Bai Hu, Liang Chen, Renjie Wang, Zhijun Zhou, Guangmei Zhang, Jinghan Ruan, Ling Xi, Yuanzhen Zhang, Qingmei Li, Bingqing Liao, Li Li, Chun Gao, Ding Ma, Min Li, Shutsung Liao
Cervical cancer progresses from high-grade squamous intraepithelial lesions driven by persistent HPV infection. Identifying the gatekeepers restraining malignant transformation could reveal strategies to prevent and treat cervical cancer. Here, we integrated single-cell transcriptomics, CRISPR-Cas9 screening, and organoid modeling to dissect this process. Single-cell analysis of patient samples revealed progressive activation of RAS and proliferation programs along epithelial differentiation paths. A focused CRISPR screen in HPV-positive pre-tumoroids identified NF1 as the top suppressor of malignant transition. NF1 loss accelerated carcinogenesis in organoids, compressing a 5~10-year process into 3~6 months, recapitulating basal cell expansion, dedifferentiation, and tumorigenicity. Mechanistically, NF1 loss enhanced RAS-MAPK and PI3K-AKT signaling, promoted proliferative programs, and remodeled chromatin accessibility at AP-1/E2F motifs. Conversely, NF1 restoration in cancer organoids induced apoptosis and suppressed malignant maintenance. AI-guided modeling was used to design a minimal NF1-mimetic peptide that engaged RAS-GTP to inhibit RAS signaling, reduce tumor burden in HPV16-driven mouse models, and restore local immune permissiveness without systemic activation. Together, these findings establish NF1 as a critical constraint on HPV-associated epithelial evolution and provide a time-compressed organoid model of cervical carcinogenesis, offering proof-of-concept for therapeutic RAS pathway interception via NF1 restoration.