Abstract 425: Characterization of TKI-induced drug-tolerant persister cells from a patient-derived cell line
Authors: Brayé Floriane , Facchinetti Francesco , Gerber Helena , Juigné Juliette , Guaitoli Giorgia , Thiery Jean-Paul , Ponce Santiago , Besse Benjamin , Olaussen Ken A. , Friboulet Luc 
Published: April 2023

Abstract
Introduction: Targeted therapies provide clinical benefits in patients harboring oncogene-driven cancers, but relapses invariably occur. Studies on resistance mechanisms are essential to guide the development of next-generation inhibitors. An increasing number of studies focus on an earlier stage of disease evolution. Resistance may develop from the selection of preexisting resistant cells or by adaptation of drug-tolerant persister (DTP) cells. In this context, our study depicts DTP cells able to survive the initial tyrosine kinase inhibitor (TKI) exposure without harboring genetic changes, in order to identify the vulnerabilities existing in this cell state. Materials and
Methods: This project focuses on a patient-derived cell line from the prospective MATCH-R clinical trial (NCT0251782). The model is a non-small cell lung cancer harboring EML4-ALK fusion and ALK C1156Y/G1269A mutation, sensitive to the 3rd generation ALK TKI lorlatinib. In vitro, fully resistant cells were generated by long-term lorlatinib exposure and the acquired resistance mechanism resulted from an epithelial-mesenchymal transition (EMT). DTP cells were generated under shorter drug exposures that eradicate sensitive carcinoma cells.
Results and Discussion: Carcinoma cell plasticity drives cell transformation towards a phenotypic state, rendering them more tolerant to drugs. To better characterize this phenotype, we first focused on EMT and observed that DTP cells co-harbored epithelial and mesenchymal markers. We noticed that DTP cells exhibited an intracellular reactive oxygen species (ROS) rate making them dependent on protective oxidation-reduction mechanisms against oxidative stress-related damage. We revealed the overexpression of p21 and p27, known to bind to cyclin-CDK complexes and induce cell-cycle arrest. This dormant phenotype of DTP cells was underlined by a global repressive chromatin state evidenced by enrichment in H3K27me3, H3K36me3 and H3K9me3 marks; the latter mark also being a component of senescence-associated heterochromatin foci. In agreement, a high level of SA-β-gal and senescence-associated secretory phenotype components (CXCL10, PAI-1), evidenced that our DTP model displayed a senescence-like phenotype. We quantified an increase in γH2AX DNA damage foci in DTP cells compared to sensitive cells, in line with the concept that TKI treatment may have a role in DNA repair modulation that ultimately promotes new mutations. Together, our data depicted DTP features that might be relevant to identify drug candidates overcoming resistance (HDAC inhibitors, senolytic drugs and DNA repair inhibitors).
Conclusion: We demonstrated potential targetable features of DTP cells. A combination of targeted therapies with DTP drug candidates could represent a therapeutic opportunity to improve the depth of response and delay the emergence of resistance in patients.
Citation Format: Floriane Brayé, Francesco Facchinetti, Helena Gerber, Juliette Juigné, Giorgia Guaitoli, Jean-Paul Thiery, Santiago Ponce, Benjamin Besse, Ken A. Olaussen, Luc Friboulet. Characterization of TKI-induced drug-tolerant persister cells from a patient-derived cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 425.