Institute of Digestive Disease and Department of Medicine and Therapeutics, State Key laboratory of Digestive Disease, Li Ka Shing Institute of Health Sciences, CUHK Shenzhen Research Institute, The Chinese University of Hong Kong, Hong Kong; Department of Anaesthesia and Intensive Care and Peter Hung Pain Research Institute, The Chinese University of Hong Kong, Hong Kong.
Institute of Digestive Disease and Department of Medicine and Therapeutics, State Key laboratory of Digestive Disease, Li Ka Shing Institute of Health Sciences, CUHK Shenzhen Research Institute, The Chinese University of Hong Kong, Hong Kong.
Gastroenterology. 2022 Oct;163(4):891-907. doi: 10.1053/j.gastro.2022.06.024. Epub 2022 Jun 11.
BACKGROUND & AIMS: N-Methyladenosine (mA) is the most prevalent RNA modification and recognized as an important epitranscriptomic mechanism in colorectal cancer (CRC). We aimed to exploit whether and how tumor-intrinsic mA modification driven by methyltransferase like 3 (METTL3) can dictate the immune landscape of CRC.
Mettl3 knockout mice, CD34 humanized mice, and different syngeneic mice models were used. Immune cell composition and cytokine level were analyzed by flow cytometry and Cytokine 23-Plex immunoassay, respectively. MA sequencing and RNA sequencing were performed to identify downstream targets and pathways of METTL3. Human CRC specimens (n = 176) were used to evaluate correlation between METTL3 expression and myeloid-derived suppressor cell (MDSC) infiltration.
We demonstrated that silencing of METTL3 in CRC cells reduced MDSC accumulation to sustain activation and proliferation of CD4 and CD8 T cells, and eventually suppressed CRC in ApcMettl3 mice, CD34 humanized mice, and syngeneic mice models. Mechanistically, METTL3 activated the mA-BHLHE41-CXCL1 axis by analysis of mA sequencing, RNA sequencing, and cytokine arrays. METTL3 promoted BHLHE41 expression in an mA-dependent manner, which subsequently induced CXCL1 transcription to enhance MDSC migration in vitro. However, the effect was negligible on BHLHE41 depletion, CXCL1 protein or CXCR2 inhibitor SB265610 administration, inferring that METTL3 promotes MDSC migration via BHLHE41-CXCL1/CXCR2. Consistently, depletion of MDSCs by anti-Gr1 antibody or SB265610 blocked the tumor-promoting effect of METTL3 in vivo. Importantly, targeting METTL3 by METTL3-single guide RNA or specific inhibitor potentiated the effect of anti-programmed cell death protein 1 (anti-PD1) treatment.
Our study identifies METTL3 as a potential therapeutic target for CRC immunotherapy whose inhibition reverses immune suppression through the mA-BHLHE41-CXCL1 axis. METTL3 inhibition plus anti-PD1 treatment shows promising antitumor efficacy against CRC.
N6-甲基腺苷（m6A）是最常见的 RNA 修饰，被认为是结直肠癌（CRC）中重要的转录后调控机制。本研究旨在探讨肿瘤内 m6A 修饰是否以及如何由甲基转移酶样 3（METTL3）驱动，从而决定 CRC 的免疫景观。
使用 Mettl3 敲除小鼠、CD34 人源化小鼠和不同同基因小鼠模型。采用流式细胞术分析免疫细胞组成，采用 Cytokine 23-Plex 免疫分析检测细胞因子水平。进行 mA 测序和 RNA 测序以鉴定 METTL3 的下游靶标和通路。使用 176 例人 CRC 标本评估 METTL3 表达与髓系来源抑制细胞（MDSC）浸润的相关性。
我们证明 CRC 细胞中 METTL3 的沉默可减少 MDSC 的积累，从而维持 CD4 和 CD8 T 细胞的激活和增殖，并最终抑制 ApcMettl3 小鼠、CD34 人源化小鼠和同基因小鼠模型中的 CRC。机制上，通过 mA 测序、RNA 测序和细胞因子阵列分析，METTL3 激活了 m6A-BHLHE41-CXCL1 轴。METTL3 以 mA 依赖性方式激活 BHLHE41 表达，随后诱导 CXCL1 转录，增强体外 MDSC 迁移。然而，在 BHLHE41 耗竭、CXCL1 蛋白或 CXCR2 抑制剂 SB265610 给药时，这种作用可以忽略不计，这表明 METTL3 通过 BHLHE41-CXCL1/CXCR2 促进 MDSC 迁移。一致地，用抗-Gr1 抗体或 SB265610 耗尽 MDSC 可阻断 METTL3 在体内的促肿瘤作用。重要的是，METTL3-sgRNA 或特异性抑制剂靶向 METTL3 可增强抗程序性细胞死亡蛋白 1（抗 PD1）治疗的效果。
本研究确定 METTL3 是 CRC 免疫治疗的潜在治疗靶点，其抑制作用通过 m6A-BHLHE41-CXCL1 轴逆转免疫抑制。METTL3 抑制联合抗 PD1 治疗对 CRC 具有有前景的抗肿瘤疗效。
