Novel Role of Chidamide in Tumor-Associated Macrophages Reprogramming and Immunotherapy Enhancement through RNA Splicing

Background

T/NK cell lymphomas (TNKCL) are aggressive malignancies with high rates of treatment failure and recurrence. Despite therapeutic advancements, including immune checkpoint inhibitors, antibody-drug conjugates (ADCs), and targeted drugs, their efficacy is limited. The tumor microenvironment (TME), particularly tumor-associated macrophages (TAMs), plays a crucial role in tumor progression and treatment response. TAMs, being highly plastic, offer a promising therapeutic target. Histone deacetylase inhibitors (HDACi) have shown efficacy against TNKCL, with five models approved. Chidamide, an HDACi targeting HDAC1/2/3/10, approved in China for relapsed or refractory peripheral T-cell lymphoma (PTCL), has shown an overall response rate of 28% and a median survival time of 21.4 months in relapsed or refractory PTCL, indicating a need to explore its mechanisms further. In this study, we investigated the effect of Chidamide on the phenotype of TAMs and its molecular mechanism, revealing the unique antitumor properties of Chidamide and expanding its clinical utility.

Methods

Macrophages from human and mouse sources were treated with varying Chidamide concentrations, and polarization was assessed by flow cytometry. A co-culture model evaluated macrophage phagocytic function in the presence of tumor cells. Inflammation-related gene and protein expressions were quantified using qPCR and Western blot. Reactive oxygen species (ROS) and metabolic indicators were measured with specific fluorescent probes. Biotinylated Chidamide was used in pull-down experiments to identify targets via mass spectrometry. Transcriptome sequencing and bioinformatics were utilized to identify variable splicing forms, confirmed by RT-PCR. T-cell lymphoma animal models were employed to test Chidamide's anti-tumor immunity and its synergistic effects with immune checkpoint inhibitors.

Results

Post-Chidamide treatment, a significant increase in M1 macrophages was observed in TNKCL patient peripheral blood mononuclear cells (PBDMs). Similar results were observed in healthy volunteer PBDMs. THP1 cells, and mouse bone marrow-derived macrophages, indicating Chidamide promotes M1 polarization. Co-culture models demonstrated enhanced macrophage phagocytosis. RNA sequencing (RNA-seq) of Chidamide-treated THP1 cells indicated activation of inflammatory pathways, indicative of a pro-inflammatory phenotype. In vivo, mice treated with Chidamide exhibited significant suppression of tumor growth, along with an increase in M1 macrophage infiltration, while the levels of CD8+ T cells and NK cells remained unchanged, indicating that the anti-lymphoma effect of Chidamide is macrophage-dependent. Comparative analysis with other HDACi showed Chidamide uniquely reprogram TAMs to M1, independent of HDAC targets. There were no significant differences in reactive oxygen species (ROS), lactate metabolism, and TAM-receptor tyrosine kinase (TAM-RTK) pathways, suggesting the involvement of a novel mechanism. The RNA splicing factor SF1 was identified as a target of Chidamide through biotin pull-down assays and mass spectrometry.SF1 knockdown abolished Chidamide's M1 polarization effect. RNA-seq showed significant reductions in exon skipping events in Chidamide-treated THP1 cells, with SETD4-L and PPWD1-L splicing variants promoting M1 polarization and stronger phagocytosis. The combination of Chidamide and anti-PD-1 antibody in mouse models showed enhanced tumor inhibition and M1 macrophage infiltration, indicating that the efficacy of immunotherapy is improved through M1 macrophage reprogramming.

Conclusion

Chidamide significantly enhances anti-lymphoma efficacy by promoting M1 macrophage polarization through a novel mechanism involving the splicing factor SF1 and specific splicing products, SETD4-L and PPWD1-L. The unique mechanism underlies Chidamide's superior clinical outcomes and its potential to enhance immunotherapy by modulating the TME.