Abstract 3394: Ponatinib, a pan-BCR-ABL inhibitor, potently inhibits key activating and drug-resistant KIT mutants found in GIST.
Authors: Garner Andrew P. , Anjum Rana , Vodala Sadanand , Schrock Alexa , Zhou Tianjun , Clackson Tim , Gozgit Joseph M. , Rivera Victor M. 
Published: August 2014

Abstract
Ponatinib (AP24534) is a multi-targeted tyrosine kinase inhibitor with potent pan-BCR-ABL activity being investigated in patients with CML. Ponatinib also inhibits the kinase activity of KIT, a clinically proven oncogenic driver. Approximately 80% of gastrointestinal stromal tumors (GIST) contain primary activating KIT mutations, the majority of which cluster in exon 11 (V560D, Δ557-8) or more rarely in exon 9 (AY502-3ins). Imatinib is approved for the 1st line treatment of GIST; however, patients frequently relapse due to the acquisition of secondary resistance mutations located in either the KIT ATP-binding pocket (T670I, V654A) or the activation (A) loop (D816H, D820A, N822K, A829P). Sunitinib is registered for 2nd line treatment of GIST but does not effectively inhibit A-loop mutants. Here we explored the activity of ponatinib against major primary and secondary KIT mutants found in GIST.
The drug sensitivity of KIT mutants was determined using engineered Ba/F3 cells harboring mutant forms of KIT. Imatinib potently inhibited the viability of cells expressing exon 11 mutant KIT (IC50&lt; 30 nM). The introduction of secondary mutations into a mutant exon 11 background (Δ557-8) dramatically reduced imatinib's activity (IC50 180 nM - 10 μM). Sunitinib was highly active (IC50&lt; 15 nM) versus KIT primary mutants and secondary ATP-binding pocket mutants. However, its activity was substantially reduced by A-loop mutations (IC50&gt; 200 nM). In contrast, ponatinib was highly potent (IC50&lt; 15 nM) across a range of primary and secondary KIT mutants, including T670I and all 4 A-loop mutants tested. The potency of ponatinib was slightly reduced versus the KIT exon 9 primary mutant (IC50= 56 nM) or when V654A was present as a secondary mutation (IC50= 59 nM). Importantly, in patients dosed once daily with 45 mg ponatinib, peak (145 nM) and trough (64 nM) plasma concentrations achieved are predicted to lead to substantial inhibition of all KIT mutants tested here.
To rationalize ponatinib's activity we solved the structure of the native KIT kinase domain co-crystallized with ponatinib. Ponatinib bound the inactive (DFG-out) conformation of KIT, filling the ATP-binding pocket as well as two adjacent hydrophobic pockets, analogous to its binding to ABL. Ponatinib can accommodate the gatekeeper T670I mutation by virtue of its triple bond circumventing the steric bulk of the larger isoleucine residue. Ponatinib's activity against A-loop mutants can be rationalized by its tight binding to the inactive conformation of KIT, even in the presence of A-loop mutations.
In conclusion, ponatinib possesses potent activity versus the major clinically relevant KIT mutants, inhibiting their activity with IC50s that fall within clinically achievable plasma concentrations. These data therefore support the evaluation of ponatinib in patients with GIST, particularly since KIT A-loop mutants represent an unmet medical need.
Citation Format: Andrew P. Garner, Rana Anjum, Sadanand Vodala, Alexa Schrock, Tianjun Zhou, Tim Clackson, Joseph M. Gozgit, Victor M. Rivera. Ponatinib, a pan-BCR-ABL inhibitor, potently inhibits key activating and drug-resistant KIT mutants found in GIST. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3394. doi:10.1158/1538-7445.AM2013-3394