The Development Of Potent, Selective Ret Inhibitors That Target Both Wild-Type Ret And Prospectively Identified Resistance Mutations To Multi Kinase Inhibitors

Introduction: RET/PTC1 was one of the first gene fusions identified from a solid tumor. In the past 3 years, oncogenic RET fusions have been identified in additional cancer types, most notably NSCLC and colon carcinomas. Activating germline RET mutations are also well established drivers of multiple endocrine neoplasia while somatic RET mutations are the most prevalent alterations in sporadic medullary thyroid cancers. In light of these findings, a number of approved multi-kinase inhibitors (mKIs) with in vitro activity against wild-type (WT) RET, such as cabozantinib, vandetanib, and lenvatinib, have been repurposed for treating RET-driven diseases. We have designed a next-generation inhibitor specifically tailored to target RET, while sparing other closely-related kinases, such as KDR/VEGFR2. Given that secondary mutations are a common resistance mechanism to kinase inhibitors, we prospectively identified resistance mutations that abrogate mKI activity and crafted our RET-selective inhibitors to also target these mutations. The structures of several mKIs bound to RET were analyzed and amino acid substitutions that would disrupt the protein-inhibitor interactions were predicted. In vitro resistance screens with cabozantinib, ponatinib, and vandetanib in a Ba/F3 KIF5B-RET cell line were conducted and confirmed these predictions. The proprietary Blueprint Medicines’ kinase inhibitor library was used to identify inhibitors of WT and resistance mutant RET as starting points for lead optimization. Results: Both structural analysis and in vitro screening revealed that only a handful of positions within the RET kinase domain enable resistance mutations to mKIs, suggesting a narrow mutational spectrum. Using our library of kinase inhibitors, we identified potent, orally bioavailable inhibitors of RET that target both the WT and resistance mutants in KIF5B-RET-driven cell lines while sparing the majority of the kinome. These inhibitors also suppressed the proliferation of thyroid cancer cell lines harboring RET fusions or activating RET mutations and demonstrated in vivo activity in xenograft models. Our analysis of the PK-PD-efficacy relationship revealed that over 70% target suppression is required for maximal efficacy. Finally, our RET-selective inhibitors induced dose-dependent tumor growth inhibition in a KIF5B-RET fusion positive lung adenocarcinoma PDX model at well-tolerated doses, further validating RET fusions as oncogenic drivers in NSCLC. Conclusion: This work describes the identification of potent inhibitors that specifically target WT RET and resistance mutations predicted to arise upon mKI treatment. By sparing kinases with known toxicity profiles, these molecules are predicted to robustly inhibit RET at tolerated doses and may provide patients with RET-driven diseases an opportunity for more durable and effective therapies. Citation Format: Rami Rahal, Erica K. Evans, Wei Hu, Michelle Maynard, Paul Fleming, Lucian DiPietro, Joseph L. Kim, Michael P. Sheets, Doug P. Wilson, Kevin J. Wilson, Nicolas Stransky, Jason D. Brubaker, Timothy Guzi, Nancy E. Kohl, Christoph Lengauer. The development of potent, selective RET inhibitors that target both wild-type RET and prospectively identified resistance mutations to multi-kinase inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2641.