Forward genetic screens in mammalian cell lines, such as RNAi and CRISPR-Cas9 screens, have made major contributions to the elucidation of diverse signaling pathways. Here, we exploited human haploid cells as a robust comparative screening platform and report a set of quantitative forward genetic screens for identifying regulatory mechanisms of mTORC1 signaling, a key growth control pathway that senses diverse metabolic states. Selected chemical and genetic perturbations in this screening platform, including rapamycin treatment and genetic ablation of the Ragulator subunit LAMTOR4, revealed the known core mTORC1 regulatory signaling complexes and the intimate interplay of the mTORC1 pathway with lysosomal function, validating the approach. In addition, we identified a differential requirement for LAMTOR4 and LAMTOR5 in regulating the mTORC1 pathway under fed and starved conditions. Furthermore, we uncovered a previously unknown “synthetic-sick” interaction between the tumor suppressor folliculin and LAMTOR4, which may have therapeutic implications in cancer treatment. Together, our study demonstrates the use of iterative “perturb and observe” genetic screens to uncover regulatory mechanisms driving complex mammalian signaling networks.

哺乳动物细胞系中的正向遗传筛选，如 RNAi 和 CRISPR-Cas9 筛选，为阐明不同的信号通路做出了重大贡献。在这里，我们利用人类单倍体细胞作为一个强大的比较筛选平台，并报告了一组定量正向遗传筛选，用于识别 mTORC1 信号传导的调节机制，这是一种感知不同代谢状态的关键生长控制途径。该筛选平台中选定的化学和遗传扰动，包括雷帕霉素处理和 Ragulator 亚基 LAMTOR4 的遗传消融，揭示了已知的核心 mTORC1 调节信号复合物以及 mTORC1 通路与溶酶体功能的密切相互作用，验证了该方法。此外，我们确定了 LAMTOR4 和 LAMTOR5 在进食和饥饿条件下调节 mTORC1 通路的不同需求。此外，我们发现了肿瘤抑制因子卵泡蛋白和 LAMTOR4 之间以前未知的“合成病”相互作用，这可能对癌症治疗具有治疗意义。总之，我们的研究证明了使用迭代“扰动和观察”遗传筛选来揭示驱动复杂哺乳动物信号网络的调节机制。