PROJECT SUMMARY Background ER+ breast cancer accounts for 2/3 of invasive breast cancer cases diagnosed in the world today. In patients, ~12% of ER+ tumors have complete loss of MLH1, a tumor suppressor and mismatch repair protein. We previously showed that MLH1 loss in ER+ breast cancer cells activates HER2 to induce resistance to standard endocrine therapy. However, the underlying mechanism remains unknown. Preliminary data support a role for MLH1 loss in altering secretion of HER ligands to mediate HER2 activation. This is of particular interest because an additional 14% of ER+ patient tumors have heterogeneous loss of MLH1. Although the functional consequence of heterogeneous MLH1 loss is as yet unknown, a role for MLH1 loss in altering the cancer secretome suggests a potentially significant impact on bulk tumor phenotypes. Hypothesis Based on our published work and preliminary data, the proposed study will test the hypothesis that MLH1 loss in ER+ breast cancer induces secretion of HER ligands, by co-opting cGAS-STING signaling, to promote HER2-dependent growth of both MLH1– and MLH1+ cells in heterogeneously MLH1– tumors. Specific Aims Aim 1 will test whether the secretion of HER ligands induced by MLH1 loss mediates HER2 activation to promote endocrine therapy resistance. Here, we will use both in vitro and in vivo approaches to determine whether the ligands secreted by MLH1- ER+ breast cancer cells promote HER2 heterodimerization and consequent activation, and thereby endocrine therapy resistant growth. Aim 2 will investigate the mechanism linking MLH1 loss to HER ligand secretion. Specifically, we will test whether inefficient DNA damage repair in the absence of MLH1 triggers the cGAS-STING secretory pathway. We will also determine how cGAS-STING activation promotes HER ligand secretion. Aim 3 will examine the therapeutic impact of heterogeneous MLH1 loss. Using in vitro and in vivo experimental models established in my lab, we will test whether HER2 activation in both MLH1+ and MLH1- cells in tumors with heterogeneous MLH1 loss renders these tumors globally susceptible to HER2 inhibitors. We will also use digital spatial profiling to assess the contribution of the secretome to signaling patterns and therapeutic response in heterogeneously MLH1- tumors. Impact The results of this study will present new therapeutic strategies to help >25,000 women diagnosed each year with ER+ breast cancer characterized by heterogeneous MLH1 loss. Our work will also provide novel insight into how MLH1 loss modulates the cGAS-STING pathway to promote a pro-growth tumor secretome. Overall, results from this study will shed new insight into the how MLH1 loss promotes cancer phenotypes.