Abstract 5543: High resolution lineage tracing for the study of cancer drug persistence

Abstract Drug-tolerant persister cells (DTPs) are a subpopulation of cancer cells that survive initial anticancer drug treatment despite lacking genetic resistance-driving alterations. DTPs have been shown to serve as an intermediate stage in the evolution of acquired drug resistance, facilitating the adaptation and survival of cancer cells under sustained therapeutic pressure. Recent studies have shed light on the biological characteristics of DTPs during and following drug treatment, describing the various adaptations that cells undergo while transforming into a DTP state. However, little is known about the biology of persistence potential — the cellular and molecular characteristics that prime certain drug-naïve cells to survive initial drug exposure. The main challenge in studying the persistence potential of drug-naïve cells lies in the need to treat cells to determine whether they will persist. To address this issue, we demonstrated the hereditary nature of persistence potential and adopted a high-resolution lineage tracing technology, to develop an assay for the study of persistence potential. This method, utilizing CRISPR-targeted evolving barcodes to reconstruct a lineage tree from a single-cell-derived clone, allows for the placement of persister cells along drug-naïve cells on the lineage tree. Utilizing this technology to study persistence to EGFR inhibitors in EGFR-positive non-small-cell lung cancer cells, we identified events on the lineage tree that alter the persistence potential of specific clades and derived the persistence potential of drug-naïve cells. Combining single-cell RNAseq data with cell persistence potential, we developed a computational pipeline to study the molecular mechanisms that govern the probability of drug-naïve cells to persist through drug treatment. We demonstrated that upregulation of genes related to oxidative phosphorylation correlates with a significant increase in persistence potential. These results agree with previous studies that demonstrate that cells in the DTP state are less dependent on glycolysis and rely more on oxidative phosphorylation for energy production and suggests that drug-naïve cells with elevated persistence potential are primed towards a high oxidative phosphorylation state. Intriguingly, we also described a module of translation-related genes that are negatively correlated with persistence potential, in agreement with recent results that show that persister cells undergo reversible remodeling of mRNA translation. This work introduces a novel approach and computational framework to study persistence potential and allows for the identification of potential drivers of persistence. A better understanding of the unique cellular and molecular traits that drive persistence potential is crucial for the study of drug persistence and development of targeted therapies preventing persistence and enhancing the likelihood of curative treatment. Citation Format: Binyanim Zhitomirsky, Jideofor Ezike, Brian P. Danysh, Gad Getz. High resolution lineage tracing for the study of cancer drug persistence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5543.