PROJECT SUMMARY/ABSTRACT Targeted immunotherapies against BRAFV600E/K-positive melanoma cancers have been efficacious at eradicating disease in >80% of patients. Nevertheless, the existence of populations of pre-resistant rare cells has posed a challenge to the efficacy of melanoma-targeted therapies, often leading to a disease relapse in the majority of patients. In the parent R01 grant, we proposed a multi-faceted approach combining new experimental techniques, statistical analysis, and theory to understand the origin of these rare, transient drug-resistant cell states and devise strategies to control them. In the current administrative supplement, our goal is to dissect the primordial cellular pathways governing cellular reprogramming that convert the transient state of resistance in the rare single cells into the permanent state of drug resistance that drive relapse. This work is related to the parent R01, but explores new concepts and experimental designs. By using a low dose exposure regimen, followed by high dose exposures, we seek to investigate whether a selective or an adaptive cellular response underlies the mechanism resulting in the conversion to permanent drug resistance. In a selection response, the permanently resistant cells share the same final resistant properties as the transient initial population. In an adaptive response, however, the final resistant properties of the permanently resistant cells could be different, most likely greater, than that of the initial transient cells. We will employ a multi-pronged approach consisting of single cell imaging, single cell barcoding, single cell RNA FISH, RNA sequencing and computational biology techniques to elucidate the primordial cellular/molecular regulators driving cellular reprogramming that results in permanently resistant cells. The mechanisms revealed by these studies will be important for the development of new targeted therapies that will be able to eradicate the relapse caused by therapy resistant cells.