Project Summary The overall goal of this proposal is to determine the mechanism by which Insulin Receptor Substrate 2 (IRS2) regulates the spindle assembly checkpoint (SAC) and to use this information to study novel therapeutic approaches for the treatment of triple negative breast cancer (TNBC). The IRS proteins are essential adaptor proteins of the insulin (IR) and insulin-like growth factor-1 (IGF1R) receptors. Dysregulation of this pathway through upregulation of receptors and ligands drives breast cancer initiation, growth and metastasis. Unfortunately, interfering with the IIS pathway for the treatment of breast cancer by receptor/ligand-targeted approaches has been unsuccessful, and efforts to improve the ability to target the IIS pathway for cancer therapy are hindered by the fact that the mechanism(s) by which this signaling pathway contributes to cancer progression are not well established. This proposal focuses on IRS2 because this adaptor protein is expressed preferentially in TNBC, and the applicant has compelling data that IRS2 plays a causal role in the aggressive nature of this breast cancer subtype. Understanding the mechanisms of action of IRS2 in TNBC is essential for exploiting IRS2 as a therapeutic target. In this regard, intriguing recent evidence from the applicant’s lab and other labs has emerged that implicates IRS2 in mitotic regulation. Specifically, IRS2 expression increases during mitosis, and it is required for cell cycle arrest at the spindle assembly checkpoint (SAC), a process that ensures precise chromosomal segregation to maintain genome integrity. Importantly, no studies to date have established the mechanistic role of IRS2 in SAC regulation. Toward this goal, the applicant has identified the cell cycle regulatory kinase PLK1, which controls the SAC, as an IRS2-specific interacting protein. Together, these findings reveal a potential IRS2-dependent vulnerability that could be exploited therapeutically, a possibility that is supported by the applicant’s preliminary data that loss of IRS2 expression sensitizes TNBC to mitotic stress drugs. To investigate the hypothesis that IRS2 regulation of the SAC sustains TNBC viability and that targeting this function of IRS2 will inhibit tumor growth at both primary and metastatic sites, the applicant will: 1) Investigate the role of IRS2 in spindle assembly checkpoint (SAC) regulation. The hypothesis that IRS2 expression and localization are essential for efficient mitotic SAC activation will be examined; 2) Establish the molecular mechanisms by which IRS2 regulates the mitotic SAC. The hypothesis that IRS2 promotes the fidelity of spindle formation through the recruitment and activation of PLK1 will be examined; 3) Assess the therapeutic efficacy of targeting IRS2-dependent cell cycle functions in TNBC. The hypothesis that acute targeting of IRS2 expression and/or its cell-cycle regulatory functions will sensitize TNBC to mitotic stress will be examined.