背景:KRAS突变是NSCLC的常见驱动因素,肺癌患者中KRASG12C和G12D突变的比例较高。KRAS以前被认为是“不可下药”的目标,但首款KRASG12C突变靶向药物AMG510,于2021年进入市场。然而,G12D突变肿瘤的治疗仍有待发现。丹酚酸F(SalF),一种来自中药丹参(SM)的单体,和KRAS有很高的结合亲和力,特别是KRASG12D.迫切需要研究针对KRASG12D驱动的NSCLC的有效和安全的新型靶向疗法。
方法:为了评估SalF的抗癌作用,我们在体外使用过表达KRAS的肺癌细胞,皮下移植肿瘤模型,和KRASG12D小鼠体内模型。然后,使用分子对接研究了SalF和KRAS的结合作用,蛋白水解测定和蛋白质热移位测定。更关键的是,我们利用RT-qPCR和Western印迹法研究了肺中PI3K/AKT信号通路.
结果:这是首次评估SalF对体内KRAS过表达的肺癌细胞或KRASG12D肺肿瘤的抗癌作用的研究。我们证明SalF抑制OE-KRASA549细胞迁移,体外增殖和促进细胞凋亡。此外,我们使用皮下移植肿瘤模型显示SalF在体内抑制肺癌细胞的生长。有趣的是,我们小组发现SalF与G12D强结合,可以通过分子对接降低稳定性并促进KRASG12D突变体的降解,蛋白水解测定和蛋白质热移位测定。进一步的研究表明,在KrasG12D小鼠模型中,经过SalF治疗,小鼠肺部肿瘤的数量和大小显著减少。更重要的是,SalF可通过抑制下游PI3K/AKT信号通路激活促进细胞凋亡。
结论:SalF激活细胞凋亡信号通路,抑制抗凋亡基因,抑制肺癌细胞生长。这些数据表明,SalF可以有效抑制KRASG12D突变的肺肿瘤的生长。SalF可能是一种新型的KRASG12D抑制剂,KRAS突变肺癌的临床治疗提供了有力的理论依据。
BACKGROUND: KRAS mutation is a common driver of NSCLC, and there is a high proportion of lung cancer patients with KRAS G12C and G12D mutation. KRAS was previously considered an \"undruggable\" target, but the first KRAS G12C mutation-targeted drug AMG510, entered the market in 2021. However, treatments for G12D mutant tumors remain to be discovered. Salvianolic acid F (SalF), a monomer derived from the traditional Chinese medicine Salvia miltiorrhiza (SM), and KRAS had high binding affinity, especially for KRAS G12D. There is an urgent need to investigate effective and safe novel targeted therapies against KRAS G12D-driven NSCLC.
METHODS: To evaluate the anticancer effect of SalF, we used KRAS-overexpressing lung cancer cells in vitro, a subcutaneous transplant tumor model, and KRAS G12D mice model in vivo. Then, the binding effect of SalF and KRAS was investigated using molecular docking, proteolytic assays and protein thermal shift assays. More critically, the PI3K/AKT signaling pathway in the lung was investigated utilizing RT-qPCR and Western Blotting.
RESULTS: This is the first study to evaluate the anticancer effect of SalF on KRAS-overexpressing lung cancer cells or KRAS G12D lung tumors in vivo. We demonstrated that SalF inhibits OE-KRAS A549 cell migration, proliferation and promotes apoptosis in vitro. In addition, we used a subcutaneous transplant tumor model to show that SalF suppresses the growth of lung cancer cells in vivo. Interestingly, our group found that SalF was strongly bound to G12D and could decrease the stability and promoted the degradation of the KRAS G12D mutant through molecular docking, proteolytic assays and protein thermal shift assays. Further research demonstrated that in the KrasG12D mice model, after SalF treatment, the number and size of mouse lung tumors were significantly reduced. More importantly, SalF can promote apoptosis by inhibiting downstream PI3K/AKT signaling pathway activation.
CONCLUSIONS: SalF activated apoptosis signaling pathways, suppressed anti-apoptotic genes, and inhibited lung cancer cell growth. These datas suggested that SalF could effectively inhibit the growth of lung tumors with KRAS G12D mutation. SalF may be a novel inhibitor against KRAS G12D, providing a strong theoretical basis for the clinical treatment of lung cancer with KRAS mutations.