Purpose KIT is the major oncogenic driver of gastrointestinal stromal tumors (GISTs). Imatinib, sunitinib and regorafenib are approved therapies; however, efficacy is often limited by the acquisition of polyclonal secondary resistance mutations in KIT, with those located in the activation (A) loop (exons 17/18) being particularly problematic. Here we explored the KIT inhibitory activity of ponatinib in preclinical models and describe initial characterization of its activity in GIST patients. Experimental Design The cellular and in vivo activities of ponatinib, imatinib, sunitinib and regorafenib against mutant KIT were evaluated using an accelerated mutagenesis assay and a panel of engineered and GIST-derived cell lines. The ponatinib-KIT co-structure was also determined. The clinical activity of ponatinib was examined in three GIST patients previously treated with all 3 FDA-approved agents. Results In engineered and GIST-derived cell lines, ponatinib potently inhibited KIT exon 11 primary mutants and a range of secondary mutants, including those within the A-loop. Ponatinib also induced regression in engineered and GIST-derived tumor models containing these secondary mutations. In a mutagenesis screen, 40 nM ponatinib was sufficient to suppress outgrowth of all secondary mutants except V654A, which was suppressed at 80 nM. This inhibitory profile could be rationalized based on structural analyses. Ponatinib (30 mg daily) displayed encouraging clinical activity in two of three GIST patients. Conclusion Ponatinib possesses potent activity against most major clinically-relevant KIT mutants, and has demonstrated preliminary evidence of activity in patients with refractory GIST. These data strongly support further evaluation of ponatinib in GIST patients.

BACKGROUND: Most patients with KIT-mutant gastrointestinal stromal tumours (GISTs) benefit from imatinib, but treatment resistance results from outgrowth of heterogeneous subclones with KIT secondary mutations. Once resistance emerges, targeting KIT with tyrosine kinase inhibitors (TKIs) sunitinib and regorafenib provides clinical benefit, albeit of limited duration. METHODS:We systematically explored GIST resistance mechanisms to KIT-inhibitor TKIs that are either approved or under investigation in clinical trials: the studies draw upon GIST models and clinical trial correlative science. We subsequently modelled in vitro a rapid TKI alternation approach against subclonal heterogeneity. RESULTS: Each of the KIT-inhibitor TKIs targets effectively only a subset of KIT secondary mutations in GIST. Regorafenib and sunitinib have complementary activity in that regorafenib primarily inhibits imatinib-resistance mutations in the activation loop, whereas sunitinib inhibits imatinib-resistance mutations in the ATP-binding pocket. We find that rapid alternation of sunitinib and regorafenib suppresses growth of polyclonal imatinib-resistant GIST more effectively than either agent as monotherapy. CONCLUSIONS: Our data highlight that heterogeneity of KIT secondary mutations is the main mechanism of tumour progression to KIT inhibitors in imatinib-resistant GIST patients. Therapeutic combinations of TKIs with complementary activity against resistant mutations may be useful to suppress growth of polyclonal imatinib-resistance in GIST.

Targeting and stabilizing distinct kinase conformations is an instrumental strategy for dissecting conformation-dependent signaling of protein kinases. Herein the structure-based design, synthesis, and evaluation of pleckstrin homology (PH) domain-dependent covalent-allosteric inhibitors (CAIs) of the kinase Akt is reported. These inhibitors bind covalently to a distinct cysteine of the kinase and thereby stabilize the inactive kinase conformation. These modulators exhibit high potency and selectivity, and represent an innovative approach for chemical biology and medicinal chemistry research.

Targeting acquired drug resistance represents the major challenge in the treatment of EGFR-driven non-small-cell lung cancer (NSCLC). Herein, we describe the structure-based design, synthesis, and biological evaluation of a novel class of covalent EGFR inhibitors that exhibit excellent inhibition of EGFR-mutant drug-resistant cells. Protein X-ray crystallography combined with detailed kinetic studies led to a deeper understanding of the mode of inhibition of EGFR-T790M and provided insight into the key principles for effective inhibition of the recently discovered tertiary mutation at EGFR-C797S.

The specific targeting of oncogenic mutant epidermal growth factor receptor (EGFR) is a breakthrough in targeted cancer therapy and marks a drastic change in the treatment of non-small cell lung cancer (NSCLC). The recurrent emergence of resistance to these targeted drugs requires the development of novel chemical entities that efficiently inhibit drug-resistant EGFR. Herein, we report the optimization process for a hit compound that has emerged from a phenotypic screen resulting in indazole-based compounds. These inhibitors are conformationally less flexible, target gatekeeper mutated drug-resistant EGFR-L858R/T790M, and covalently alkylate Cys797. Western blot analysis, as well as characterization of the binding kinetics and kinase selectivity profiling, substantiates our approach of targeting drug-resistant EGFR-L858R/T790M with inhibitors incorporating the indazole as hinge binder.