317 - Lactate-Induced DNMT1 Lactylation in Dendritic Cells Epigenetically Silences STAT1 to Suppress Antigen Presentation and Drive Radioresistant Tumor Immune Evasion
Presenter(s)
F. Sun1, W. Wen1, J. Wang1, X. Zhang1, Y. Lin1, D. Chen2, and J. Yu2; 1Department of Radiation Oncology and Shandong Provincial Key Laboratory of Precision Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, Shandong, China, 2Department of Radiation Oncology and Shandong Provincial Key Laboratory of Precision Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, China
Purpose/Objective(s): Radioresistance in solid tumors often leads to immune evasion and metastasis, with lactate accumulation in the tumor microenvironment as a key feature. However, the mechanisms connecting lactate to immune suppression in radioresistant tumors remain unclear. This study investigates how lactate produced by radioresistant tumors, epigenetically silences dendritic cell (DC) function to facilitate radioresistant tumor immune evasion.
Materials/Methods: Radioresistant tumor cell lines were established via in vivo selection in immunocompetent murine models. Flow cytometry characterized tumor immune infiltration. Western blotting assessed LDHA levels. Metabolomics quantified lactate in tumor fluid and conditioned media. Primary bone marrow-derived dendritic cells (BMDCs) and DC2.4 cell lines were treated with lactate or conditioned media from radioresistant cells in vitro, after which lactylome profiling was used to identify lactylated proteins, followed by RNA-Seq to assess transcriptional changes. Additionally, bisulfite sequencing was performed to analyze DNA methylation patterns in DCs. Antigen presentation capacity was assessed by co-culturing OVA-pulsed DCs with OT-I CD8+ T cells in vitro, followed by measuring T cell proliferation and cytokine secretion using CFSE dilution and ELISpot. Site-directed mutagenesis (DNMT1 K1247R) was implemented to validate lactylation-dependent epigenetic regulation in vitro. In mice bearing radioresistant tumors, DNMT1 inhibitor 5-azacytidine was administered to assess therapeutic effects.
Results: Radioresistant tumors exhibited reduced MHC-I+ DCs and impaired CD8+ T cell cytotoxicity, correlating with elevated LDHA-driven lactate secretion. Conditioned media or lactate treated DCs showed defective antigen presentation, impairing OT-I T cell proliferation and IFN-?/GzmB secretion in vitro. Additionally, conditioned media or lactate treatment increased STAT1 promoter methylation and suppressed STAT1 transcription in DCs. Lactylome analysis identified DNMT1 as a key target of lactylation, with K1247 as the modified residue, which enhanced its methyltransferase activity. DNMT1 K1247R mutation reversed STAT1 silencing and restored MHC-I expression. In vivo, 5-azacytidine treatment suppressed radioresistant tumor growth and metastasis, concomitant with restored MHC-I+ DCs and enhanced CD8+ T cell cytotoxicity.
Conclusion: Lactate from radioresistant tumors induces DNMT1 K1247 lactylation in DCs, epigenetically silencing STAT1 to suppress MHC-I-dependent antigen presentation. This impairs CD8+ T cell immunity, contributing to radioresistant tumor immune evasion. Targeting DNMT1 or lactate metabolism offers a promising strategy to restore immune function and overcome radioresistance.