Summary Recent studies show that whole genome duplication (WGD) is a frequent event in cancer evolution that promotes chromosomal instability and aneuploidy. WGD tumors have worse prognosis, elevated drug resistance, and increased metastatic potential when compared with near-diploid counterparts. However, the mechanisms driving WGD during cancer evolution remain unclear. This is because the events that lead to WGD occur asynchronously and at low frequency, making them difficult to capture using traditional approaches such as fixed endpoint approaches. My lab has pioneered genetically encoded biosensors and image analysis techniques to biochemically characterize thousands of individual cells for multiple days as they go through normal or aberrant cell cycles. Using these approaches, we have found that WGD occurs in response to common stress conditions such as osmotic stress, DNA damage, and ribosome collisions. This process involves two steps: first cells go from G2 to G0 without entering into mitosis (i.e. mitotic bypass). Second, some cells escape cell cycle arrest and enter S- phase, thereby re-duplicating their genome. Furthermore, our data show that DNA damage caused by commonly used chemotherapeutics promotes WGD in cancer cells raising questions about the role of chemotherapy- induced WGD in acquired drug resistance. This is particularly important in metastatic Triple Negative Breast Cancer (mTNBC), for which DNA damaging agents are still a mainstream treatment, and the development of resistance continues to be a devastating health care problem (median survival rate <18 months). Here I propose to use our live single-cell approaches to characterize the causes and consequences of WGD in cancer progression and acquired drug resistance. In Aim 1, we will combine live cell imaging and single-cell sequencing to uncover how physiological stresses trigger mitotic bypass and WGD in non-transformed cells. In Aim2, we will study the mechanisms of chemotherapy-induced WGD in cancer cells. In Aim 3, we will use human breast cancer organoids and patient derived xenografts to define the role of chemotherapy-induced WGD in acquired drug resistance. Overall, our work will pave the way toward characterizing cell-cycle dynamics in cancer cells to identify unique vulnerabilities that target the aberrant cell cycles that drive tumorigenesis.