Structure-guided dual targeting of EZH2 and dopamine D1 receptor suppresses TNBC growth and metastasis

Triple-negative breast cancer (TNBC) is an aggressive subtype with high relapse rates and limited therapies, emphasizing the need for mechanism-based strategies. EZH2, the catalytic subunit of PRC2, drives TNBC proliferation, metastasis, and chemoresistance, while dopamine D1 receptor (D1R) activation induces apoptosis, autophagy, and invasion inhibition. Immunohistochemistry confirmed strong EZH2 expression and elevated H3K27me3, but catalytic inhibition alone is insufficient due to non-enzymatic functions. We hypothesized that dual targeting of EZH2 and D1R would synergistically suppress TNBC by disrupting the EZH2-D1R axis. Computational modeling using a full-length AlphaFold EZH2 structure and three ligands (GSK126, A77636, SKF38393) identified canonical and cryptic binding pockets via PocketMiner and Fpocket, revealing the SET domain as the primary high-affinity site. DiffDock docking showed stable, high-affinity binding of GSK126, while A77636 and SKF38393 displayed dynamic, less-specific interactions. Molecular dynamics simulations over 100 ns confirmed GSK126's stability and persistent contacts at key residues (624, 684, 686), providing mechanistic insight. Immunoprecipitation validated an EZH2-D1R interaction, and D1R knockdown reduced combination efficacy. Experimentally, combination therapy reduced 2D cell viability (CI = 0.24). In droplet-based tumor spheroids (TSIMS), GSK126 + A77636 decreased spheroid diameter by 62% and induced necrosis without apoptosis, with confocal imaging showing EZH2 degradation, H3K27me3 loss, and cytoskeletal disruption. EZH2 knockout cells failed to form spheroids. In a vascularized SynTumor organ-on-chip model, the combination reduced circulating tumor cells by 50% within 96 hours, demonstrating suppression of metastatic spread. Together, these silico-guided and experimental findings establish a translationally actionable dual-target strategy that synergistically inhibits TNBC growth and metastasis. (Supported by DOD: W81XWH2010065, Eswar Shankar.).