Introduction: Ponatinib was designed as a highly potent inhibitor of native BCR-ABL as well as the T315I gatekeeper mutant that is resistant to all approved therapies, and has demonstrated substantial activity in patients with native or mutant BCR-ABL in a phase 1 trial. Activity against T315I is due to a key design feature, the triple bond linker, which enforces ligand linearity in this sterically demanding region of the binding site. Ponatinib also inhibits the in vitro kinase activity of a number of other clinically relevant kinases (FLT3, RET, KIT, PDGFRα, PDGFRα, and FGFR1; IC50s of 0.2 to 13 nM), many of which have developed resistance to existing therapies via mutation of the gatekeeper residue. Here we assessed whether ponatinib might represent a more general solution to gatekeeper mutant inhibition of these and other clinically important kinases by virtue of its design features. Results: The cellular activity of ponatinib was assessed using Ba/F3 cells transformed to IL-3 independence by expression of an activated kinase (via fusion to a TEL dimerization domain). Ponatinib potently inhibited viability of cells expressing activated FLT3, RET, KIT, PDGFRα, PDGFRα and FGFR1 with IC50s of 15, 8, 7, 1, 2 and 21 nM, respectively. In contrast, ponatinib did not affect viability of parental Ba/F3 cells grown in the presence of IL-3 (IC50 >1000 nM). Kinase variants in which the gatekeeper residue was mutated to isoleucine, leucine or methionine (all by a single nucleotide change from the native sequence) were then transformed into Ba/F3 cells. All of these variants have been shown previously, or in our studies, to confer substantial resistance (i.e. >50-fold) to other targeted agents. Ponatinib maintained substantial activity against all variants tested. The smallest potency reduction (2- to 9-fold) was seen in variants mutated to isoleucine, with FLT3F691I, KITT670I, PDGFRαT674I, and PDGFRαT681I being inhibited with IC50s of 62, 26, 2 and 17 nM, respectively. A slightly greater potency reduction (10- to 22-fold) was seen in sterically more demanding leucine or methionine mutations, with RETV804M, RETV804L and FGFR1V561M being inhibited with IC50s of 84, 178 and 202 nM, respectively. In all cell lines, cell viability effects were accompanied by inhibition of receptor phosphorylation. Conclusion: Ponatinib is a potent inhibitor of activated variants of FLT3, RET, KIT, PDGFRα, PDGFRα, and FGFR1, in addition to BCR-ABL. Ponatinib also maintains substantial activity against gatekeeper variants shown to mediate resistance to other targeted agents, inhibiting their activity with IC50s that are within range of known clinically achievable plasma concentrations. These results suggest that ponatinib may offer a more general solution to gatekeeper mutant inhibition and support clinical testing in a variety of cancers driven by activated kinases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 853. doi:1538-7445.AM2012-853