Abstract 7027: Multi-lineage evolution of drug resistance via a novel keratin 17+ drug tolerant persister population in <i>EGFR</i> -mutant NSCLC.

Abstract Background: EGFR mutant lung cancers initially respond to frontline targeted therapy. However, resistance inevitably develops through diverse mechanisms. Resistant tumors universally emerge from drug tolerant persister cells (DTPCs) that survive initial treatment. Despite this knowledge, the biology of EGFR mutant DTPCs is not well understood. This study was conducted to interrogate DTPC phenotypes that enable evolution of therapy resistance. Methods: DTPC models were generated by treating EGFR mutant cell lines with &amp;gt;IC90 concentrations of osimertinib for 14 days. After 14 days, cells were profiled with 10X 3’ single cell RNA sequencing (scRNAseq). Seurat was used to integrate data, normalize data, cluster cells, and conduct differential expression analyses. UCell was used to score cells for activity of biology pathways. scanpy was used to analyze scRNAseq data from the Gardner et al. ERPMT mouse model. Human patient samples were collected from lung cancer patients treated at MD Anderson Cancer Center using IRB approved protocols. Samples were profiled with 10X 5’ scRNAseq. scanpy was used to integrate data, normalize data, and cluster cells. Palantir was used to perform trajectory analyses. Results: Differential expression (DE) analysis comparing DTPCs and control cells were conducted for cell line pairs. Using DE results, we generated consensus gene signatures capturing genes universally upregulated or downregulated across DTPC models. Review of these genes identified that KRT17 was highly upregulated in osimertinib DTPCs. We found KRT17+ DTPCs upregulate epithelial-to-mesenchymal (EMT), stemness, and aberrant basaloid signatures compared to KRT17- DTPCs. Subclustering showed that KRT17 DTPCs are heterogeneous and label non-overlapping EMT, proliferative, and MET expressing populations. To validate our in vitro findings, we analyzed scRNAseq datasets from multiple in vivo sources. In GEMM models, Krt17+ cells populate minimal residual disease (MRD) following suppression of mutant EGFR. Krt17+ cells were a stem-like population that emerged as residual tumor cells lost alveolar epithelial identity prior to neuroendocrine transformation. Lastly, we confirmed the clinical relevance of our findings by investigating human patient clinical specimens. Compared to treatment naïve samples, KRT17+ cells were significantly enriched in osimertinib-treated MRD samples. Trajectory analyses demonstrated that KRT17 expression characterized cell populations that preceded development of resistance through several known mechanisms, including MET amplification and histologic transformation (HT). Conclusions: Our data demonstrate that EGFR-mutant cells surviving initial treatment converge onto a novel KRT17+ DTPC state which serves as a multipotent progenitor population capable of leveraging diverse mechanisms to resist therapy, including HT. Citation Format: Benjamin B. Morris, Monique B. Nilsson, Ethan Earlie, Eric E. Gardner, Hong Chen, Santiago G. Trevino, Alexa J. Halliday, Yuanxin Xi, Jing Wang, Natalie Vokes, Don Gibbons, Jianjun Zhang, Ashley M. Laughney, Yasir Y. Elamin, Xiuning Le, John V. Heymach. Multi-lineage evolution of drug resistance via a novel keratin 17+ drug tolerant persister population in EGFR-mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7027.