Abstract 509: Synergistic inhibition of TNBC via enhancer of zeste homologue 2 (EZH2) inhibition and D1 receptor agonism

Abstract EZH2, a histone methyltransferase linked to poor outcomes in cancer, is overexpressed in triple-negative breast cancer (TNBC), disproportionately affecting African-American and Hispanic women. EZH2 regulates the tumor immune microenvironment (TIME) by inhibiting MHC-I antigen presentation, upregulating PD-L1, leading to reduced antitumor immune responses. Current EZH2 inhibitors target its catalytic activity; however, the development of resistance mechanisms in cancer cells frequently undermines their effectiveness. The neurotransmitter dopamine, through activation of its D1 receptor, promotes apoptosis and autophagy while suppressing cancer cell invasion. Dopamine D1 receptor signaling mitigates the immunosuppressive effects of myeloid-derived suppressor cells (MDSCs) on T cell proliferation and IFN-γ production. Using in silico approaches such as cryptic pocket identification, docking, and molecular dynamics (MD) simulations, initially we assessed the binding affinity of the DRD1 agonist to EZH2. Our in-silico analyses uncovered stable binding profiles, indicating novel interactions at previously uncharacterized sites within the CXC domain and the EED-binding region for both GSK126 and A77636. Next, we performed in vivo experiments combining GSK126 and A77636 to evaluate their effect on tumor growth and the TIME using an NSG mouse model. MDA-MB-231 cells were injected into the breast fat pads of 4-6-week-old female NSG mice. Once tumors were palpated, the mice were randomized into four groups: vehicle treatment, GSK126 (2 mg/kg), A77636 (50 mg/kg), and the combination of both, with treatments administered intraperitoneally for four weeks. Tumor growth was monitored throughout the treatment period, and at the termination, blood, tumor, and spleen were collected and processed into single-cell suspensions for high-dimensional flow cytometry analysis. GSK126 and A77636 treatment in combination significantly reduced tumor volume and decreased EZH2 expression in monocytes and neutrophils within the blood and tumor microenvironment and lowering the levels of pro-inflammatory cytokine IL-1β. In summary, this approach unveils new possibilities for treating TNBC by concurrently targeting EZH2 and DRD1 through a combinatorial strategy. (This work is supported by DOD: W81XWH2010065, for Eswar Shankar). Citation Format: Rajni Kant Shukla, Kate Ormiston, Gautam Sarathy, Shivani Dhekne, Sanjay Gupta, Daniel Stover, Christian Rolfo, Eswar Shankar. Synergistic inhibition of TNBC via enhancer of zeste homologue 2 (EZH2) inhibition and D1 receptor agonism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 509.