PO.ET03.05 · 实验与分子治疗
多组学鉴定EMT和PI3K/AKT通路为黄芪多糖介导逆转osimertinib耐药的机制
Multi-omics identifies EMT and PI3K/AKT pathways as mechanisms for Astragalus membranaceus polysaccharide-mediated reversal of osimertinib resistance
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景与目的：Osimertinib是唯一获批用于EGFR突变晚期NSCLC患者一线治疗的第三代表皮生长因子受体（EGFR）酪氨酸激酶抑制剂。然而，耐药严重阻碍其疗效。osimertinib失败后治疗选择有限。黄芪（Astragalus membranaceus，AM）是一种长期用于传统中医的药用植物。与化疗联用时，AM已被证明可增强抗癌疗效并减少副作用。AM的有益生物学效应主要归因于其多糖（AMP）。然而，其潜在机制仍不明确。本研究采用多组学方法探究AMP克服osimertinib耐药的机制，聚焦于关键基因异常和关键信号通路的改变。
方法：从AM干燥根中提取AMP的方法已经过优化和标准化。粗药材和AMP根据《中国药典》基于形态和化学特性进行了鉴定。在NSCLC细胞系体外以及NSG小鼠患者来源的肿瘤异种移植（PDX）中研究了AMP对osimertinib耐药的规避。在显示出有前景药物联合效应的PDX中，对从肿瘤组织提取的总RNA进行RNA测序，以评估动态基因表达谱变化。此外，进行了基于质谱（MS）的磷酸化蛋白质组学分析，以鉴定与AMP规避osimertinib耐药相关的蛋白质组学特征和信号通路改变。
结果：在测试的osimertinib耐药NSCLC细胞系中，AMP在增效osimertinib方面对EGFR T790M突变和MET扩增的H820细胞最为有效。AMP还显著抑制肿瘤迁移和侵袭。AMP进一步被证明能在两个药物难治性PDX模型中显著增强osimertinib的抗肿瘤效应，且不引起显著毒性。通过RNA-seq分析，GSEA结果表明AMP处理的PDX肿瘤中EMT相关基因集显著富集。使用连接图谱（connectivity mapping），发现AMP与PI3K/mTOR抑制剂共享高度相似的转录组学特征，提示一种可能的作用机制。全局定量MS揭示了AMP逆转EMT的特征性蛋白质组学特征。磷酸化蛋白质MS进一步揭示，AMP处理后ErbB和Rap 1（KEGG数据库）以及PI3K/AKT信号通路（REACTOM数据库）的富集。综上所述，我们的研究在转录组、全局蛋白质组和磷酸化蛋白质组水平上鉴定了AMP克服osimertinib耐药的新机制。
结论：这些发现支持对AMP用于治疗osimertinib难治性NSCLC进行临床评估。
查看英文原文 English abstract
Background and Aim: Osimertinib is the only 3 rd generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor approved for the 1 st line therapy of advanced NSCLC patients with EGFR mutations. However, drug resistance severely hinders its efficacy. There is limited therapeutic option after osimertinib failure. Astragalus membranaceus (AM) is a medicinal plant long used in traditional Chinese medicine. When combined with chemotherapy, AM has been shown to enhance anticancer efficacy and reduce side effects. The beneficial biological effects of AM are mainly attributed to its polysaccharides (AMP). However, the underlying mechanisms remain elusive. This study employed a multi-omics approach to investigate the mechanism by which AMP overcome osimertinib resistance, focusing on key genetic abnormalities and alterations in critical signaling pathways.
Method: The extraction of AMP from the dry roots of AM has been optimized and standardized. The crude herb and AMP were authenticated based on morphological and chemical properties as per the Chinese Pharmacopoeia. The circumvention of osimertinib resistance by AMP was investigated in NSCLC cell lines in vitro and patient-derived tumor xenograft (PDX) in NSG mice. In PDXs that demonstrated promising drug combination effect, RNA sequencing was conducted on total RNA extracted from the tumor tissues to evaluate the dynamic gene expression profile changes. Moreover, mass spectrometry (MS)-based phosphoproteomic analysis was conducted to identify the proteomic signatures and signaling pathway alterations associated with the circumvention of osimertinib resistance by AMP.
Results: Among the tested osimertinib-resistant NSCLC cell lines, AMP was the most effective at potentiating osimertinib in EGFR T790M mutated and MET amplified H820 cells. AMP also significantly inhibited tumor migration and invasion. AMP was further shown to remarkably potentiate the antitumor effect of osimertinib in two drug refractory PDX models, without causing notable toxicity. By RNA-seq analysis, GSEA results indicate significant enrichment of EMT-related gene sets in AMP-treated PDX tumors. Using connectivity mapping, AMP was found to share a highly similar transcriptomic signature with PI3K/mTOR inhibitors, suggesting a putative mechanism of action. Global quantitative MS revealed a characteristic proteomic signature of EMT reversal by AMP. MS for phosphorylated proteins further revealed enrichment of ErbB and Rap 1 (KEGG database) and PI3K/AKT signaling pathways (REACTOM database) following AMP treatment. Taken together, our study identified novel mechanisms by which AMP overcome osimertinib resistance at transcriptomic, global proteomic, and phosphoproteomic levels.
Conclusion: The findings support clinical evaluation of AMP for treating osimertinib refractory NSCLC.
利益披露 Disclosure
K. K. To, None..
L. Wang, None..
Z. Zuo, None..
W. C. Cho, None.