Carm1 Inhibition Potentiates Irradiation-Induced Antitumor Immunity via Tumor Intrinsic STING Pathway Activation

Abstract

Radiotherapy is commonly applied in multiple cancer types. Besides irradiation induced direct cell death, radiotherapy stimulated significant immune responses for tumor control. Intact and functional cGAS-STING pathway in both tumor cells and host cells is indispensable for efficient irradiation-induced anti-tumor effects. Coactivator-associated arginine methyltransferase 1 (Carm1) is emerging as an attractive therapeutic target and a biomarker for prognosis in various types of cancer. It has been reported that Carm1 inhibition could improve immunotherapy induced anti-tumor effects. However, it remains unclear how tumor cell intrinsic Carm1 affects irradiation-induced anti-tumor immunity. Carm1 deficient cell lines were established in MC38 and B16F10 murine cancer cells using the CRISPR/Cas9 technology. To verify the effects of tumor `subcutaneous tumor mouse models were established and one fraction of 15Gy was administrated when the tumor volume reached 200mm3, followed by flow cytometry assays. Transcriptome sequencing, protein mass spectrometry, single-cell sequencing, Digital Spatial Profiling (DSP), real-time qPCR, western blotting, immunofluorescence and co-immunoprecipitation were carried out to explore and verify possible molecular mechanisms. Here we found Carm1 deficiency in tumor cells dramatically enhanced irradiation-induced anti-tumor immune responses. Transcriptome sequencing of irradiated tumor cells and further experiments then validated that cGAS-STING pathway was significantly activated after irradiation in the absence of Carm1 in tumor cells, which contributed to enhance anti-tumor immunity after irradiation. Mechanistically, Carm1 deficiency in tumor cells attenuated autophagy, resulting in increased cytoplasmic mtDNA enrichment and enhanced cGAS-STING pathway activation. On the other hand, we also found that Carm1 caused asymmetric arginine methylation (ADMA) modification of TBK1 with reduced phosphorylation level, and Carm1 deficiency could activate cGAS-STING pathway by reducing AMDA modification and enhancing phosphorylation of TBK1. Finally, Carm1 inhibitor EZM2302 was applied in combination with radiotherapy in vitro, and it's indicated that combination therapy resulted in intensive anti-tumor immunity and prominent abscopal effects. In this study, we identified that Carm1 ablation in tumor cells could promote irradiation-induced antitumor immunity through tumor cell intrinsic STING pathway activation. Mechanically, Carm1 deficiency directly activated the cGAS-STING pathway by interacting with TBK1 and increased mtDNA accumulation in cytoplasm by inhibiting autophagy. These findings provided new strategies for targeting Carm1 to boost the efficacy of radiotherapy.