Abstract Activating mutations in EGFR are key drivers in a subset of non-small cell lung cancer (NSCLC) patients, as well as effective predictors of response to first-generation reversible EGFR tyrosine kinase inhibitor (TKI) therapy. However, resistance develops in many patients due to acquisition of a T790M mutation in EGFR. Rociletinib and osimertinib target the T790M mutation, and have been evaluated in previously treated EGFR mutant lung cancer patients. Osimertinib recently received FDA approval for treatment of patients with EGFR T790M mutation-positive NSCLC whose disease has progressed on or after EGFR TKI therapy. However, there is limited information on the mechanisms of resistance to these drugs, as well as limited in vivo models for acquired resistance to examine signaling pathways and/or possible treatments to abrogate resistance. We treated an EGFR-L858R-T790M transgenic lung adenocarcinoma mouse model daily with rociletinib to examine sensitivity and acquired resistance. Rociletinib exhibited potent antitumor activity as a single agent, resulting in complete tumor regression and suppression of phospho-EGFR. Despite the success of treatment, after long-term dosing (4-5 months) tumors acquired resistance to the drug, as evidenced by tumor regrowth on MRI. DNA from multiple tumor samples representing four individual drug-resistant mice was sequenced, and several novel mutations in EGFR were observed that may account for the resistant outgrowth of tumors in these mice. These included an insertion and a deletion mutation as well as missense mutations. Interestingly, in some cases different mutations arose in individual tumor nodules within the same mouse, suggesting that each tumor is an individual resistant clone. All mutations were located in the kinase domain of EGFR. For example, a de novo G796C mutation was one of the potential mechanisms of resistance to rociletinib found in two isolated nodules from one mouse lung. A mutation at the same amino acid has been found in a lung cancer patient after treatment with osimertinib; moreover, this mutation was found in a cell line mutagenesis screen to study the resistance to different EGFR TKIs. We generated Ba/F3 cell lines carrying each de novo identified EGFR mutation (along with the activating L858R and T790M mutations) and compared the potency of EGFR TKIs to cell lines carrying only the activating mutations. The cells that expressed putative acquired resistance mutations proved to be resistant to both rociletinib and osimertinib in vitro, suggesting that these mutations indeed mediated acquired resistance in vivo. Tumor nodules from drug-resistant transgenic mice were transplanted into recipient mice and expanded for further evaluation. Mice with transplanted resistant tumors did not respond to rociletinib or osimertinib treatment. Pathway analysis showed that phospho-EGFR was no longer suppressed in drug-resistant tumors carrying a deletion mutation, confirming that novel mutations result in drug resistance. We discovered that resistance to third-generation EGFR inhibitors may occur through novel mutations in EGFR. Heterogeneity of mutations in different nodules within the same lung is akin to intra-patient heterogeneity and highlights the utility of mouse models for examining acquired resistance to drugs. Citation Format: Rajaa El Meskini, Anthony Iacovelli, Alan Kulaga, Michelle Gumprecht, Myla Spencer, Lilia Ileva, Andrew Simmons, Zoë Weaver Ohler. Continuous treatment with rociletinib in EGFR transgenic mice results in acquired resistance to both rociletinib and osimertinib and intra-“patient” tumor heterogeneity [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 A162.