Aneuploidy is a hallmark of cancer with tissue-specific prevalence patterns that suggest it plays a driving role in cancer initiation and progression. However, the contribution of aneuploidy to tumorigenesis depends on both cellular and genomic contexts. Whole-genome duplication (WGD) is a common macroevolutionary event that occurs in more than 30% of human tumors early in tumorigenesis. Although tumors that have undergone WGD are reported to be more permissive to aneuploidy, it remains unknown whether WGD also affects aneuploidy prevalence patterns. Here we analyzed clinical tumor samples from 5,586 WGD− tumors and 3,435 WGD+ tumors across 22 tumor types and found distinct patterns of aneuploidy in WGD− and WGD+ tumors. WGD+ tumors were characterized by more promiscuous aneuploidy patterns, in line with increased aneuploidy tolerance. Moreover, the genetic interactions between chromosome arms differed between WGD− and WGD+ tumors, giving rise to distinct cooccurrence and mutual exclusivity aneuploidy patterns. The proportion of whole-chromosome aneuploidy compared with arm-level aneuploidy was significantly higher in WGD+ tumors, indicating distinct dominant mechanisms for aneuploidy formation. Human cancer cell lines successfully reproduced these WGD/aneuploidy interactions, confirming the relevance of studying this phenomenon in culture. Finally, induction of WGD and assessment of aneuploidy in isogenic WGD−/WGD+ human colon cancer cell lines under standard or selective conditions validated key findings from the clinical tumor analysis, supporting a causal link between WGD and altered aneuploidy landscapes. We conclude that WGD shapes the aneuploidy landscape of human tumors and propose that this interaction contributes to tumor evolution. Significance: These findings suggest that the interactions between whole-genome duplication and aneuploidy are important for tumor evolution, highlighting the need to consider genome status in the analysis and modeling of cancer aneuploidy.

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.

Aneuploidy is a hallmark of human cancer, yet the cellular mechanisms that allow cells to cope with aneuploidy-induced cellular stresses remain largely unknown. Such coping mechanisms may present cellular vulnerabilities that can be harnessed for targeting cancer cells. Here, we induced aneuploidy in non-transformed RPE1-hTERT cells and derived multiple stable clones with various degrees of chromosome imbalances. We performed an unbiased genomic profiling of 6 isogenic clones, using whole-exome and RNA sequencing. We then functionally interrogated their cellular dependency landscapes, using genome-wide CRISPR/Cas9 screens and large-scale drug screens. We found that aneuploid clones activated the DNA damage response (DDR), and were consequently more resistant to further DNA damage induction. Interestingly, aneuploid cells also exhibited elevated RAF/MEK/ERK pathway activity, and were more sensitive to several clinically-relevant drugs targeting this pathway, and in particular to genetic and chemical CRAF inhibition. CRAF activity was functionally linked to the resistance to DNA damage induction, as CRAF inhibition sensitized aneuploid cells to DNA damage-inducing chemotherapies. The association between aneuploidy, RAF/MEK/ERK signaling, and DDR was independent of p53. The increased activity and dependency of aneuploid cells on the RAF/MEK/ERK pathway was validated in another isogenic aneuploid system, and across hundreds of human cancer cell lines, confirming their relevance to human cancer. Overall, our study provides a comprehensive resource for genetically-matched karyotypically-stable cells of various aneuploidy states, and reveals a novel therapeutically-relevant cellular dependency of aneuploid cells.

Aneuploidy, an abnormal chromosome composition, results in a stoichiometric imbalance of protein complexes, which jeopardizes the fitness of aneuploid cells. Aneuploid cells thus need to compensate for the imbalanced DNA levels by regulating their RNA and protein levels, a phenomenon known as dosage compensation. However, the molecular mechanisms involved in dosage compensation in human cells - and whether they can be targeted to selectively kill aneuploid cancer cells - remain unknown. Here, we addressed this question via molecular dissection of multiple diploid vs. aneuploid cell models. Using genomic and functional profiling of a novel isogenic system of RPE1-hTERT cells with various degrees of aneuploidy, we found that aneuploid cells cope with both transcriptional burden and proteotoxic stress. At the mRNA level, aneuploid cells increased RNA synthesis, but concomitantly elevated several RNA degradation pathways, in particular the nonsense-mediated decay (NMD) and the microRNA-mediated mRNA silencing pathways. Consequently, aneuploid cells were more sensitive to the genetic or chemical perturbation of several key components of these RNA degradation pathways. At the protein level, aneuploid cells experienced proteotoxic stress, resulting in reduced translation and increased protein degradation, rendering them more sensitive to proteasome inhibition. These findings were recapitulated across hundreds of human cancer cell lines and primary tumors, confirming that both non-transformed and transformed cells alter their RNA and protein metabolism in order to adapt to the aneuploid state. Our results reveal that aneuploid cells are dependent on the over- or under-activation of several nodes along the gene expression process, identifying these pathways as clinically-actionable vulnerabilities of aneuploid cells.

Germline BRCA–associated pancreatic ductal adenocarcinoma (glBRCA PDAC) tumors are susceptible to platinum and PARP inhibition. The clinical outcomes of 125 patients with glBRCA PDAC were stratified based on the spectrum of response to platinum/PARP inhibition: (i) refractory [overall survival (OS) <6 months], (ii) durable response followed by acquired resistance (OS <36 months), and (iii) long-term responders (OS >36 months). Patient-derived xenografts (PDX) were generated from 25 patients with glBRCA PDAC at different clinical time points. Response to platinum/PARP inhibition in vivo and ex vivo culture (EVOC) correlated with clinical response. We deciphered the mechanisms of resistance in glBRCA PDAC and identified homologous recombination (HR) proficiency and secondary mutations restoring partial functionality as the most dominant resistant mechanism. Yet, a subset of HR-deficient (HRD) patients demonstrated clinical resistance. Their tumors displayed basal-like molecular subtype and were more aneuploid. Tumor mutational burden was high in HRD PDAC and significantly higher in tumors with secondary mutations. Anti–PD-1 attenuated tumor growth in a novel humanized glBRCA PDAC PDX model. This work demonstrates the utility of preclinical models, including EVOC, to predict the response of glBRCA PDAC to treatment, which has the potential to inform time-sensitive medical decisions. Significance: glBRCA PDAC has a favorable response to platinum/PARP inhibition. However, most patients develop resistance. Additional treatment options for this unique subpopulation are needed. We generated model systems in PDXs and an ex vivo system (EVOC) that faithfully recapitulate these specific clinical scenarios as a platform to investigate the mechanisms of resistance for further drug development.