ABSTRACT While it is well established that tumors evolve as they adapt to environmental cues, exactly how this process occurs remains under investigation. Precision medicines impose a relatively uniform selective pressure on the tumor and have well-described mechanisms of action, which enable a unique opportunity to study the determinants of evolutionary selection. By utilizing such treatments in conjunction with newly established methodologies that enable single-cell sequencing and prospective tracking of subclonal populations, this proposal aims to provide insight into the principles that govern the selection of resistance-causing alterations. BRAF mutant tumors were selected as an experimental model system because these are found in approximately 7% of cancer patients, including those with malignant melanoma, thyroid, colorectal and lung cancers. While RAF and/or MEK inhibitors are clinically effective active against these tumors, resistance is inevitable and almost all patients die from their disease, indicating the need to identify improved therapies. In recent work, we have established a conceptual framework that explains the selection and propagation of resistance-causing alterations and identified an intermittent three-drug combination therapy, which has a potent antitumor effect in BRAF V600E mutant lung cancer and melanoma patient-derived xenograft models. Based on these advances, we now proposed to study the effect of this treatment on intratumoral clonal heterogeneity and the emergence of resistance. In aim 1 we will determine genetic alterations that confer resistance to the intermittent combination treatment and determine the effect of this therapy on the intratumoral heterogeneity. In aim 2 we will prospectively evaluate the effect of therapy on clonal selection. In aim 3 we will investigate the mechanisms that confer fitness to the subclones that are selected during therapy. The impact of this proposal centers on advancing our understanding of the dynamics that drive the selection of subpopulations harboring resistance-causing alterations and on the identification of novel and more effective treatments for patients with BRAF mutant tumors.