Abstract Aneuploidy, the state in which cells carry an incorrect number of chromosomes, is a hallmark of human cancers. Over 85% of cancers are aneuploid, and higher rates of aneuploidy are often associated with poor patient prognosis. Aneuploid tumors can display heterogeneous karyotypes, which underlie heterogeneity in cellular phenotypes. This presents a specific challenge in treating aneuploid tumors, because they can rapidly evolve mechanisms to evade treatment. Given the high incidence of aneuploidy in diverse cancer types, an attractive strategy would be to selectively sensitize cells to the aneuploid state itself, especially in combination with chemotherapy drugs. This has been challenging, in large part because it remains unclear how aneuploid cancer cells can tolerate extra DNA content in the first place. We have taken a novel perspective to this challenge, by studying wild strains of budding yeast Saccharomyces cerevisiae that are naturally tolerant to extra chromosomes. Yeast is a powerful model for dissecting cellular biology, because many of the mechanisms and defense strategies are conserved in humans. By comparing aneuploidy-tolerant wild strains to a well-studied laboratory strain that is unusually sensitive to aneuploidy, we discovered a single gene that, when deleted, produces little to no phenotype in euploid strains, but renders cells very sensitive to extra chromosomes. Thus, we can sensitize cells to aneuploidy without producing major phenotypes in the normal euploid cells. The gene – Ssd1 – has been implicated in mRNA localization, translational control, and chromosome maintenance among other things, but the mechanisms remain unclear. We believe that the process of aneuploidy tolerance is conserved between yeast and humans. The human ortholog of Ssd1, hDis3L2, shares several features with Ssd1, including links to translational regulation, P-body localization, and proper chromosome segregation. The goal of this proposal is two fold: 1) to identify the mechanism through which SSD1 deletion sensitizes yeast to aneuploidy and 2) to use this information to test if knockdown of orthologous functions sensitizes cancer cell lines to aneuploidy, with and without chemotherapy treatment. Aim 1 will use genomics, proteomics, single-molecule RNA fluorescence in situ hybridization (FISH), and singe, live-cell imaging to test the role of Ssd1 in aneuploidy tolerance. Aim 2 will leverage these insights to test if orthologous mechanisms, including knockdown of the human ortholog hDis3L2, can sensitize breast and colon cancer cell lines to aneuploidy, with and without paclitaxel treatment. This aim will use a powerful system to produce isogenic sets of euploid and aneuploid human cells, enabling sensitive dissection of phenotypes that are specific to the aneuploid state. Results of this work will expand our understanding of the function of Ssd1/hDis3L2 and could pave the way to new thera...