Single-cell transcriptomic analysis of treatment-persistent residual disease in BRAF-mutant lung adenocarcinoma

Despite the encouraging results of BRAF-targeted therapies (BRAF-TT) in BRAF -mutant lung adenocarcinoma (LUAD), most tumor responses are only partial and limited in time, and ultimately all patients develop resistance to treatment. The limited number of pertinent preclinical models of BRAF -mutant LUAD has hindered the identification of molecular determinants of BRAF-TT response. In this context, treatment resistance may emerge from a subpopulation of drug-tolerant persister cells (DTP) that survive to treatment through early, non-genetic tumor cell adaptation, and might constitute the source of incomplete tumor responses and disease recurrence. Here, we assessed the prevalence and dynamics of cell states associated with DTP in patient-derived LUAD models and established LUAD cell lines representing each of the three BRAF mutation classes. Upon treatment with BRAF-TT (either with combined BRAF/MEK inhibitors or EGFR/MEK inhibitors), DTP exhibit different transcriptomic cell states, compared to treatment-naïve (TN) populations, in V600E, G469A and G466V-mutant cell lines and in a patient-derived organoid (PDO), generated from a G469A-mutant LUAD resected specimen. A small fraction of cycling DTP was observed in V600E and G469A cell lines. In both established cell lines and PDO, DTP systematically display features of phenotypic plasticity depicted by the enrichment of gene signatures related to epithelial-to-mesenchymal transition (EMT) and cell adhesion. Moreover, gene signatures associated with a dedifferentiation phenotype (i.e., alveolar and embryonic diapause) were increased in G469A treatment-residual organoids and cell lines but unchanged V600E-mutant cells. In V600E-mutant cells and G469A-mutant PDO we identified a transition-like cluster, including subpopulations of DTP and TN cells, characterised by enriched EMT and cell migration signatures, down-regulation of G2/M checkpoint and intermediate “drug-tolerant_up” scores, suggesting that these constitute early events of BRAF-TT adaptation. Of note, in a BRAF-TT resistant patient-derived xenograft (PDX), scRNAseq revealed the presence of six different transcriptomic cell states, including a DTP-like cluster: increased “drug-tolerant_up” and embryonic diapause scores, and deregulation of G2/M checkpoint, denoting the dynamic nature of the DTP-like phenotype under treatment. Trajectory and gene regulatory network bionformatic analyses are ongoing, together with functional experiments, to determine the role of phenotypic plasticity in treatment-persistent residual disease in BRAF-mutant LUAD. Our results show evidence on how BRAF-mutant LUAD DTP remodel their molecular portraits at the single-cell level, and provide insights into the pertinence of these models to study treatment-residual disease in BRAF -mutant LUAD.