Embryonic Ectoderm Development (EED) Inhibitor APG-5918 Demonstrates Robust Antitumor Activity in Preclinical Models of T-Cell Lymphomas (TCLs)

Introduction

TCLs encompass a heterogeneous group of hematologic neoplasms derived from clonal proliferation of T lymphocytes and are often characterized by aggressive clinical behavior and poor prognosis. Enhancer of zeste homolog 2 (EZH2), as a crucial histone methyltransferase and the catalytic subunit of the polycomb repressive complex 2 (PRC2), mediates trimethylation of histone H3 at lysine 27 (H3K27me3) to silence gene expression. EZH2 is aberrantly overexpressed with very poor prognosis in TCLs. EED, another core subunit of the PRC2, is vital for maintaining histone methyltransferase activity. Targeting of EED has emerged as a promising strategy to inhibit PRC2 function. APG-5918 is an investigational potent, selective, small-molecule EED inhibitor. The aim of this study was to evaluate antitumor activity and molecular mechanisms of APG‑5918 monotherapy in preclinical models of TCLs.

Methods

The in vitro antiproliferative activity of APG-5918 was evaluated in four commonly used TCL cell lines: HH, HuT102, HuT78, and H9. Cell viability was assessed using the CellTiter-Glo^® luminescent assay. Cellular apoptosis was examined by annexin V-FITC assay via flow cytometry. Cell cycle assays were used to determine the proportion of cells at different stages by flow cytometry. Western blotting was employed to investigate protein expression in cell lines. A subcutaneous HuT102 CDX model was established to evaluate the antitumor activity of APG‑5918 in vivo.

Results

APG-5918 demonstrated potent inhibitory effects on cellular proliferation as a single agent in vitro, with half-maximal concentration (IC₅₀) values ranging from 0.1 to 7.2 μM. The antiproliferative activity of APG-5918 was superior to that of dual EZH1/2 inhibitor valemetostat and EZH2-selective inhibitor tazemetostat under the same experimental conditions. Specifically, IC₅₀ values for APG-5918 were 2.75 µM in HH, 2.53 µM in H9, and 7.2 µM in HuT78 cells, whereas HuT102 exhibited the highest sensitivity, with an IC₅₀ value of 0.1 µM, representing 3.8- and 5.7-fold more potent activity than valemetostat and tazemetostat, respectively. APG‑5918 induced apoptosis in HuT102 cells in a dose-dependent manner, with approximately 23.7% of cells at 10 nM, 48.4% at 100 nM, and 67.8% at 1 μM. Furthermore, cell cycle analysis revealed that APG‑5918 dose‑dependently increased the proportion of HuT102 cells in the G0/G1-phase, whereas it decreased percentages in the S- and G2/M-phases; these findings indicate a cell cycle arrest effect. In the HuT102 CDX model, APG-5918 was administered orally at doses of 1, 3, 10, or 30 mg/kg once daily for 28 days. Significant and dose-dependent antitumor activities were observed. On day 29, T/C values were 58.98% for 1 mg/kg, 31.80% for 3 mg/kg (p < 0.05 vs. vehicle), and 8.72% for 10 mg/kg (p < 0.01 vs. vehicle). Notably, administration of APG-5918 at 30 mg/kg led to complete tumor regression (p < 0.01 vs. vehicle), achieving a 100% overall response rate. No significant differences in body weight changes were observed between treatment groups and the vehicle group, indicating a favorable safety profile (all p values > 0.1 vs. vehicle). Mechanistically, western blot analyses revealed that APG-5918 dose‑dependently downregulated key on-target pharmacodynamic markers, including H3K27me3, EED, and EZH2. In addition, APG-5918 markedly suppressed phosphorylated Rb (pRb) and CDK6, which are two crucial regulators involved in cell cycle regulation. APG-5918 also upregulated expression of proapoptotic proteins Bcl-2-interacting mediator of cell death (BIM) and Noxa. The apoptogenic activity of APG-5918 was further supported by increased levels of cleaved poly(ADP-ribose) polymerase-1 (PARP-1) and caspase-3.

Conclusions

Our results suggest that APG-5918 inhibits tumor growth of TCL both in vitro and in vivo. Mechanistically, APG-5918 likely exerts its antitumor activity by modulating the PRC2 complex, leading to apoptosis induction and cell cycle arrest. These findings provide a strong scientific rationale for the future clinical development of APG-5918 for TCLs.