PROJECT SUMMARY Genome instability (GIN) is a hallmark of cancer and a leading cause of therapeutic resistance. This is especially true for pancreatic ductal adenocarcinoma (PDAC), where an estimated 95% of PDAC cells exhibit GIN. GIN is characterized by both a failure to accurately repair DNA in interphase and high rates of chromosome mis- segregation in mitosis. It is not fully understood how these pathways work in concert throughout the cell cycle to defend against GIN. Our previous work has demonstrated a link between the DNA damage response (DDR) protein ATR and the mitotic machinery in promoting faithful chromosome segregation. This observation has prompted me to hypothesize that other components of the DDR promote faithful chromosome segregation and genome stability. My preliminary work has elucidated a novel mitotic function for Chk2, an important DDR kinase that is activated following DNA double-stranded breaks. I have shown that Chk2 is active in a DNA damage-independent manner in mitosis. Moreover, I have demonstrated that Chk2 promotes proper activity of the major mitotic kinase PLK1. In this application, I propose to investigate the mechanism by which Chk2 promotes faithful chromosome segregation (Aim 1), determine how Chk2 activity is regulated in mitosis (Aim 2), and use PLK1 inhibition to specifically target Chk2-deficient cancer cells (Aim 3). My proposed studies will define the mitotic and DNA damage-independent role for Chk2. This will further our understanding of how the DDR pathway and mitotic machinery work in concert to promote faithful chromosome segregation and genome stability. Lastly, experiments from this proposal may develop a targeted therapeutic strategy to specifically kill Chk2-deficient cancers. This is critically important, as Chk2 loss-of-function mutations make up a significant portion of familial PDAC cases, and Chk2 mutations render cells insensitive to current front-line treatments. My career goal is to obtain a research faculty position at a leading institution where I will dissect the noncanonical and understudied mechanisms by which DDR proteins promote genome stability. My scientific development will be bolstered by successful completion of this project, including gaining expertise in cancer biology and mouse models. I will use these acquired skills to investigate whether PLK1 inhibitors can be used to specifically target Chk2-deficient cancer cells. Importantly, funding for this application will also enhance my training in science communication, writing, teaching, mentoring, and career development, all of which will be essential for successfully running my own laboratory. This project is supported by an outstanding network of sponsors and collaborators that will ensure this project is successfully completed. Receipt of this award will allow me to expand my research plan and establish myself as a primary investigator in the field of cancer biology and genome stability.