Parameters that determine cell fate during mitotic arrest

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Prolonged activation of the spindle assembly checkpoint (SAC) due to mitotic spindle disruption can result in p53 activation, centriole disengagement and cell death. Indeed, one chemotherapeutic strategy frequently applied to aggressive and hormone-independent cancers is to target the mitotic spindle, a microtubule-based structure that is required for proper chromosome segregation and cell division. Drugs such as vinblastine or Paciltaxel suppress the normal microtubule assembly dynamics, leading to mitotic arrest and eventual cell death by apoptosis. However, despite their decades-long implementation in the clinic, the mechanisms by which prolonged mitotic delay results in cell death remains unclear. Further, despite the universality of the requirement of the mitotic spindle for cell division, there is still a great deal of heterogeneity in how cells respond to spindle disruption, which may reduce the efficacy of anti- mitotic chemotherapeutic strategies. Using a combination of biochemical and live cell imaging approaches, our preliminary data reveals that targeting both Kinesin Spindle Protein (KSP), a molecular motor required for spindle bipolarity, and the Phosphatidylinositide 3-kinase (PI3K)/Akt/mTOR signaling pathway dramatically accelerates the kinetics of mitotic cell death relative to mitotic arrest alone. Moreover, it elicits a more homogeneous response from the treated cells. PI3K signaling is involved in a variety of regulatory pathways that regulate cell survival, metabolism and proliferation, but the mechanism by which PI3K activity promotes cell viability during mitotic arrest is unknown. To better understand how PI3K signaling is involved in the timing of cell death and variability of cellular responses of mitotic delay, we will continue to apply high-throughput timelapse imaging, high-resolution 4D imaging and biochemical approaches to a battery of cell lines differ in their sensitivity to mitotic delay as well as dependence on PI3K signaling. The Specific Aims of this project will 1) Define the protective role of PI3K in normal and cancer cells; and 2) Determine the mechanism by which PI3K promotes cell survival during mitotic delay. If successful, these studies will lay the foundation for future translational studies to further develop adjuvant therapies that will target mitotically active tumor cells without the side effects associated with other microtubule disruptors.