Inhibiting EZH2 Targets Atypical Teratoid Rhabdoid Tumor by Triggering Viral Mimicry via Both RNA and DNA Sensing Pathways [RNA-Seq]

Loss of function mutations in SMARCB1 are prevalent in pediatric atypical teratoid rhabdoid tumors (ATRTs) and confer an oncogenic dependency on EZH2, providing a compelling rationale for treating these genetically defined cancers via EZH2 inhibition (EZH2i). EZH2i results in tumor regression in SMARCB1-deficient tumors in preclinical studies, but the molecular mechanism has not been fully elucidated. Here we found that the sensitivity of SMARCB1-deficient tumors to EZH2i is associated with the viral mimicry response that depends on both double-stranded RNA (dsRNA) and cytoplasmic DNA sensing pathways. Unlike other epigenetic therapies targeting transcriptional repressors, viral mimicry by EZH2i in SMARCB1-deficient tumors is not triggered by crypt initiation of endogenous retroelements, but rather mediated by increased expression of genes enriched for intronic inverted-repeat Alu (IR-Alu) elements. Interestingly, we found that interferon-stimulated genes (ISGs) are highly enriched for dsRNA-forming intronic IR-Alu elements, suggesting a positive feedback loop whereby interferon response leads to dsRNA formation from intronic ISGs and activation of viral mimicry. Moreover, EZH2i in ATRT cells also enhances the expression of full-length LINE-1 elements, leading to genomic instability and cGAS/STING response in a process dependent on reverse transcriptase activity. Supporting this mechanism, co-depletion of dsRNA sensing and cytoplasmic DNA sensing completely rescues the viral mimicry response to EZH2i in SMARCB1-deficient tumors. UNC1999 and UNC24000 treatments. Day 4 and Day 6. 3 replicates.

SMARCB1功能丧失突变在儿童非典型畸胎样横纹肌样瘤（ATRTs）中高发，并赋予肿瘤细胞对EZH2的致癌依赖，这为通过EZH2抑制剂（EZH2i）治疗这类具有明确遗传特征的癌症提供了坚实的理论依据。临床前研究表明，EZH2i可使SMARCB1缺陷型肿瘤发生退缩，但相关分子机制尚未完全阐明。本研究发现，SMARCB1缺陷型肿瘤对EZH2i的敏感性与依赖双链RNA（dsRNA）和胞质DNA感知通路的病毒模拟应答密切相关。与其他靶向转录阻遏物的表观遗传疗法不同，EZH2i在SMARCB1缺陷型肿瘤中引发的病毒模拟应答并非由内源性逆转录元件的隐秘起始所触发，而是由富含内含子反向重复Alu（IR-Alu）元件的基因表达上调所介导。有趣的是，我们发现干扰素刺激基因（ISGs）高度富集可形成双链RNA的内含子IR-Alu元件，这提示存在一条正反馈环路：干扰素应答可通过内含子干扰素刺激基因形成双链RNA，进而激活病毒模拟应答。此外，在ATRT细胞中，EZH2i还可上调全长长散在核元件1（LINE-1）的表达，进而导致基因组不稳定性并激活cGAS/STING应答，这一过程依赖于逆转录酶活性。为验证该机制，我们对dsRNA感知通路和胞质DNA感知通路进行共敲除，结果完全挽救了SMARCB1缺陷型肿瘤中EZH2i诱导的病毒模拟应答。实验采用UNC1999与UNC24000进行处理，分别于第4天、第6天取样，设置3次生物学重复。