Abstract The sustainable activation of the RAS/MAPK and PI3K/AKT signaling pathways in cancer is promoted by a reduction in the activity of the tumor suppressor protein phosphatase 2A (PP2A). Therefore, a novel therapeutic strategy consists of directly activating PP2A, leading to the simultaneous inhibition of these oncogenic pathways. PP2A is a heterotrimeric complex, consisting of a scaffolding A subunit, catalytic C subunit, and one of many regulatory B subunits responsible for substrate specificity. Our lab has successfully developed first-in-class Small Molecule Activators of PP2A (SMAPs), which induce tumor growth inhibition in both transgenic and patient derived xenograft mice models. However, alterations to the drug binding site is one of the most common mechanisms of acquired resistance, and we hypothesized that point mutations of the drug binding amino acid residues of PP2A would lead to decreased sensitivity to SMAP treatment. Determining potential mechanisms responsible for the development of resistance to SMAPs would guide the development of novel therapeutic strategies to improve patients’ prognosis. In silico docking calculations, photo affinity labeling, and hydroxyl radical footprinting studies were used to identify K194, E197, and L198 as the putative residues of PP2A-Aα that were interacting with SMAPs. K194R, E197K, and L198V mutations were generated by site-directed mutagenesis. H358, a KRAS-driven lung adenocarcinoma cell line, was used to create isogenic cell lines stably overexpressing mutated and wild type PP2A-Aα. These mutations did not affect the phosphatase activity or its ability to form holoenzymes in a cell-free system. SMAP response was investigated in vivo using a xenograft model of H358 isogenic cell lines and it was determined that tumors harboring mutant K194R and L198V PP2A-Aα were resistant to SMAPs treatment. Together, our results suggest that residues K194 and L198 are required for drug binding and subsequent target engagement. These findings shed light on possible mechanisms of acquired resistance to SMAPs in patients and have potential to guide the design of second-generation drugs. Citation Format: Rita Tohme, Jaya Sangodkar, Janna Kiselar, Daniel Leonard, Caitlin O'Connor, Sai Gandhe, Wenqing Xu, David Brautigan, Mark Chance, Michael Ohlmeyer, Goutham Narla. Drug target mutations as a mechanism of acquired resistance to small molecules activators of protein phosphatase 2a [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4173. doi:10.1158/1538-7445.AM2017-4173