PROJECT SUMMARY Whole-genome duplication (WGD) is increasingly appreciated as a defining moment for enabling chromosome instability in solid tumors. This proposal investigates chemotherapy-induced WGD that promotes the formation of polyploid cancer cells that appear senescent but in the absence of functional p53 are able to escape the senescent state and generate heterogenous daughter cells. The focus on high-grade serous ovarian carcinoma (HGSOC) derives from the urgent clinical need to improve therapy outcomes and the fact that p53 mutations are the most prevalent genetic abnormality (≥90%) associated with aneuploidy in this malignancy. Polyploid cancer cells exhibit a high level of plasticity and are thought to be a driving force in repopulating a tumor with highly aggressive, therapy-resistant, metastasis-prone progeny. Currently, there is a gap in knowledge that connects clinical descriptions of polyploid cells in tumor specimen with laboratory observations on the formation of polyploid cancer cells. We hypothesize to close this gap through a CRISPR-Cas9 screen to identify genes that are functionally necessary for formation of polyploid cancer cells and their daughter cells. We further hypothesize that polyploid cancer cells in clinical specimen can be identified by their large polynuclear morphology and one or more putative markers (cytoplasmic p21, acid ceramidase, VEGF-R2) resulting from an unbiased RNAseq analysis. Our specific aims are to (1) Define essential genes for the polyploid lifecycle critical transition stages and (2) to determine expression of putative markers of polyploid cancer cells in archived HGSOC samples. The proposed studies will serve as the foundation for future larger mechanistic, drug development, or prognostic studies with the potential to improve outcomes in ovarian cancer patients. An immediate impact will include prioritizing patients for the angiogenesis inhibitor Bevacizumab, a common HGSOC option, which may work better in VEGF-R2 expressing PGCC tumors. Of particular interest will also be therapeutic targeting of the polyploid population through re-purposing of existing drugs that would be combined with neo-adjuvant chemotherapy or used as a maintenance strategy following surgical debulking. Results obtained in HGSOC likely have broader relevance, since polyploid cells are observed in many other solid malignancies.