Aneuploidy is a hallmark of human cancer, yet the molecular mechanisms to cope with aneuploidy-induced cellular stresses remain largely unknown. Here, we induce chromosome mis-segregation in non-transformed RPE1-hTERT cells and derive multiple stable clones with various degrees of aneuploidy. We perform a systematic genomic, transcriptomic and proteomic profiling of 6 isogenic clones, using whole-exome DNA, mRNA and miRNA sequencing, as well as proteomics. Concomitantly, we functionally interrogate their cellular vulnerabilities, using genome-wide CRISPR/Cas9 and large-scale drug screens. Aneuploid clones activate the DNA damage response and are more resistant to further DNA damage induction. Aneuploid cells also exhibit elevated RAF/MEK/ERK pathway activity and are more sensitive to clinically-relevant drugs targeting this pathway, and in particular to CRAF inhibition. Importantly, CRAF and MEK inhibition sensitize aneuploid cells to DNA damage-inducing chemotherapies and to PARP inhibitors. We validate these results in human cancer cell lines. Moreover, resistance of cancer patients to olaparib is associated with high levels of RAF/MEK/ERK signaling, specifically in highly-aneuploid tumors. Overall, our study provides a comprehensive resource for genetically-matched karyotypically-stable cells of various aneuploidy states, and reveals a therapeutically-relevant cellular dependency of aneuploid cells.

非整倍体是人类癌症的标志，但应对非整倍体诱导的细胞应激的分子机制在很大程度上仍然未知。在这里，我们在未转化的 RPE1-hTERT 细胞中诱导染色体错误分离，并衍生出具有不同程度非整倍体的多个稳定克隆。我们使用全外显子组 DNA、mRNA 和 miRNA 测序以及蛋白质组学，对 6 个同基因克隆进行系统的基因组、转录组学和蛋白质组学分析。同时，我们使用全基因组 CRISPR/Cas9 和大规模药物筛选对它们的细胞脆弱性进行功能询问。异倍体克隆激活 DNA 损伤反应，并且对进一步的 DNA 损伤诱导更具抵抗力。无倍体细胞还表现出升高的 RAF/MEK/ERK 通路活性，并且对靶向该通路的临床相关药物更敏感，尤其是对 CRAF 抑制。重要的是，CRAF 和 MEK 抑制使非整倍体细胞对诱导 DNA 损伤的化疗和 PARP 抑制剂敏感。我们在人类癌细胞系中验证了这些结果。此外，癌症患者对奥拉帕尼的耐药性与高水平的 RAF/MEK/ERK 信号传导有关，特别是在高度非整倍体肿瘤中。总体而言，我们的研究为各种非整倍体状态的遗传匹配核型稳定细胞提供了全面的资源，并揭示了非整倍体细胞的治疗相关细胞依赖性。