Wee1激酶SUMO化修饰在KRAS/TP53双突变肺癌协同致死药物治疗中的作用机制

Lung cancer is the a malignant disease with highest morbidity and mortality in China. KRAS mutant lung cancer still lacks effective therapy. The precise classification of KRAS mutant lung cancer will promote the development of fine treatments. Our Preliminary work showed that KRAS / TP53 mutant lung cancer possesses Wee1 kinase SUMOylation obstacles, and is more sensitive to Wee1 inhibitor. This project intends to genrate KRAS / TP53 mutant lung cancer cell lines using CRISPR / Cas9 genome editing, explore the obstacle of WEE1 kinase Sumoylation , and clarify the Wee1 SUMOylation Lysine sites. The regulation of Wee1 kinase activity by SUMOylation and the possible SUMO modified E3 ligase of Wee1 will also be studied. The synergistic effects of Wee1 inhibitor and DNA damage drugs in the treatment of KRAS / TP53 mutant lung cancer will be elucidated. Gene activation PiggyBac transposon system is used to screen the synthetic lethal targeted drugs for Wee1 inhibitor in KRAS / TP53 mutant lung cancer. All the findings in this project will be verified in the classical Kras (G12D) / p53 - / - transgenic mouse model of lung adenocarcinoma. The project will elucidate the function and significance of Wee1 SUMO modification in KRAS / TP53 mutant lung cance, and provide preclinical evidence for the development of precision treatment of KRAS / TP53 mutant lung cancer.

肺癌是中国发病率，死亡率最高的肿瘤，KRAS肺癌至今仍缺乏有效治疗方法，KRAS肺癌精准分型将推动精细化治疗方法的发展。前期工作表明KRAS/TP53双突变肺癌亚型具有Wee1激酶 SUMO修饰障碍，并对Wee1激酶抑制剂和基因毒药物更敏感。本项目拟采用CRISPR/Cas9法构建KRAS/TP53肺癌双突变细胞株，研究WEE1激酶SUMO修饰障碍的特征表现，阐明Wee1 SUMO修饰的赖氨酸位点及其对激酶活性的调控作用，探索Wee1 SUMO修饰的E3 连接酶。研究Wee1抑制剂与基因毒药物治疗此型肺癌的协同疗效；并采用基因激活PiggyBac转座子系统筛选Wee1抑制剂的协同致死靶向药物。最后利用Kras(G12D)/ p53-/-转基因小鼠肺腺癌模型进行结论验证。本项目将初步阐明Wee1 SUMO修饰障碍在此型肺癌中功能及意义，为KRAS/TP53双突变肺癌的精准治疗提供临床前依据。

肺癌是中国乃至全球死亡率最高的恶性肿瘤之一，晚期肺癌的驱动基因分型（driver mutation）是制定药物治疗方案的最重要依据。KRAS突变在肺腺癌东亚人中占到11-16%，在西方人则达到26-30% 。研究KRAS驱动突变肺癌的发生机制及精准治疗方法具有重要的理论和临床应用价值。其中KRAS/TP53突变的亚型也被广泛提及，在非小细胞肺癌中，KRAS/TP53双突变亚型成为了潜在的生物靶点之一。我们发现在KRAS/TP53双突变的NSCLC中WEE1激酶出现72kd表达增多。我们研究发现WEE1激酶存在依赖TP53的SUMO化修饰。当KRAS/TP53双突变时，细胞内WEE1激酶SUMO化修饰障碍导致其原型形式增多，而这与TP53发生突变相关。WEE1激酶SUMO化修饰不仅影响了激酶活性，同时影响了细胞周期G2/M期阻滞的发生，这使得WEE1成为潜在得的肿瘤治疗靶点。当WEE1抑制剂联合顺铂作用于KRAS/TP53双突变非小细胞肺癌时，表现出协同抑制作用。因此，本研究为KRAS/TP53双突变的非小细胞肺癌的治疗提供了新的临床前证据，使得WEE1激酶成为KRAS/TP53双突变非小细胞肺癌的潜在靶点。