Project Summary Aneuploidy is a cellular state in which cells contain extra or missing chromosomes. Over 90% of solid tumors are aneuploid. Aneuploidy has been shown to contribute to drug resistance and metastasis, and aneuploid cancers have a worse patient survival rate than euploid cancers. Despite aneuploidy’s role in cancer, aneuploidy itself causes growth defects and induces several ongoing stressors within the cell. Gaining or losing chromosomes leads to transcriptomic and proteomic stress, metabolic deregulation, an altered secretome, and induces further chromosome mis-segregation. We hypothesize that aneuploidy-induced cellular stresses can be targeted to specifically eliminate aneuploid cells, and we aim to discover genetic dependencies that are specific to aneuploid cells. We will use CRISPR to screen multiple near-euploid human cell lines and aneuploid clones that we derive from these near euploid cell lines. We will then compare the effect of aneuploidy on gene dependency, independent of cell line-specific effects. Toward this goal, we have generated 54 aneuploid clones from nine near euploid human cancer cell lines, and we have access to an additional ten aneuploid clones from two human cell lines. We will screen these aneuploid clones, and their near-euploid controls, with a domain- specific CRISPR library. This library targets multiple druggable protein domains, including all kinase, ubiquitinase, transcription factor, epigenetic modulator, “royal family” epigenetic factor, protease, and ubiquitin ligase genes. In preliminary work, we have screened multiple euploid and aneuploid cell lines with a smaller kinase domain-focused CRISPR library, and we identified several potential aneuploid-specific dependencies. More aneuploid cancer cell lines and their corresponding controls will be screened to confirm these potential hits. Cancer aneuploidy is not entirely random, as specific chromosome gains and losses are selected for in certain cancer types. In addition to uncovering general aneuploid dependencies, we aim to uncover chromosome specific dependencies. Once we have uncovered potential aneuploidy dependencies, we will validate their aneuploid-specificity, and then we will use cDNA to rescue gene knockout and rule out off-target effects. We will use IP mass spectrometry to uncover any differences in hit protein binding between euploid and aneuploid conditions, or to identify the protein binding partners of poorly characterized genes. Next, we will perform RT- qPCR and IF to screen for aneuploidy-associated phenotypes including chromosome missegregation, proteomic stress, senescence, and apoptosis. Additional follow up experiments will be performed to uncover their aneuploidy-targeting mechanisms and better understand the targetable stressors induced by aneuploidy. During this period I will be trained in CRISPR screening, bioinformatic analysis, and mass spectrometry techniques. Aneuploid dependencies and chromosome-specific aneuploid...