Lung cancer is the leading cause of cancer-related deaths among men and women worldwide. Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are effective therapies for advanced non-small-cell lung cancer (NSCLC) patients harbouring EGFR-activating mutations, but are not curative due to the inevitable emergence of resistances. Recent in vitro studies suggest that resistance to EGFR-TKI may arise from a small population of drug-tolerant persister cells (DTP) through non-genetic reprogramming, by entering a reversible slow-to-non-proliferative state, before developing genetically derived resistances. Deciphering the molecular mechanisms governing the dynamics of the drug-tolerant state is therefore a priority to provide sustainable therapeutic solutions for patients. An increasing number of molecular mechanisms underlying DTP survival are being described, such as chromatin and epigenetic remodelling, the reactivation of anti-apoptotic/survival pathways, metabolic reprogramming, and interactions with their micro-environment. Here, we review and discuss the existing proposed mechanisms involved in the DTP state. We describe their biological features, molecular mechanisms of tolerance, and the therapeutic strategies that are tested to target the DTP.

Purpose: Drug-tolerant “dormant” cells (DTC) have emerged as one of the major non-genetic mechanisms driving resistance to targeted therapy in lung cancer, although the sequence of events leading to entry and exit from dormancy remain poorly described. Here, we provide a step-by-step phenotypic and molecular characterization of the different processes involved during the adaptive response to osimertinib using several EGFR-mutated lung cancer models. This strategy led to the identification of a common vulnerability of drug-tolerant cells which could be efficiently and safely targeted by a clinical stage drug. Experimental design: We used the FUCCI (fluorescence ubiquitination cell cycle indicator) system to determine the cell cycle dynamics in real time during the adaptive response to osimertinib in a panel of EGFR-mutated lung cancer cell lines. We performed scRNAseq on untreated and osimertinib-treated G1 and S/G2 sorted cells during early relapse to determine the molecular mechanisms underlying entry and exit from dormancy. We validated our observations in several in vitro and in vivo models as well as in publicly available patient data. Results: FUCCI labelling allowed the identification of a rare population of S/G2 cycling cells (referred to as early escapers) that emerged in the first hours of treatment amongst a majority of stably arrested and progressively dying G1 cells. scRNAseq data revealed that early escapers emerged from a differentiated alveolar type 1 (AT1) phenotype which was invariably associated with an increase in contractile-related gene signatures, F-actin polymerization and Rho/ROCK pathway activation. Using a screen of Rho-pathway inhibitors, we found that tipifarnib, a farnesyltransferase inhibitor (FTi), induced a complete clearance of DTC in vitro. Co-treatment with tipifarnib, a clinically active FTi, safely and durably prevented relapse to osimertinib in a PC9-xenograft model as well as in a PDX model of EGFRL858R/T790M lung cancer for up to 6 months with no evidence of toxicity. Several farnesylated targets were identified in both G1 and S/G2 treated cells, which could explain the high efficiency of tipifarnib in preventing the adaptive response to osimertinib. Finally, we observed that osimertinib + tipifarnib co-treatment completely suppressed the emergence of the AT1 phenotype, prevented mitosis of S/G2-treated cells and increased the apoptotic response through activation of the unfolded protein response (UPR) pathway. Conclusion: Our data strongly support the use of tipifarnib in combination with osimertinib in the clinic to effectively, durably and safely prevent relapse. Citation Format: Sarah Figarol, Célia Delahaye, Rémi Gence, Raghda Asslan, Sandra Pagano, Jacques Colinge, Jean-Philippe Villemin, Antonio Maraver, Isabelle Lajoie-Mazenc, Estelle Clermont, Anne Casanova, Anne Pradines, Julien Mazières, Olivier Calvayrac, Gilles Favre. Tipifarnib prevents emergence of resistance to osimertinib in EGFR-mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB080.