Genetic and Pharmacologic Targeting of Eya3 in Macrophages Drives Anti-Tumor Immunity in Triple-Negative Breast Cancer

Abstract

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer that remains difficult to treat despite its relatively high immunogenicity, as tumors frequently evade immune destruction through mechanisms that are not yet fully understood. Here, we discover a previously unrecognized role for Eya3 within macrophages in the tumor immune microenvironment, where its expression is elevated and correlated with increased aggressiveness in TNBC. Using macrophage-targeted Eya3 knockdown and conditional knockout mouse models, we show that Eya3 depletion induces coordinated transcriptional and functional changes in macrophages, enhancing migration, antigen processing, and inflammatory signaling associated with anti-tumor immunity. Strikingly, macrophage-targeted deletion of Eya3 reprograms the immune response in TNBC, increasing CD8+ T cell infiltration, suppressing primary tumor growth, and prolonging survival. Pharmacologic inhibition of Eya1-3 tyrosine phosphatase activity with a newly derived allosteric inhibitor, LG1-34, mirrors this effect, dramatically reducing primary TNBC growth through immune-mediated mechanisms that likely act both though targeting tumor and immune cells. Importantly, TNBC patients treated with pembrolizumab exhibit high expression of Eya3 in both tumor and immune compartments, and increased macrophage specific Eya3 expression correlates with worsened response to anti-PD1 therapy. These findings identify Eya3 as a macrophage-intrinsic checkpoint on anti-tumor immunity, identifying a new potential vulnerability in TNBC.