The vast majority (>80%) of patients with BRAFV600E/K melanomas initially respond to highly specific BRAFV600E/K inhibitors as monotherapy or BRAF/MEK inhibitor combination therapy, but at varying levels. Nearly all patients relapse after three months to two years, despite that all of their tumor cells are carrying the BRAF mutations. In this application, we focus on the biological dynamics of small populations within BRAFV600E/K melanomas that are a priori resistant or rapidly adapt upon treatment and drive eventual relapse. In the first aim we will follow the dynamics of mutant BRAFV600E/K melanoma cells treated with BRAF and MEK inhibitor combinations. We have identified a slow-cycling subpopulation that is dynamically changing as the cells survive the initial treatment, then persist and finally regrow with acquired resistance to a variety of drugs. We will follow the cells in in vitro and in vivo models of human melanoma using bar codes and single cell RNA analyses. These studies will not only determine whether minor subpopulation(s) evolve under drug treatment stochastically or hierarchically, but also will provide us with information on their pre-existence and/or whether malignant cell plasticity is the major reason for intra-tumor heterogeneity to drug responses. In the second aim, we are testing the hypothesis that intrinsically resistant subpopulations of cells present new therapeutic opportunities that, if targeted, will specifically inhibit the onset of resistance. Our preliminary studies using CRISPR/Cas9 have allowed us to functionally dissect the pathways that form these rare subpopulations and, separately, the pathways that allow those subpopulations to reprogram. We will validate hits in 3D culture systems and in in vivo mouse models. We expect that targeting separate aspects of the resistance process in a rationally designed way (in vitro and then in preclinical studies) will enable the reduction of resistance onset upon BRAF/MEK inhibitor therapy.