Expressed barcoding enables high resolution tracking of the evolution of drug tolerance.

For a majority of non-small cell lung cancer (NSCLC) patients with EGFR mutations, treatment with EGFR inhibitors (EGFRi) induces a clinical response. Despite this initial reduction in tumor size, residual disease persists that leads to disease relapse. Elucidating the pre-existing biological differences between sensitive cells and surviving drug-tolerant persister cells and deciphering how drug-tolerant cells evolve in response to treatment could help identify strategies to improve the efficacy of EGFRi. In this study, we tracked the origins and clonal evolution of drug tolerant cells at a high resolution by using an expressed barcoding system coupled with single cell RNA-sequencing. This platform enabled longitudinally profiling of gene expression and drug sensitivity in response to EGFRi across a large number of clones. Drug tolerant cells had higher expression of key survival pathways such as YAP and EMT at baseline and could also differentially adapt their gene expression following EGFRi treatment compared to sensitive cells. In addition, drug combinations targeting common downstream components (MAPK) or orthogonal factors (chemotherapy) showed greater efficacy than EGFRi alone, which is likely attributable to broader targeting of the multiple EGFRi tolerance mechanisms present in tumors. Overall, this approach facilitates thorough examination of clonal evolution in response to therapy that could inform the development of improved diagnostic approaches and treatment strategies for targeting drug tolerant cells.