A novel dual-target strategy against TNBC: Combining EZH2 inhibition and dopamine D1 receptor activation to restore immune balance

Triple-negative breast cancer (TNBC) remains one of the most aggressive and therapeutically challenging breast cancer subtypes, characterized by high heterogeneity, intrinsic resistance, and poor immune infiltration. There is a critical unmet need for less toxic approaches that can both suppress tumor growth and remodel the tumor microenvironment (TME). EZH2, a histone methyltransferase, is frequently overexpressed in TNBC, associated with immunosuppression, and disease progression. We previously reported combining GSK126 (an EZH2 inhibitor) with A77636 (a dopamine D1 receptor [DRD1] agonist) produced superior antitumor effects compared to monotherapies. We hypothesized synergistic targeting of EZH2 and DRD1 would convert the immunologically “cold” TNBC microenvironment into a “hot,” immune-permissive state, thereby enhancing therapeutic efficacy and overcoming resistance mechanisms. Female NSG mice (4-6 weeks old) were orthotopically implanted with MDA-MB-231 cells, randomized into four treatment groups (n=8): vehicle, GSK126 (2 mg/kg), A77636 (50 mg/kg), or combination. Treatments were administered intraperitoneally weekly for four weeks. Tumor volume was measured weekly and at endpoint. Tumors and immune tissues were analyzed by flow cytometry. Combination therapy significantly reduced tumor weight (mean difference = 0.278g, 95% CI: 0.109-0.446, p =0.0018) and volume (mean difference = 101 mm³, 95% CI: 51.7-151, p <0.0001) compared with vehicle and single agents. Notably, the combination markedly decreased monocyte populations in both blood and tumor tissue and downregulated EZH2 expression in tumor-associated monocytes and neutrophils. Kinetic profiling revealed a biphasic monocyte response-initial Ly6C^hi recruitment followed by Ly6C^lo transition. The combination suppressed Ly6C^hi infiltration (0.32 vs. 0.92; 65% decrease, p =0.0138) while promoting Ly6C^lo accumulation (2.5 vs. 1.5; 1.67-fold increase, p =0.5126). By using a combinational treatment strategy to redirect monocyte phenotypes, we were able to suppress the pro-inflammatory IL-1beta environment while simultaneously enhancing anti-inflammatory IL-10 signaling-ultimately creating a tumor-suppressive immune milieu. In summary, dual modulation of EZH2 and DRD1 effectively halts TNBC progression by reshaping the immunological landscape. These findings uncover a distinctive therapeutic avenue that integrates epigenetic regulation with dopaminergic activation to restore immune responsiveness in TNBC. (This work was supported by DOD W81XWH2010065 to Eswar Shankar).