, or both, beyond SNF5 ejection, and (3) Determine the drivers of SS18-SSX oncogenesis by defining the genomic locations of the SS18-SSX fusion oncoprotein (and partners) by ChIP-seq, and their impact at occupied loci by RNA-seq. Reverse genetic experiments in an otherwise fully penetrant model of synovial sarcomagenesis in the mouse will test the necessity of central members of each mechanism. Both conditional and temporally inducible expression of SS18-SSX will test the impact of its addition to Snf5-loss induced tumorigenesis in the mouse. Synovial sarcoma will specifically be induced in mice by expression of a novel V5-tagged SS18-SSX fusion, to correct for the difficult interpretation of prior experiments with antibodies non-specific for the fusion, thus enabling genome-wide localization of the fusion and co-localization of partnering proteins at silenced and activated loci. These genomic localization assessments will therefore be performed in an idealized experimental tumorigenesis setting with comparison control assessments available in the tissue most enriched for the pre-transformation cell of origin. Data from this idealized experimental paradigm in the model will then be prioritized and validated with comparative genomics from human synovial sarcoma samples.