2892 - Targeting A-to-I RNA Editing and Radiation-Induced CD8+ T Cell Dysfunction in Non-Small-Cell Lung Cancer

C. Tian^1,2, F. Wang¹, J. Zhang¹, S. Bao¹, J. Ma¹, Z. Zhang³, S. Shang¹, M. Wu⁴, J. Yu⁵, and D. Chen¹; ¹Department of Radiation Oncology and Shandong Provincial Key Laboratory of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, China, ²Department of Medical Oncology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China, ³Shandong Cancer Hospital and Institute, Jinan, Shandong, China, ⁴Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, Shandong, China, ⁵Department of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, China

Purpose/Objective(s): Radiotherapy is one of the classic and widely recommended treatment strategies for non-small cell lung cancer (NSCLC). It is known that radiotherapy has immunological effects reshaping the tumor microenvironment. Understanding the mechanism of immune microenvironment remodeling after radiotherapy is of great significance to improve the local and systemic efficacy of radiotherapy. Adenosine-to-inosine (A-to-I) RNA editing, primarily mediated by adenosine deaminase acting on RNA 1 (ADAR1), is a common post-translational modification. ADAR1 has been proved to play an important role in the development of various tumors and is associated with immune infiltration in melanoma. The purpose of this study was to explore the potential role of ADAR1-mediated A-to-I editing in immune microenvironment reconstruction after radiotherapy, and to identify novel targets for radiotherapy sensitization. Materials/

Methods: High-throughput sequencing combined with RNA editing analysis was utilized to detect A-to-I RNA editing sites after radiotherapy. RNA sequencing and 4D label-free proteomics were conducted to screen altered proteins and mRNAs. ADAR1 shRNAs and 8-Azaadenosine were used for genetic and pharmacological inhibition of A-to-I editing, respectively. The abundance and function of tumor infiltrating immune cells in vivo and in vitro were analyzed by flow cytometry. TCR_OT-1 CD8⁺ T cells were co-cultured with OVA-coated mouse NSCLC cells to observe the direct effect of tumor cells on CD8⁺ T cells. Actinomycin D treatment was applied to assess mRNA stability, while RIP was performed to confirm the binding of ADAR1 with target mRNA. CoIP-MS was carried out to detect potentially interacting proteins of ADAR1.
Results: A-to-I editing level and ADAR1 protein expression increased after radiotherapy. Inhibition of ADAR1-mediated A-to-I editing in vivo enhanced tumor response to radiotherapy, promoted CD8⁺ T cells’ infiltration and cytotoxic activity and partly reversed CD8⁺ T cell exhaustion. Based on the comprehensive analysis of the altered editing sites, mRNAs and proteins after radiation or ADAR1 knockdown, CD70 was screened as downstream candidate gene. Inhibition of A-to-I editing dampened CD70 expression on tumor cells and reduced radiation-induced CD8⁺T cells exhaustion in co-culture experiment. Mechanistically, ADAR1 directly bound to CD70 mRNA and promoted its stability through RNA editing. In addition, radiotherapy stabilized ADAR1 protein expression by decreasing E3 ubiquitin ligase TRIM21 mediated ubiquitin degradation of ADAR1.
Conclusion: Our research demonstrates that ADAR1-mediated A-to-I editing remodels radiotherapy-induced CD8⁺ T cell exhaustion by regulating CD70, and provides novel potential targets to improve the efficiency of radiotherapy.