Abstract LB-A006: Impact of pharmacokinetic profiles on the <i>in vivo</i> antitumor efficacy of EHMT2 inhibitors combined with immune checkpoint blockade

Abstract Aberrant epigenetic regulation contributes to tumor progression and immune evasion. Euchromatic histone-lysine N-methyl transferase 2 (EHMT2; G9a), a key mediator of H3K9 methylation–dependent transcriptional repressor, is frequently overexpressed in multiple cancers and has emerged as a promising epigenetic target. However, the pharmacological properties of EHMT2 inhibitors remain insufficiently characterized. Here, we compared the cellular exposure, pharmacokinetics, and antitumor and immunomodulatory efficacies of two widely used EHMT2 inhibitors, BIX-01294 (BIX) and UNC0642 (UNC). Although UNC exhibited stronger EHMT2 inhibition in enzymatic assays, it was less effective in reducing the viability of colorectal and pancreatic cancer cell lines. Western blot analysis confirmed that BIX induced H3K9me2 reduction, p62 degradation, and LC3-II accumulation more effectively than UNC in MIA PaCa-2 cells. Moreover, BIX exhibited consistently higher intracellular levels than UNC, with about 3–4-fold differences observed across concentrations (1 or 4 μM) and time points (2, 6, or 16 h). These results indicate that enhanced cellular uptake or retention contributes to the superior cellular efficacy of BIX, despite its lower EHMT2 inhibitory effect. In vivo pharmacokinetic studies in ICR mice demonstrated that BIX achieved approximately two-fold higher systemic exposure than UNC following intravenous administration. Both compounds exhibited poor oral bioavailability, consistent with their low Caco-2 permeability. In contrast, intraperitoneal (i.p.) administration resulted in the highest systemic exposure, with BIX achieving an area under the concentration-time curve extrapolated to infinity of 115,662.0 ± 7,872.7 ng·h/mL, compared with 58,106.3 ± 8,018.9 ng·h/mL for UNC. Accordingly, the i.p. route was selected for efficacy studies. In the MIA PaCa-2 xenograft model, BIX was administered at 20 and 40 mg/kg, and UNC was administered at 5 and 8 mg/kg. For UNC, further dose escalation was limited by its toxicity. Under these conditions, BIX showed greater tumor growth inhibition than did UNC. Therefore, BIX was used in an immuno-oncology combination study at 30 mg/kg to mitigate weight loss observed at 40 mg/kg. In the MC38 syngeneic model, combination treatment with BIX (30 mg/kg) and α–PD-L1 antibody (10 mg/kg) produced significantly greater tumor growth inhibition than either monotherapy. Consistently, immunofluorescence analysis of tumor tissues demonstrated increased infiltration of CD8α+ cytotoxic T cells and NK1.1+ natural killer cells in the combination treatment group. Collectively, these findings demonstrate that cellular exposure and systemic pharmacokinetics are key determinants of EHMT2 inhibitor–mediated antitumor and immune-enhancing effects. BIX showed superior in vitro and in vivo efficacy and functioned as a promising partner for immune checkpoint blockade. Nevertheless, dose-limiting toxicity and suboptimal pharmacokinetics highlight the need for additional preclinical evaluations and optimization of its pharmacokinetic properties. Citation Format: Sang Eun Park, Ji-Yoon Lee, Unju Lee, Seoyeong Kim, Soo Jin Oh, Jung-Jin Hwang. Impact of pharmacokinetic profiles on the in vivo antitumor efficacy of EHMT2 inhibitors combined with immune checkpoint blockade [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr LB-A006.