Human aneuploid cells depend on the RAF/MEK/ERK pathway for overcoming increased DNA damage.

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.

Increased RNA and protein degradation is required for counteracting transcriptional burden and proteotoxic stress in human aneuploid cells.

Aneuploidy results in a stoichiometric imbalance of protein complexes that jeopardizes cellular fitness. Aneuploid cells thus need to compensate for the imbalanced DNA levels by regulating their RNA and protein levels, but the underlying molecular mechanisms remain unknown. Here, we dissected multiple diploid vs. aneuploid cell models. We found that aneuploid cells cope with transcriptional burden by increasing several RNA degradation pathways, and are consequently more sensitive to the perturbation of RNA degradation. At the protein level, aneuploid cells mitigate proteotoxic stress by reducing protein translation and increasing protein degradation, rendering them more sensitive to proteasome inhibition. These findings were recapitulated across hundreds of human cancer cell lines and primary tumors, and aneuploidy levels were significantly associated with the response of multiple myeloma patients to proteasome inhibitors. Aneuploid cells are therefore preferentially dependent on several key nodes along the gene expression process, creating clinically-actionable vulnerabilities in aneuploid cells.

Short-term molecular consequences of chromosome mis-segregation for genome stability.

Chromosome instability (CIN) is the most common form of genome instability and is a hallmark of cancer. CIN invariably leads to aneuploidy, a state of karyotype imbalance. Here, we show that aneuploidy can also trigger CIN. We found that aneuploid cells experience DNA replication stress in their first S-phase and precipitate in a state of continuous CIN. This generates a repertoire of genetically diverse cells with structural chromosomal abnormalities that can either continue proliferating or stop dividing. Cycling aneuploid cells display lower karyotype complexity compared to the arrested ones and increased expression of DNA repair signatures. Interestingly, the same signatures are upregulated in highly-proliferative cancer cells, which might enable them to proliferate despite the disadvantage conferred by aneuploidy-induced CIN. Altogether, our study reveals the short-term origins of CIN following aneuploidy and indicates the aneuploid state of cancer cells as a point mutation-independent source of genome instability, providing an explanation for aneuploidy occurrence in tumors.