Identification of potential drug targets for tuberous sclerosis complex by synthetic screens combining CRISPR-based knockouts with RNAi

Combining targeted CRISPR-mediated gene editing with an RNAi-mediated screen identifies candidate drug targets. Double screening for drug targets The tumor suppressors TSC1 and TSC2 form a complex that limits the activity of mTORC1, a multiprotein complex that couples nutrient availability to cell proliferation and growth. Individuals with mutations in either of the TSC-encoding genes develop tumors in various organs. Housden et al. used CRISPR to generate Drosophila cell lines that lacked either TSC1 or TSC2 and performed RNAi screens directed against kinase- and phosphatase-encoding genes in these cell lines to identify those genes that limited the growth of cells lacking either TSC1 or TSC2. Candidate genes that, when knocked down, suppressed the growth of both of the TSC1- and TSC2-deficient Drosophila cell lines (but not that of normal cells) were validated in TSC2-deficient human cells. Because these candidates are evolutionarily conserved, they are more likely to be bona fide drug targets, and this combination of techniques and multispecies screening could be used to identify drug targets for other diseases. 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.