PROJECT SUMMARY BRAFV600E mutation, the most common BRAF mutation, is found in approximately 3% of lung adenocarcinomas and 50% of melanomas and presents a significant disease burden. While RAF inhibitors, alone or in combination with MEK inhibitors, have improved survival, resistance eventually arises and patients progress on therapy. More effective treatments are urgently needed, especially for patients with advanced disease. Our previous work showed that propagation of resistance-causing alterations, such as BRAFV600E-amplification, is due to an inadequate fitness threshold imposed by therapy, where fitness threshold refers to the barrier subclones must overcome for continued growth. This suggests that a combination therapy consisting of multiple-drugs would be more effective. We designed an intermittent RAF, MEK, and ERK inhibitor regimen that inhibited tumor growth in a panel of lung adenocarcinoma and melanoma patient-derived xenograft (PDX) models, even those with known resistance-causing alterations to single agents. The goal of this project is to assess the durability of response to the intermittent three-drug combination, and to elucidate tumor clonal interactions that may facilitate the evolution of resistance. Specifically, in Aim 1, I will a) evaluate the long-term treatment response to the intermittent three-drug combination in PDX models; b) identify resistance mechanisms through deep targeted sequencing; and c) evaluate the effect of treatment on tumor clonal architecture using novel single cell DNA sequencing. In Aim 2, using BRAFV600E-amplification as a model and fluorescently barcoded single cell clonal expansions as my experimental system, I plan to a) identify clonal interaction mechanisms that support the growth of resistant clones and b) prospectively track the effect of therapy on tumor clonal composition. These studies will improve understanding of the determinants of evolution of targeted therapy resistance and enable the design of more effective treatments, as well as identify a therapeutic combination that may be curative for some BRAFV600E-mutant cancers. !