An aneuploidy epistasis map reveals metabolic vulnerabilities associated with supernumerary chromosomes in cancer

Despite the general detriment of aneuploidy to cellular fitness, >90% of solid tumors carry an imbalanced karyotype. Regardless of this existing paradox, our understanding of the molecular responses to aneuploidy remains limited. Here, we explore these cellular stresses and unique vulnerabilities in aneuploid human mammary epithelial cells (HMECs) enriched for breast cancer-associated copy number alterations (CNAs). To uncover the genetic dependencies specific to aneuploid cells, we conducted a comprehensive, genome-wide CRISPR knockout screen targeting isogenic diploid and aneuploid HMEC lines. Our study reveals that aneuploid HMECs exhibit an increased reliance on pyrimidine biosynthesis and mitochondrial oxidative phosphorylation genes, and demonstrate heightened fitness advantages upon loss of tumor suppressor genes. Using an integrative multi-omic analysis, we confirm nucleotide pool insufficiency as a key contributor to widespread cellular dysfunction in aneuploid HMECs with net copy number gain. While diploid cells can switch seamlessly between pyrimidine synthesis and salvage, cells with increased chromosomal content exhibit p53 activation and S-phase arrest when relying on salvage alone, and exhibit increased sensitivity to DNA-damaging chemotherapeutics. This work advances our understanding of the consequences of aneuploidy and uncovers potential avenues for patient stratification and therapeutic intervention based on tumor ploidy.