Identification of potential drug targets for tuberous sclerosis complex by synthetic screens combining CRISPR-based knockouts with RNAi.
Identification of potential drug targets for tuberous sclerosis complex by synthetic screens combining CRISPR-based knockouts with RNAi.
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DOI：
10.1126/scisignal.aab3729
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发表时间：
2015-09-08
影响因子：
7.3
通讯作者：
Perrimon N
中科院分区：
文献类型：
--
作者：
Housden BE;Valvezan AJ;Kelley C;Sopko R;Hu Y;Roesel C;Lin S;Buckner M;Tao R;Yilmazel B;Mohr SE;Manning BD;Perrimon N
肿瘤抑制因子的结节性硬化症复合体（TSC）家族，TSC 1和TSC 2，在进化上保守的蛋白质复合体中一起起作用，该蛋白质复合体是调节mTOR复合体1（mTORC 1）的主要细胞信号传导途径的会聚点。TSC复合物的突变或异常抑制在各种人类肿瘤综合征和癌症中是常见的。因此，发现选择性靶向具有该复合物功能丧失的细胞的新型治疗策略对于患有非恶性TSC和患有散发性癌症的患者具有临床意义。我们开发了一种基于CRISPR的方法来产生同质突变果蝇细胞系。通过将TSC 1或TSC 2突变细胞系与针对所有激酶和磷酸酶的RNAi筛选相结合，我们鉴定了与TSC 1和TSC 2的合成相互作用。单独敲除三个候选基因（mRNA-cap、Pitslre和CycT;人类RNGTT、CDK 11和CCNT 1的直系同源物）降低了缺乏TSC 1或TSC 2的果蝇细胞的群体增长率，但对野生型细胞没有影响。此外，这三个基因的单独敲除在哺乳动物TSC 2缺陷细胞系（包括人肿瘤衍生细胞）中具有相似的生长抑制作用，说明了这种跨物种筛选策略识别潜在药物靶点的能力。
The tuberous sclerosis complex (TSC) family of tumor suppressors, TSC1 and TSC2, function together in an evolutionarily conserved protein complex that is a point of convergence for major cell signaling pathways that regulate mTOR complex 1 (mTORC1). Mutation or aberrant inhibition of the TSC complex is common in various human tumor syndromes and cancers. The discovery of novel therapeutic strategies to selectively target cells with functional loss of this complex is therefore of clinical relevance to patients with nonmalignant TSC and those with sporadic cancers. We developed a CRISPR-based method to generate homogeneous mutant Drosophila cell lines. By combining TSC1 or TSC2 mutant cell lines with RNAi screens against all kinases and phosphatases, we identified synthetic interactions with TSC1 and TSC2. Individual knockdown of three candidate genes (mRNA-cap, Pitslre, and CycT; orthologs of RNGTT, CDK11, and CCNT1 in humans) reduced the population growth rate of Drosophila cells lacking either TSC1 or TSC2 but not that of wild-type cells. Moreover, individual knockdown of these three genes had similar growth-inhibiting effects in mammalian TSC2-deficient cell lines, including human tumor-derived cells, illustrating the power of this cross-species screening strategy to identify potential drug targets.