Mutant IDH1 inhibition induces dsDNA sensing to activate tumor immunity [ChIP-Seq]

Isocitrate Dehydrogenase 1 (IDH1) is the most commonly mutated metabolic gene across human cancers. Mutant IDH1 (mIDH1) generates the oncometabolite (R)-2-hydroxyglutarate, disrupting enzymes involved in epigenetics and other processes. A hallmark of IDH1-mutant solid tumors is T cell exclusion, whereas mIDH1 inhibition in preclinical models restores anti-tumor immunity. Here, we define a cell-autonomous mechanism of mIDH1-driven immune evasion. IDH1-mutant solid tumors show striking, selective hypermethylation and silencing of the cytoplasmic dsDNA sensor, CGAS, compromising innate immune signaling. mIDH1 inhibition restores DNA demethylation, derepressing CGAS and transposable element (TE) subclasses. dsDNA produced by TE-reverse transcriptase activates cGAS, triggering viral mimicry and stimulating anti-tumor immunity. Thus, we demonstrate that mIDH1 epigenetically suppresses innate immunity and link endogenous reverse transcriptase activity to the mechanism of action of an FDA-approved oncology drug. Overall design: This study used the mIDH inhibitor, AG120 (Servier Pharmaceuticals). Murine ICC (2205) cells were treated for 10 days with either DMSO or 1 µM mIDH inhibitor (AG120). Where indicated, 20ng/mL IFN? was added for the final two days of culturing. 20 million cells per replicate for each condition (DMSO, AG120, IFN?, AG120 + IFN?) were cross-linked using 1% formaldehyde for 10 minutes at room temperature and quenched using glycine for 5 minutes. ChIP experiments were performed using SimpleChIP Enzymatic Chromatin IP Kit based on manufacturer's protocol. Sequencing libraries were prepared using the NEBNext Ultra II DNA library preparation kit (New England Biolabs) and were sequenced using NovaSeq 6000 (Illumina) with paired-end 150 bp reads.

异柠檬酸脱氢酶1（Isocitrate Dehydrogenase 1, IDH1）是人类癌症中突变最为频发的代谢基因。突变型IDH1（mutant IDH1, mIDH1）可产生促癌代谢物(R)-2-羟基戊二酸，干扰参与表观遗传调控及其他生理过程的酶活性。IDH1突变实体瘤的典型特征为T细胞浸润缺失，而临床前模型中抑制mIDH1可恢复抗肿瘤免疫应答。本研究阐明了mIDH1介导免疫逃逸的细胞自主机制：IDH1突变实体瘤可出现胞质双链DNA（double-stranded DNA, dsDNA）感受器CGAS的显著选择性高甲基化与基因沉默，进而削弱先天免疫信号转导。抑制mIDH1可恢复DNA去甲基化，解除CGAS与转座子元件（transposable element, TE）亚型的转录抑制。由TE逆转录酶产生的双链DNA可激活cGAS，引发病毒模拟效应并激活抗肿瘤免疫。综上，本研究证实mIDH1可通过表观遗传机制抑制先天免疫，并将内源性逆转录酶活性与一款获美国食品药品监督管理局（FDA）批准的肿瘤治疗药物的作用机制关联起来。
实验设计概况：本研究使用mIDH抑制剂AG120（施维雅制药Servier Pharmaceuticals）。将小鼠肝内胆管癌（intrahepatic cholangiocarcinoma, ICC）2205细胞分别用二甲基亚砜（dimethyl sulfoxide, DMSO）或1 μM mIDH抑制剂AG120处理10天。如实验设计所示，部分组别在培养的最后两天加入20 ng/mL 干扰素γ（interferon-γ, IFNγ）。每个实验组（DMSO组、AG120组、IFNγ组、AG120联合IFNγ组）各设置3次生物学重复，每次重复使用2000万细胞，于室温下用1%甲醛交联10分钟，随后用甘氨酸淬灭反应5分钟。染色质免疫沉淀（chromatin immunoprecipitation, ChIP）实验按照生产商说明书，使用SimpleChIP酶解染色质免疫沉淀试剂盒完成。测序文库采用NEBNext Ultra II DNA文库制备试剂盒（纽英伦生物New England Biolabs）构建，使用Illumina NovaSeq 6000平台进行双端150 bp读长测序。