新型抑癌基因YLPM1的失活促进胃肠间质瘤增殖的分子机制及靶向策略研究

胃肠间质瘤(GIST)是胃肠道最常见的间叶源性肿瘤，KIT或者PDGFRA激活突变是形成GIST的初始分子事件，但不足以形成临床GIST。前期，我们通过全外显子测序发现10.4%的GIST含有YLPM1基因失活突变，蛋白水平的失活高达46.8%。体内外实验证明YLPM1失活促进GIST的增殖，但其失活的原因和促癌的分子机制尚不明确。伊马替尼耐药仍是GIST研究和临床的难题。近半数GIST中存在YLPM1失活，若开发YLPM1失活的用药靶点，可为克服伊马替尼耐药提供新的策略。本课题拟（1）通过亚硫酸盐测序和蛋白质谱研究YLPM1突变以外的失活机制；（2）利用体外GIST模型、体内移植鼠模型和遗传修饰小鼠自发GIST模型证明YLPM1失活促进增殖和肿瘤形成；（3）多组学研究YLPM1失活促癌的分子机制；（4）CRISPR筛选YLPM1失活特异的易感弱点靶基因并探索其在GIST中的潜在治疗前景。

Gastrointestinal stromal tumors (GISTs) are the most common human sarcoma, which are mostly initiated by activating mutations of the receptor tyrosine kinase KIT (75-80%) or PDGFRA (5-10%). The fact that micro-GISTs are common in general individuals, found in one third of the general population, without clinical symptoms indicates additional genetic alterations contribute to the progression of clinical GISTs. Recently, using whole exome sequencing, we discovered recurrent genomic inactivated YLPM1 gene mutations in GISTs (10.4%, 8 of 77 patients). Strikingly, the frequency of YLPM1 protein loss was even as high as 46.8%. The demonstration of clonal YLPM1 inactivation mutations in longitudinal specimens and in multiple metastases from individual patients suggests that these mutations have tumorigenic roles in GIST progression. Both in vivo nude mice and in vitro experiments demonstrate that YLPM1 inactivation promotes GIST proliferation, suggesting that YLPM1 is a novel tumor suppressor. However, the mechanisms of YLPM1 inactivation and the biological mechanisms by which YLPM1 loss contributes to cancer promotion is unclear. Furthermore, intratumor and intretumor resistance heterogeneity to kinase inhibition in GIST makes it challenging to overcome tyrosine kinase inhibitor (TKI) resistance. The ability to perturb genes by Clustered Regularly Interspaced Short Palindromic repeats (CRISPR) in human cells holds great potential for elucidating gene function and finding novel therapeutic targets. Considering the high frequency of YLPM1 inactivation in GIST, this vulnerability can be exploited as a therapeutic advantage in order to delay the establishment of large resistant cell populations. Four aims are proposed for this research plan: (1) To explore the inactivation mechanisms of YLPM1 through bisulfite sequencing and protein profiling; (2) To demonstrate that YLPM1 inactivation promotes GIST progression with various in vitro and in vivo GIST models; (3) To dissect the molecular mechanisms of YLPM1 inactivation in GIST proliferation through RNA-seq, etc; (4) To systemically identify genetic vulnerabilities in YLPM1-inactivated GIST using a high-complexity second-generation CRISPR screen on our unique repository of GIST resources, and validate their therapeutic potentials with various GIST models. The goal of the project is to explore the molecular mechanisms of YLPM1 inactivation mediating tumorgenesis and to discover small compounds that reverse the YLPM1 deficiency in advanced GIST. The research could potentially lead to the development of novel agents against advanced GIST.