Targeting CLEC4E in immunosuppressive tumour-associated macrophages via BET inhibition

Abstract

Background: Immunosuppressive tumour-associated macrophages (TAMs) represent a promising target for cancer immunotherapy; however, existing TAM-directed therapies have shown limited clinical efficacy. C-type lectin domain family 4 member E (CLEC4E), a pro-inflammatory molecule expressed on macrophages, was recently found to be highly enriched in TAMs. This study aims to elucidate the role of CLEC4E in TAMs and identify potential therapeutic agents targeting CLEC4E, and to clarify the mechanism of Bromodomain and extraterminal domain (BET) inhibitor NHWD-870 in downregulating CLEC4E.

Methods: We first assessed the correlation between CLEC4E expression and survival in melanoma patients. Clec4e^flox/flox Lyz2-cre (knockout) and Clec4e^flox/flox (control) mice were generated and implanted with melanoma or ovarian cancer models. Single-cell RNA sequencing was performed to characterise macrophage phenotypic changes following CLEC4E knockout, with validation via RT-PCR, flow cytometry and proteomic sequencing. A drug screen identified BET inhibitors targeting CLEC4E, and their mechanisms were further investigated using RNA silencing, Chromatin Immunoprecipitation (ChIP)-seq and luciferase reporter assays.

Results: In melanoma patients, high CLEC4E⁺ TAM infiltration was associated with poor prognosis. CLEC4E knockout significantly suppressed tumour growth compared to control mice. TAMs from knockout mice exhibited downregulated proliferation markers and upregulated genes related to antigen presentation and pro-inflammatory responses. Mechanistically, CLEC4E deletion inhibited TAM proliferation via the Erk signalling pathway, enhanced TAM‒T cell interactions, and increased granzyme B expression in T cells. The BET inhibitor NHWD-870 was shown to disrupt BRD4‒CEBPβ interaction, leading to downregulation of CLEC4E expression.

Conclusions: CLEC4E⁺ TAMs promote an immunosuppressive microenvironment by enhancing their own proliferation and impairing anti-tumour functions, thereby limiting T-cell cytotoxicity. Targeting the BRD4/CEBPβ/CLEC4E axis with BET inhibitors represents a promising therapeutic strategy for reprogramming TAMs and enhancing anti-tumour immunity.

Keywords

BET inhibitor / CLEC4E / conditional knockout mice / single-cell RNA sequencing / tumour-associated macrophage

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2025 The Author(s). Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.