Dissecting the mechanisms of drug resistance development post neoadjuvant osimertinib treatment in EGFR-mutant non-small lung cancer

Introduction: Despite treatment with osimertinib in EGFR -mutant non-small cell lung cancer (NSCLC), drug-tolerant persisters (DTPs) survive through reversible adaptations. Early intervention may be an effective strategy to delay or prevent the emergence of acquired resistance to osimertinib. In this study, we characterized DTPs that survived neoadjuvant osimertinib treatment via single-cell RNA sequencing, and identified DTPs at an early transition stage before they acquire resistance. Methods: Neoadjuvant osimertinib was administered daily for two 28-day cycles followed by surgical resection and adjuvant osimertinib for 3 years. We performed single-cell RNA sequencing on matched pre- and post- treatment samples from 17 patients with NSCLC harboring EGFR mutations (E19del, N=7; L858R, N=10). The analysis was performed based on the Python Scanpy package. Results: Tumor epithelial cells were divided into 11 subclusters. Based on the increase in modules, signatures, and regulon activity, we subclustered and annotated them accordingly. High DTP features included the Multi Adaptive, EMT, Immune Evasion, ERS/UPR, Cycling, AT1, and Hyper Ciliated clusters. The AT2, Chromatin Accessible, Intermediate, and Detox Ciliated clusters exhibited weak DTP features. After neoadjuvant osimertinib, the proportion of Multi Adaptive, ERS/UPR, and EMT clusters were significantly increased. The Multi Adaptive cluster had the highest DTP signature whereas the Immune Evasion cluster expressed genes known for recruiting immunosuppressive cells. The ERS/UPR cluster showed increase in XBP1 regulon activity as well as endoplasmic reticulum stress and unfolded protein response signature. The Cycling cluster was characterized by increased activity of E2F regulons with upregulated proliferation genes. The AT1 cluster had strong Wnt signaling and increased TEAD4 regulon activity. The Hyper Ciliated cluster showed increased expression of ciliogenesis genes. Trajectory analysis revealed that EMT, Immune Evasion, Cycling, and AT1 clusters which commonly express alveolar markers, originated from AT2 and then diverge into three lineages at the Intermediate cluster. Association analysis of gene expression with pseudotime revealed that type I interferon and TGFbeta pathways were significantly augmented in all lineages. Conclusion: Our analysis shows the diversity and heterogeneity of the DTPs population and the evolutionary trajectories of DTPs after neoadjuvant osimertinib treatment in EGFR -mutant NSCLC. Targeting these DTPs features may potentially be applied for development of novel therapeutic strategies to overcome resistance to osimertinib.