P71.02 Molecular and Cellular Dynamics of Drug-Tolerant Persister (DTP) Cells During Osimertinib Therapy in EGFR Mutant Lung Adenocarcinoma

Abstract

The acquisition of drug resistance to molecular targeted therapies is a major obstacle in the treatment of EGFR mutant lung cancer. One proposed mechanism to develop resistance to EGFR-tyrosine kinase inhibitor (TKI) involves subpopulations of tumor cells to enter a drug-tolerant persister (DTP) state while on the therapy, leading to eventual acquisition of diverse resistance mechanisms. Thus, understanding and targeting this mechanism may provide a therapeutic opportunity to prevent tumor relapse. However, properties of DTPs, including genetic and transcriptomic architecture and intra-tumor heterogeneity, leading to the generation of DTPs remains unknown. The aim of this study is to investigate key properties and molecular characteristics of DTPs in 2 patient-derived xenograft (PDX) models. DTPs were generated through chronic exposure to osimertinib in a lung adenocarcinoma PDX models with EGFR exon-19 deletion and exon-19 deletion+T790M mutation, respectively. Histological analysis, genomic clonal structure analysis using whole-exome sequencing (WES), transcriptomic analysis with single-cell RNA-seq were conducted on DTPs and compared to pre-treatment baseline (BL) tumors. To examine the reversibility of DTP feature and the detail clonal changes, DTPs were repopulated upon drug discontinuation and the regrowth tumors were compared to BL and DTPs. EGFR-mutant PDXs showed significant response to osimertinib. However, all PDX tumors regrew after drug release, indicating EGFR-TKI cannot eradicate EGFR-mutant lung cancer cells. The regrowth tumors consistently showed sensitivity to repeat treatment. Although DTPs exhibited distinct histologic features from BL, with small number of persister tumor cells and significantly increased stromal fibrosis, the histologic findings of regrowth tumors closely resembled the BL, indicating DTPs are phenotypically reversible. WES demonstrated no new known resistant mutations arising in DTPs and regrowth tumors. In genomic clonal structure analysis, dynamic clonal change was not observed across the treatment state and most of genomic subclones observed in BL were retained in DTPs and regrowth tumors. single-cell RNA-sequencing revealed that DTPs consisted of several transcriptomically-defined clusters, with majority of DTPs formed distinct clusters from BL, while some overlapped with BL, demonstrating DTPs are generated through mixed process of transcriptomic reprogramming and clonal selection of pre-existing cells in BL. Regrowth tumors demonstrated mostly overlapped transcriptomic feature with BL tumors, indicating DTPs have reversible biological capacity. In EGFR-mutant PDX models, DTPs have phenotypically and transcriptomically reversible feature. Genetic clonal selection does not mainly contribute to the generation of DTP. DTPs consist of transcriptomically heterogeneous subpopulations, and each subclone could potentially survive EGFR-TKI therapy through different molecular mechanisms.