Tumors evolve as they adapt to environmental cues. The principles governing evolution of tumors under the selective pressure of targeted therapy are not well understood. We aimed to evaluate the evolution of resistance and to identify therapeutic modalities that prevent this process in BRAF V600E -mutant tumors. We modeled the selection and propagation of BRAF V600E amplification (BRAF amp ) in patient-derived tumor xenografts (PDX) treated with a direct ERK inhibitor. Single-cell sequencing and multiplex-fluorescence in situ hybridization mapped the emergence of extra-chromosomal amplification in multiple subclones of the same tumor shortly after treatment. The evolutionary selection of BRAF amp is determined by the fitness threshold, the barrier subclonal populations need to overcome to regain fitness in the presence of therapy. This differed for ERK signaling inhibitors, and single-cell sequencing of a melanoma PDX model showed that drugs of the same pathway do not necessarily select for the same subclones. These data suggest that sequential monotherapy is not optimal, but concurrent targeting of RAF, MEK, and ERK, however, imposes a sufficiently high fitness threshold to prevent the propagation of subclones with high-level amplification. Administered on an intermittent schedule, this treatment inhibited tumor growth without apparent toxicity in 11/11-lung cancer and melanoma PDX models with various additional alterations. Thus, gene amplification can be acquired and expanded through parallel evolution, enabling tumors to adapt while maintaining their intratumoral heterogeneity. Treatments that impose a high fitness threshold, such as our intermittent triple therapy, will likely prevent the evolution of resistance-causing alterations and merit testing in patients. Citation Format: Yaohua Xue, Luciano Martelotto, Timour Baslan, Alberto Vides, Martha Solomon, Kalyani Chadalavada, Elisa DeStanchina, Gouri Nanjangud, Michael Berger, Scott Lowe, Jorge S. Reis-Filho, Neal Rosen, Piro Lito. An approach to suppress the evolution of resistance in BRAF V600E -mutant cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B015.