Abstract Background: Approximately 80% of GISTs contain primary activating mutations in KIT, the majority of which cluster in exon 11. Imatinib is approved for treatment of metastatic and/or unresectable GIST; however patients often relapse due to the acquisition of secondary resistance mutations in KIT, typically located in the ATP-binding pocket or activation loop (A-loop). Sunitinib is approved for 2nd line treatment, but does not effectively inhibit A-loop mutants. Ponatinib is a multi-targeted tyrosine kinase inhibitor (TKI) with potent activity against BCR-ABL that has been approved for treatment of CML and Ph+ ALL patients resistant or intolerant to prior TKIs. We have previously shown that ponatinib also potently inhibits both activated KIT and KIT containing imatinib- or sunitinib-resistance mutations, including those at the gatekeeper residue and within the A-loop. V654A was identified as the most recalcitrant mutant, although ponatinib was still capable of significantly inhibiting tumor growth in a V654A mouse model. To survey more broadly for mutations that might confer substantial resistance to ponatinib, an accelerated KIT mutagenesis screen was performed. Results: Ba/F3 cells dependent on exon 11 mutant KIT (Δ557-8) were chemically mutagenized to introduce random mutations and grown in the presence of TKIs to identify secondary resistance mutations. Importantly, resistant clones that grew in the presence of imatinib or sunitinib recapitulated the spectrum of mutations observed clinically. In the presence of imatinib, mutations in the ATP pocket and A-loop were observed, with the T670I gatekeeper mutation conferring the highest level of resistance. In the presence of sunitinib, mutations were observed primarily in the A-loop (D816, D820, N822 and Y823), but not the ATP pocket. In contrast, in the presence of 40 nM ponatinib, only the V654A mutation persisted, in a small number of clones. In the presence of 80 nM ponatinib the development of resistance mutations was completely suppressed. Importantly, levels of ponatinib achieved in patients dosed once daily with 45 mg ponatinib (145 nM peak; 64 nM trough) exceed these target levels. The results of mutagenesis studies using other primary mutant backgrounds, and the newly approved TKI regorafenib, will also be described. Conclusion: Preclinical analysis suggests that, in an exon 11 mutant KIT background, ponatinib can inhibit development of a broad spectrum of potential resistance mutations, including multiple problematic mutations within the A-loop. Thus far, the only secondary mutation identified that is not completely suppressed at clinically achievable trough concentrations is V654A. These studies provide further support for an ongoing phase 2 study of ponatinib in patients with TKI-resistant GIST (NCT01874665). Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B266. Citation Format: Alexa B. Schrock, Tzu-Hsiu Chen, Victor M. Rivera, Joseph M. Gozgit. Ponatinib, a potent KIT inhibitor, suppresses the emergence of secondary resistance mutations in a gastrointestinal stromal tumor (GIST) model system. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B266.