Multi-omics identifies EMT and PI3K/AKT pathways as mechanisms for Astragalus membranaceus polysaccharide-mediated reversal of osimertinib resistance

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.