Project Summary Ploidy variation is a cancer hallmark and is frequently associated with poor prognosis in high-grade cancers. Such variation can include an increase in chromosome set (polyploidy), a difference of chromosomes in a set (aneuploidy), or regional ploidy changes and copy number variations (CNVs). Genome-wide studies of multiple cancer specimens have shown that whole genome doubling is one of the most common molecular abnormalities in human cancers and is closely linked to other copy number alterations. However, how exactly polyploidy contributes to tumor growth, progression and malignancy remains largely unclear. Using a simple and highly reproducible Drosophila tumor model, where an active form of Notch (NICD) drives tumorigenesis in an epithelial transition zone (TZ), our preliminary studies indicate that tumor progression is driven by occurrences of polyploid mitosis, endoreplication, and ploidy reduction divisions. Both polyploid mitosis and depolyploidization are error-prone and can lead to chromosome abnormalities such as CNVs and polyaneuploidy, resulting in intratumoral heterogeneity in DNA ploidy. Comparative RNA-Seq analyses revealed that DNA damage response (DDR) genes are upregulated in these NICD-TZ tumors. Genetic epistasis studies have further shown that some of these DDR genes are required for the ploidy reduction division. Additionally, these tumors show sexual dimorphism regarding tumor growth and progression. Based on these findings, we hypothesize that polyploidy and associated cell-cycle variants are critical for continued tumor growth and increased tumor-cell genome instability during tumor progression. To test this hypothesis, we will carry out studies proposed in the following two aims: (1) to determine how polyploid cell divisions contribute to tumor growth and progression; (2) to characterize the sexual dimorphism of the NICD-TZ tumor model. The successful execution of the proposed studies will lead to a better understanding of intratumor ploidy heterogeneity and cancer evolution, as well as improved strategies in cancer prevention and treatment in a sex-dependent manner.