ID3 Enhances PD-L1 Expression by Restructuring MYC and Promotes Colorectal Cancer Immune Evasion [RNA-seq]

The inhibitor of DNA-binding protein ID3 is a vital component of immune cells and has been associated with the progression of colorectal cancer (CRC). Despite its significance, its specific role in the immune evasion strategies utilized by CRC remains unclear. RNA-seq analysis revealed that ID3 is associated with the PD-L1 immune checkpoint. We further demonstrated that ID3 modulates PD-L1 expression, suppresses the infiltration and activation of CD8+ T cells, and facilitates the immune escape of CRC cells. Additionally, we found that the knockdown of ID3 significantly enhanced the effectiveness of PD-L1 antibody treatment in combating CRC, reduced the upregulation of PD-L1 induced by the antibody, and altered the immune microenvironment within CRC. Mechanistically, our biological and structural analyses demonstrated that ID3 reconstructed the four-dimensional structure of MYC, thereby enhancing its binding affinity to the PD-L1 promoter and augmenting PD-L1 transcriptional activity. By integrating analysis of ChIP-seq, RNA-seq, and ImmPort gene sets, we found that ID3's DNA-assisted binding function was widespread and could either enhance or suppress gene transcription, not only affecting tumor immune escape through immune checkpoints, but also regulating various cytokines and immune cells involved in tumor immunity. In conclusion, our study uncovered a new mechanism by which ID3 promotes immune evasion in CRC and targeting ID3 may improve the efficacy of anti-PD-1/PD-L1 immunotherapy. To investigate the role of the inhibitor of DNA-binding protein ID3 in immune escape of colorectal cancer, we established ID3-knockout colorectal cancer cell line HCT116 using CRISPR/Cas9 technology to study the gene expression regulated by ID3. We then performed gene expression profiling analysis using data obtained from RNA-seq of sgID3 and sgCtrl. Comparative gene expression profiling of RNA-seq data is sgID3_1, sgID3_2, sgCtrl_1 and sgCtrl_2.

DNA结合抑制蛋白ID3（inhibitor of DNA-binding protein ID3）是免疫细胞的关键组分，且与结直肠癌（colorectal cancer, CRC）的进展密切相关。尽管其重要性已得到广泛认可，但ID3在结直肠癌所采用的免疫逃逸策略中的具体作用仍未明晰。RNA-seq分析显示，ID3与PD-L1免疫检查点存在关联。我们进一步证实，ID3可调控PD-L1的表达，抑制CD8+ T细胞的浸润与活化，并促进结直肠癌细胞的免疫逃逸。此外，我们发现敲低ID3可显著增强PD-L1抗体治疗结直肠癌的疗效，降低该抗体诱导的PD-L1上调，并改变结直肠癌内部的免疫微环境。从机制层面而言，我们的生物学与结构分析表明，ID3可重构MYC的四维结构，进而增强其与PD-L1启动子的结合亲和力，提升PD-L1的转录活性。通过整合分析ChIP-seq、RNA-seq及ImmPort基因集数据，我们发现ID3的DNA辅助结合功能具有普遍性，既可增强也可抑制基因转录；其不仅通过免疫检查点影响肿瘤免疫逃逸，还可调控参与肿瘤免疫的多种细胞因子与免疫细胞。综上，本研究揭示了ID3促进结直肠癌免疫逃逸的全新机制，靶向ID3或可提升抗PD-1/PD-L1免疫治疗的疗效。为探究DNA结合抑制蛋白ID3在结直肠癌免疫逃逸中的作用，我们利用CRISPR/Cas9技术构建了ID3敲除的结直肠癌细胞系HCT116，以研究ID3所调控的基因表达。随后，我们基于sgID3与sgCtrl的RNA-seq数据开展基因表达谱分析。本次RNA-seq数据的对比基因表达谱分析样本为sgID3_1、sgID3_2、sgCtrl_1及sgCtrl_2。