Abstract PS5-03-11: Combinatorial EZH2 Inhibition and Dopamine D1 Agonism Suppresses Pro-Inflammatory Monocytes and TNBC Tumor Progression

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive disease and lacks effective therapies due to tumor heterogeneity, resistance, and poor immune infiltration. There is a critical need to develop novel and less toxic therapies to improve TNBC survival outcomes. Treatment with Enhancer of Zeste Homolog 2 (EZH2) inhibitors have shown improved efficacy suggesting that these inhibitors play a pivotal role in TNBC progression. We have previously demonstrated that combined treatment with GSK126, an EZH2 inhibitor and dopamine D1 receptor agonist (A77636) exhibit higher efficacy in inhibiting tumor growth and metastasis of TNBC cells both in vitro and in vivo. Here, we examined the immune phenotype/function by which EZH2 and A77636 combination reprogram the TNBC microenvironment. Methods: Female NSG mice (4-6 weeks old) were orthotopically injected with MDA-MB-231 cells into the mammary fat pads. Once tumors became palpable, mice were randomized into four treatment groups (n = 8 per group): Vehicle control, GSK126 (2 mg/kg body weight), A77637 (50 mg/kg body weight), and the combination of GSK126 and A77637. Treatments were administered intraperitoneally once per week for four weeks. Tumor size was measured weekly using calipers, and tumor volume was calculated using the formula: (length × width2)/2. At the end of the treatment period, mice were euthanized. Tumors were excised and weighed. Tumor tissue, bone, blood, and spleen were harvested for flow cytometry analysis. Results: The combination of GSK126 and A77636 demonstrated a synergistic antitumor effect, significantly reducing both tumor weight and volume compared to vehicle or single-agent treatments. Tumor weight was significantly lower in the combination group compared to vehicle (mean difference = 0.278 g, 95% CI: 0.109-0.446, p = 0.0018). Similarly, tumor volume was significantly reduced in the combination group relative to vehicle (mean difference = 101 mm3, 95% CI: 51.7-151, p 0.0001). This combinatorial treatment also led to a marked reduction in monocyte populations within both the peripheral blood and tumor microenvironment. EZH2 expression was significantly decreased in tumor-associated monocytes and neutrophils in the combination group. Kinetic analysis of tumor-infiltrating monocytes revealed a biphasic response: initial recruitment of pro-inflammatory Ly6Chi monocytes followed by a transition to anti-inflammatory Ly6Clo monocytes. Notably, the combination therapy significantly reduced Ly6Chi monocyte infiltration compared to the vehicle control (0.32 vs. 0.92, representing a 65% decrease; p = 0.0138). In contrast, it increased Ly6Clo monocyte levels relative to vehicle control (2.5 vs. 1.5), showing a 1.67-fold increase, (p = 0.5126) compared to single-agent and single-dose treatments. Furthermore, the combination group showed a trend toward increased production of the anti-inflammatory cytokine IL-10, while reduction of pro-inflammatory cytokine IL-1β production in both Ly6Chi and Ly6Clo monocyte. Conclusion: These findings demonstrate that dual targeting of EZH2 and A77636 not only suppresses TNBC tumor growth but also reprograms the tumor immune repertoire by shifting monocyte dynamics toward an anti-inflammatory phenotype, offering a promising strategy to overcome resistance and improve therapeutic outcomes. (This work is supported by DOD: W81XWH2010065, for Eswar Shankar) Citation Format: R. Shukla, K. Ormiston, G. Sarathy, S. Dhekne, D. Quiroga, S. Gupta, D. Stover, P. Giglio, C. Rolfo, E. Shankar. Combinatorial EZH2 Inhibition and Dopamine D1 Agonism Suppresses Pro-Inflammatory Monocytes and TNBC Tumor Progression abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS5-03-11.