基于CRISPR/Cas9系统在肾透明细胞癌（ccRCC）中筛选VHL合成致死基因

Von Hippel-Lindau（VHL）肿瘤抑制基因的纯合缺失是肾透明细胞癌的标志性起始事件。VHL蛋白失活导致缺氧诱导因子HIF的组成性激活，进而引起下游血管内皮生长因子（VEGF）和哺乳动物雷帕霉素靶蛋白（mTOR）等促血管生成和促增殖蛋白的表达上调。VEGF和mTOR抑制剂因此被用于肾透明细胞癌的治疗，但有效率低和抗药性仍然限制着患者的预后和生存。合成致死指两个基因，其中任何一个基因的单独丢失不会致死，但两个基因的同时丢失是致死的。识别癌症特异性的突变作为合成致死伙伴可以特异性清除肿瘤细胞，由于VHL突变在肾透明细胞癌中的普遍性，针对VHL突变的合成致死相互作用作为肾透明细胞癌的治疗靶点存在巨大潜力。

本文通过先后稳定转染Cas9蛋白表达载体与VHL过表达载体或相应的空载对照，在携带VHL突变的786-O肾透明细胞癌细胞系上构建了高敲除效率的VHL等基因细胞。大型基因组学研究揭示，表观遗传调控因子基因经常与VHL共同缺失，并且是肾透明细胞癌形成的重要共同驱动因素。本文设计了针对449个表观遗传调控因子基因的EPI-KO sgRNA文库用于筛选肾透明细胞癌标志性表观遗传异常的VHL合成致死伙伴。通过不同的生物信息学分析得到两组共10个候选VHL合成致死基因，并通过细胞混合增殖实验检验了第一组6个基因的合成致死效果，结果显示TRIM28基因与VHL存在合成致死效果。本文中构建的适用于CRISPR/Cas9高通量筛选的VHL合成致死筛选模型将为肾透明细胞癌药物靶点的发现做出一定贡献。

Homozygous loss of function of the Von Hippel-Lindau tumor suppressor gene (VHL) is a hallmark event in clear cell renal cell carcinoma (ccRCC). The inactivation of the von Hippel-Lindau (VHL) protein causes constitutive activation of the hypoxia-inducible factor (HIF), leading to the upregulation of downstream pro-angiogenic and pro-proliferative proteins. Vascular endothelial growth factor (VEGF) and mammalian target of rapamycin (mTOR) are such downstream targets, and their inhibitors are effective in suppressing ccRCC. However, easily developed drug resistance of both inhibitors and other side effects hindered their efficacy. Synthetic lethality between two genes refers to a phenomenon where perturbing either gene alone is viable, while simultaneous perturbation of both genes results in loss of viability. Targeting cancer-specific mutations for synthetic lethal partner identification is a great treatment strategy for precision tumor cell elimination. Due to its prevalence in ccRCC, VHL mutation holds significant potential as a target for synthetic lethal treatment strategies.

In this paper, we created an isogenic cell pair with high knockout efficiency. We first stably transfected the Cas9 protein expression vector into the VHL deficient 786-O cell line. Then, we introduced either the VHL overexpression vector or the corresponding empty vector to the Cas9-expressing cell. Large genomic studies reveal that epigenetic regulator genes are often co-deleted with VHL and are important co-drivers of ccRCC. Therefore, we developed an EPI-KO sgRNA library targeting 449 epigenetic regulator genes for CRISPR screening of VHL synthetic lethal partners. Through bioinformatical analysis of screening results, two groups of 10 candidate VHL synthetic lethal genes were obtained. Six of these genes were tested through mixed cell proliferation experiments to validate their synthetic lethal effects. The results showed that TRIM28 and VHL have synthetic lethal effects. The CRISPR/Cas9-based high-throughput synthetic lethality screening model developed in this study contributed to the discovery of drug targets for ccRCC.

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