PROJECT SUMMARY The outcome for patients with HER2-positive breast cancer (BC) has been significantly improved with the use of HER2-directed agents; however, hormone receptor (HR)+/HER2+ BCs that express both HR and HER2 are less responsive to anti-HER2 therapies as compared with HR-/HER2+ BC. Combined endocrine therapy plus anti-HER2-targeted therapy have been clinically investigated, but only a small subset of patients benefit from the treatment, indicating that HR+/HER2+ tumors have different biological characteristics, such as ER and HER2 signaling crosstalk. Dual HER2-blockade induces a Luminal A-like phenotype both in patients’ tumors and in in vitro models. However, the molecular mechanism underlying activation of ER signaling by anti-HER2 agents remains unclear. Using single cell RNA-sequencing (scRNA-seq), we identified Bromodomain Containing Protein 8 (BRD8) as an essential hub bridging HER2 and ER signaling pathways. We found that, BRD8, a component of p400 histone acetylase complex, was rapidly induced by treatment with a variety of anti-HER2 agents including neratinib, lapatinib and trastuzumab and that BRD8 is at the high hierarchy of ER activation in responsive to HER2 blockade. Furthermore, BRD8 regulates other growth promoting pathways in addition to ER. BRD8 knockout significantly impairs growth of a fulvestrant-resistant cell line and xenografts, and BRD8 knockdown enhances the sensitivity of HR+/HER2+ cells to HER2-targeting agents. These preliminary data lead to the hypothesis that BRD8 is an essential hub linking ER and HER2 pathways and BRD8 mediates ER activation in response to HER2 blockade in HER2+ cells. Therefore, combinatory BRD8 ablation with HER2 blockade should be effective for treating HR+/HER2+ BC. We propose three aims to test our hypothesis: (1) to elucidate the mechanism of BRD8 activation by anti-HER2 agents in HER2+ cells at the single cell level; (2) to define the roles of BRD8 in ER signaling activation and ER-independent functions upon HER2 blockade; (3) to test whether ablation of BRD8 sensitizes HR+/HER2+ BC to anti-HER2 blockade. The studies will reveal, at single-cell level, the cell-cell variability of BRD8 induction by anti-HER2 agents, and the novel functions of BRD8 in mediating ER/HER2 signaling crosstalk and ER-independent growth promoting functions. The mechanistic insights will lay foundation for developing combinatory BRD8 ablation and HER2 blockade therapy as a new treatment regimen for HR+/HER2+ BC.