Targeting bromodomain and extra-terminal proteins reprograms macrophages and inhibits breast cancer progression.

Introduction: Breast cancer (BC) remains a leading cause of cancer-related deaths, with resistance to therapy driving tumor progression. While immune checkpoint inhibitor (ICI) therapy is effective in some breast cancers, the heterogeneity of treatment responses is partly driven by cancer stem cells (CSCs) and an immunosuppressive tumor microenvironment (TME). Bromodomain and extra-terminal (BET) proteins regulate tumor and immune biology, but their role in innate anti-tumor immunity remains poorly understood. We investigated the role of BET proteins in modulating innate immunity using the potent BET degrader (BETd) ZBC260. Methods: ZBC260 efficacy was evaluated in immunocompetent BALB/c mice bearing D2A1 tumors. NK cells or macrophages were depleted to determine their contribution to BETd response. Results and discussion: ZBC260 inhibited tumor growth in SCID mice but not in NOD-SCID mice, with impaired macrophages and partially compromised NK cells, implicating the innate immune system. To assess the contribution of innate immune cells to BETd efficacy, we depleted NK cells or macrophages in immunocompetent BALB/c mice with D2A1 tumors and treated with ZBC260 or vehicle. Immune depletion studies revealed macrophage loss partially attenuated anti-tumor and anti-CSC effects, highlighting their critical role. To understand the impact of BETd on TME populations, we performed single-cell RNA sequencing, which identified myeloid cells as the predominant population. BETd reduced MRC1⁺immunoregulatory tumor-associated macrophages and enriched CD74+ antigen-presenting macrophages, with upregulated phagosome pathways and enhanced Stat1 activation. Conclusion: Collectively, these findings demonstrate that BETd ZBC260 reshapes the TME and reprograms macrophages toward an anti-tumor phenotype, highlighting its translational potential for breast cancer therapy.