Chidamide reprograms AML-associated macrophages via HDAC3 inhibition to boost CD8+ T cell immunity

Abstract Introduction: Acute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by frequent relapse and treatment resistance. While therapeutic options have expanded to include molecularly targeted agents, clinical responses remain suboptimal. Emerging evidence highlights that tumor-associated macrophages (TAMs) within the tumor microenvironment (TME) are closely linked to AML progression. Targeting TAMs thus emerges as a compelling therapeutic strategy for AML immunotherapy. Chidamide is a histone deacetylase (HDAC) inhibitor approved in China for relapsed/refractory peripheral T-cell lymphoma. Emerging evidence suggests that HDAC inhibitors may possess immunomodulatory properties, with clinical reports indicating potential efficacy in relapsed/refractory AML and post-transplant AML settings. These findings underscore the importance of investigating its impact on the immunosuppressive microenvironment of AML and its underlying mechanisms. This study explores the effects of chidamide on AML-associated macrophages (AAMs) and elucidates its molecular mechanisms of action. Methods： We analyzed published datasets (GSE116256 scRNA-seq and GSE114868 transcriptome) to evaluate M1/M2-related gene expression patterns and their prognostic significance in AML. An AML-educated macrophage model was established. qRT-PCR measured expression changes of immune/inflammatory markers in macrophages after chidamide treatment. CFSE-labeled murine CD8+T cells were co-cultured with conditioned bone marrow-derived macrophages (BMDMs), with proliferation assessed by flow cytometry. HDAC-specific molecular inhibitors and siRNA knockdown were performed to identify chidamide's molecular targets, and transcriptomic sequencing was applied to screen core regulatory pathways. AML in vivo models were employed to test the antitumor immune effects of chidamide, especially its impact on AAMs. Results: Single-cell transcriptomic analysis of AML reveals heterogeneity in AAMs, with AML-enriched macrophages exhibited a propensity towards M2-type polarization. Further, analysis of the TCGA database demonstrates that M2-related markers are associated with poor prognosis. To explore the potential of immune activation in AAMs, we investigated the effects of chidamide on macrophage function. Following chidamide treatment, both RAW264.7 macrophages and BMDMs exhibited an immune-activated phenotype, as evidenced by upregulated expression of CD80, IL-6, TNFα, CXCL1 and CCL4. Furthermore, chidamide reversed the AAMs' suppressive effect on the proliferation of co-cultured CD8+ T cells. In vivo, mice treated with Chidamide exhibited significant suppression of tumor growth, along with an increase in M1 macrophage infiltration. RNA sequencing (RNA-seq) of Chidamide-treated RAW264.7 cells indicated activation of inflammatory pathways, including the TNF-α/NF-κB signaling pathway. The TNF-α inhibitor R7050 blocked the chidamide-mediated activation of anti-tumor immunity in AAMs, confirming its dependence on the TNF-α pathway. Chidamide is a histone deacetylase (HDAC) inhibitor targeting HDAC1, 2, 3, and 10. To investigate its mechanism, we treated AAMs with inhibitors specific to HDAC1, 2, 3, and 10. Results showed that only the HDAC3 inhibitor recapitulated the immune-activated phenotype observed with chidamide treatment. This effect was further confirmed by HDAC3 knockdown using siRNA. Conclusion：Chidamide reprograms AAMs toward an immunostimulatory phenotype by selectively inhibiting HDAC3 and activating the downstream TNF-α/NF-κB pathway. This reprogramming potentiates CD8+ T cell proliferation, thereby providing a novel mechanistic basis for enhancing AML immunotherapy.