Discovery of XNW5004 as a novel EZH2 inhibitor that enhances anti-tumor immunity and synergizes with PD-1 blockade immunotherapy in lung adenocarcinoma

Abstract

Synergistic strategies are urgently needed to enhance the efficacy of immunotherapy in lung cancer. Recent evidence highlights Enhancer of zeste homolog 2 (EZH2) as a pivotal epigenetic regulator that fosters an immunosuppressive tumor microenvironment, thereby driving immunotherapy resistance. We hypothesized that EZH2 pharmacological inhibition could increase immunotherapy susceptibility. This study aimed to investigate the potential of a novel EZH2 inhibitor, XNW5004, to sensitize lung adenocarcinoma (LUAD) to programmed cell death protein 1 (PD-1) blockade. In vitro, colony formation and apoptosis assays assessed direct cytotoxicity of XNW5004 on tumor cells at 0–12 µM. A co-culture system of tumor cells and peripheral blood mononuclear cells evaluated immune-mediated killing. In vivo, immunodeficient nude mice and immunocompetent C57BL/6 mice were randomly assigned to the control, XNW5004, anti-PD1, and combination groups to assess the tumor suppressive effect. Underlying mechanisms were explored through RNA sequencing alongside comprehensive cellular and molecular assays. In vitro, pre-treating tumor cells with 1.5 µM XNW5004 enhanced their sensitivity to immune cell attack, resulting in fewer residual cells and increased apoptosis, an effect further potentiated by PD-1 blockade. In vivo, XNW5004 suppressed tumor growth in immunocompetent C57BL/6 mice but showed minimal effect in immunodeficient nude mice. Mechanistically, XNW5004 stimulated chemokine-mediated recruitment of dendritic cells and T cells into tumor sites. Additionally, it upregulated the antigen presentation molecule major histocompatibility complex class I (MHC-I), while simultaneously augmenting the expression of co-signaling molecules programmed death ligand 1(PD-L1) and intercellular adhesion molecule-1 (ICAM-1). These alterations contributed to the augmented cytotoxic activity of both CD8+ T cells and natural killer cells, as evidenced by increased interferon-γ and granzyme B. The STING-TBK1-NF-κB axis functions as a pivotal regulatory signaling pathway driving these phenotype alterations. The EZH2 inhibitor XNW5004 enhances anti-tumor immunity and synergistically interacts with PD-1 blockade immunotherapy in LUAD, establishing this combinatorial approach as a promising therapeutic strategy.