TOPK Suppresses the CD8⁺ T Cell Antitumor Immunity via Modulation of IRF5 Expression

Background: T-LAK cell-originated protein kinase (TOPK), a serine/threonine kinase, is aberrantly overexpressed in human tumors and promotes malignant proliferation. Melanoma is a highly immunogenic tumor in which CD8⁺ T cell-mediated cytotoxicity is usually less effective in tumor control and responsive to immune checkpoint blockade. It is unclear whether the expression and functional characterization of TOPK within the immune cells affect the tumor microenvironment (TME) in patients with melanoma. This study aims to elucidate the expression pattern and immunoregulatory function of TOPK in CD8⁺ T lymphocytes during antitumor responses. Methods: Public single-cell RNA-sequencing (scRNA-seq) dataset analysis and flow cytometry assessed TOPK in tumor-infiltrating CD8⁺ T cells from patients with melanoma. Genetic deletion and pharmacological inhibition of TOPK using HI-TOPK-032 tested T cell-mediated melanoma control. Flow cytometry and tumor cell coculture killing assays measured effector release and target-cell apoptosis. Mechanistic analyses included assessment of interferon regulatory factor 5 (IRF5) expression, together with combination therapy using a programmed cell death protein 1 (PD-1)-blocking antibody in vivo. scRNA-seq of tumor-infiltrating lymphocytes (TILs) from Topk ^fl/fl and Cd8 ^Cre Topk ^fl/fl mice was also performed to define TOPK-dependent immune programs within the melanoma TME. Results: Single-cell transcriptomes identified a TOPK⁺ subset of tumor-infiltrating CD8⁺ T cells in melanoma, which was higher than that in normal lymph nodes (LNs), and exhibited suppressed cytotoxic and cytokine programs. CD8⁺ T cell-specific Topk deletion increased granzyme B (GzmB), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) secretion and improved tumor control. TOPK-deficient CD8⁺ T cells showed elevated activation-associated signaling pathways and immune effector gene expression. In murine TIL scRNA-seq, Cd8 ^Cre Topk ^fl/fl tumors exhibited increased effector and activation programs, reduced exhaustion and dysfunction programs, and enhanced immune crosstalk in the TME. Mechanistically, TOPK suppressed IRF5 expression and HI-TOPK-032 restored CD8⁺ T cell cytotoxicity in vitro and, with anti-PD-1, further inhibited tumor growth and increased intratumoral cytokine production. In human CD8⁺ T cells, enforced TOPK expression impaired cytotoxicity and cytokine secretion, reversed by IRF5 coexpression. Conclusions: These findings establish TOPK as the immune checkpoint limiting CD8⁺ T cell functionality in tumors and indicate the potential of TOPK inhibition as a strategy to augment T cell-based immunotherapies.