新型抑癌基因dystrophin失活促进胃肠道间质瘤转移的分子机制和小分子化合物的筛选

Gastrointestinal Stromal Tumors (GISTs) are the most common gastrointestinal soft tissue malignancies. Gain-of-function KIT or PDGFRA mutations are initiating genetic events in GISTs. Somatic mutations presumably contribute to tumorigenic progression in GIST, e.g. cell cycle dysregulation by CDKN2A or TP53 inactivation, but few genetic progression mechanisms in GIST have been characterized. More recently, for the first time, we (Wang et al, Nat Genet 2014) demonstrated that dystrophin is a genomically-inactivated tumor suppressor in human cancers with myogenic programs, including leiomyosarcoma, rhabdomyosarcoma, and GIST. For example, in GISTs, the major dystrophin 427kDa isoform was expressed strongly (and had no demonstrable genomic mutations) in each of 11 low-risk (indolent) GISTs, whereas dystrophin expression was undetectable in 26 of 27 (96%) metastatic GISTs, most of which had inactivating intragenic DMD mutations. Furthermore, we established immortal cell lines from several of the dystrophin-mutant clinical specimens, and showed in these in vitro models that dystrophin inactivation had profound cell response consequences, increasing cell invasion, migration and anchorage-independent growth. Our data also suggest that dystrophin could serve as a potential target of therapeutic attack. Although dystrophin inactivation during GIST progression is convincing and well documented, the biological mechanisms by which dystrophin inactivation contributes to carcinogenesis is unclear. In the Duchenne muscular dystrophy field, there are several genetic approaches targeting dystrophin inactivation in development (preclinical and clinic trial stage). Unfortunately, none of them have been approved by FDA after a decade of research. Given recent advances in chemical genomics such as gene expression-based high-throughput screening and the Connectivity Map, we hypothesized that the gene expression signature resulting from dystrophin restoration in GIST could be used to search for similar signatures in publicly available gene expression profiles. With the overarching goal of characterize the pathways dysregulation by dystrophin inactivation and to discover small compounds that reverse the dystrophin deficiency in advanced GIST, we will perform systematic screens utilizing state-of-the-art technology on our unique repository of GIST resources. Two aims are proposed for this research plan: (1) Systematic screens to identify pathways dysregulated by dystrophin inactivation in advanced GIST. (2) To reverse dystrophin deficiency in advanced GIST with small molecules. The goal of the project is to explore the molecular mechanisms of dystrophin inactivation mediated tumorgenesis and to discover small compounds that reverse the dystrophin deficiency in advanced GIST. The research could potentially lead to the development of novel agents against advanced GIST.

胃肠道间质瘤(GIST)是最常见的肉瘤，KIT 或者PDGFRA激活突变是GIST的初始事件，CDKN2A 和TP53 失活突变促进细胞增殖，但这些突变在原发GIST就已经存在，而GIST治疗最棘手的难题之一就是转移，我们首次报道了dystrophin在GIST演变过程中发生失活突变，是一个新型抑癌基因。dystrophin在原发性低危GIST中高表达，而在约96%转移GIST中由于失活突变导致表达丢失。通过一系列功能试验证明dystrophin失活促进GIST细胞侵袭、迁移、非锚定依赖性生长和细胞invadopodia形成。尽管从遗传学角度证明了dystrophin失活促进了GIST转移，但具体分子机理尚不清楚。该研究拟（1）解析dystrophin失活促进GIST转移的分子机制；（2）寻找能恢复dystrophin功能的小分子化合物并证明在转移GIST 中的潜在应用前景。

胃肠间质瘤(GIST)是最常见的肉瘤，KIT或者PDGFRA激活突变是GIST的初始分子事件，而GIST治疗最棘手的难题之一就是恶性进展最终转移，在本项目资助下，本研究围绕GIST的恶性进展分子机制开展了系列研究：（1）证明GIST转移相关基因dystrophin的失活促进MLC2磷酸化的水平，进而增强细胞侵袭、迁移能力，从而促进GIST转移；筛选到小分子化合物Sildenafil具有类似dystrophin回补功能的作用；为靶向dystrophin失活治疗转移GIST提供了实验依据。（2）前期RNA-seq筛选dystrophin失活的转移GIST时意外发现DEPDC5在转移GIST中表达显著降低，利用一系列基因组学、细胞生物学、肿瘤生物学、动物实验首次鉴定了位于22号染色体的新型抑癌基因DEPDC5，16.4%的GIST患者中DEPDC5蛋白由于其编码基因突变（无义突变、移码突变、纯合缺失等）而处于失活状态，并且GIST恶性程度越高，该蛋白表达量越低。DEPDC5的失活通过激活mTOR通路促进GIST的恶性进展，DEPDC5的失活降低了GIST对KIT靶向药（如格列卫）的敏感性，最后为DEPDC5失活的病人应联合使用格列卫和mTOR抑制剂的治疗策略提供了实验依据。（3）在本项目资助下已发表SCI论文2篇(PNAS 2019; Carcinogenesis 2019。均标注NSFC资助和项目批准号)，修回中SCI论文2篇(标注NSFC资助和项目批准号)，申请中国发明专利2项，培养硕士/博士研究生10名（1名毕业，9名在读）。