Loss of NOTCH2 Creates a TRIM28-Dependent Vulnerability in Small Cell Lung Cancer [bulk_murine_RNA_seq]

Small cell lung cancer (SCLC) is a highly aggressive malignancy that lacks effective targeted therapies, in part due to frequent loss-of-function mutations in tumor suppressors and the absence of recurrent oncogenic drivers. Approximately 15% of SCLCs harbor inactivating mutations in NOTCH1 or NOTCH2, and most neuroendocrine-high SCLCs exhibit low NOTCH activity. Using CRISPR/Cas9 screening in primary cell lines derived from NOTCH1/2-isogenic SCLC genetically engineered mouse models, we identified TRIM28 as a synthetic lethal dependency in NOTCH2-inactivated SCLCs. Loss of TRIM28 in this context robustly induced expression of endogenous retroviruses, activated viral sensing pathways, and triggered a type I interferon response. Mechanistically, NOTCH2 inactivation increased reliance on TRIM28-mediated ERV silencing, creating a hyper-dependence on TRIM28 via the STING–MAVS–TBK1 axis. Notably, TRIM28 was essential for tumor growth only in the setting of NOTCH2 loss. These findings identify TRIM28 as a potential therapeutic target in NOTCH2-deficient or low-NOTCH2-expressing SCLC. Overall design: Bulk RNA-seq profiling of 1). Neuroendocrine suspension and non-neuroendocrine adherent NOTCH-WT (K93) and NOTCH2-Mutant (K60) SCLC cell lines derived from RPR2 genetically engineered mouse model and genetic inactivation of TRIM28 with two sgRNA and non-targeting control using CRISPR-Cas9. 2). SCLC cell line 1014 cells derived from RPR2 genetically engineered mouse model with TRIM28-dTAG-HA knock-in and genetic inactivation of NOTCH2 with two sgRNA and non-targeting control using CRISPR-Cas9 and treated with 100 nM dTAG-V1 for 72 hours to degrade TRIM28 or with DMSO as a control.

小细胞肺癌（Small Cell Lung Cancer, SCLC）是一种高侵袭性恶性肿瘤，目前尚无有效的靶向治疗手段，部分原因在于其肿瘤抑制基因频发功能丧失性突变，且缺乏复发性致癌驱动因子。约15%的小细胞肺癌存在NOTCH1或NOTCH2的失活性突变，多数高神经内分泌分型的小细胞肺癌表现出较低的NOTCH通路活性。本研究针对源自NOTCH1/2同基因工程小细胞肺癌小鼠模型的原代细胞系开展CRISPR/Cas9筛选，鉴定出TRIM28是NOTCH2失活型小细胞肺癌的合成致死依赖靶点。在此细胞背景下敲除TRIM28可显著诱导内源性逆转录病毒（Endogenous Retrovirus, ERV）的表达，激活病毒感知通路，并触发I型干扰素应答。机制层面分析显示，NOTCH2失活会增强细胞对TRIM28介导的内源性逆转录病毒沉默的依赖，进而通过STING–MAVS–TBK1信号轴形成对TRIM28的高度依赖。值得注意的是，仅在NOTCH2缺失的细胞背景中，TRIM28才对肿瘤生长至关重要。上述研究结果表明，TRIM28可作为NOTCH2缺陷型或低表达型小细胞肺癌的潜在治疗靶点。

实验整体设计：
1. 源自RPR2基因工程小鼠模型的神经内分泌悬浮型与非神经内分泌贴壁型NOTCH野生型（K93）及NOTCH2突变型（K60）小细胞肺癌细胞系，采用CRISPR-Cas9系统通过两条单导RNA（single guide RNA, sgRNA）靶向敲除TRIM28，并以非靶向单导RNA作为对照；
2. 源自RPR2基因工程小鼠模型的SCLC细胞系1014，该细胞系经基因编辑引入TRIM28-dTAG-HA敲入突变；采用CRISPR-Cas9系统通过两条单导RNA靶向敲除NOTCH2，并以非靶向单导RNA作为对照；随后用100 nM dTAG-V1处理72小时以降解TRIM28，同时以二甲基亚砜（Dimethyl Sulfoxide, DMSO）处理作为对照。