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Drug resistance profiles of mutations in the RET kinase domain.

ABSTRACT: BACKGROUND AND PURPOSE:Alterations in the tyrosine kinase enzyme RET are found in thyroid and lung cancer. While RET TK inhibitors (TKIs) are used to treat thyroid cancer and are in clinical trials for RET fusion-positive non-small cell lung cancer, the impact of mutations in the RET kinase domain on drug sensitivity is largely uncharacterized. EXPERIMENTAL APPROACH:We identified and analysed mutations in the RET kinase domain that conferred resistance to the TKIs cabozantinib, lenvatinib, vandetanib and nintedanib using RET kinase-dependent BaF3/KIF5B-RET (BaF3/KR) cells. We also examined the sensitivity of RET (M918T), a RET mutation prevalent in aggressive multiple endocrine neoplasia type 2B, to these TKIs in the context of BaF3/KR cells. KEY RESULTS:Fourteen mutations were analysed. Pan resistance to the four TKIs was found in six RET kinase domain mutations (L730I, V738A, V804L/M, Y806N, G810S). Seven RET kinase domain mutations (L730V, E732K, A807V, G810A, V871I, M918T, F998V) displayed selective resistance to one or more of these drugs. L730I/V and G810A/S had different drug resistance profiles. V871I, M918T and F998V mutations are located at distant sites away from the TKI binding pocket. CONCLUSIONS AND IMPLICATIONS:A panel of TKI-resistant RET mutations were identified, and their drug sensitivities were cross-profiled. The results provide a reference for selecting appropriate TKIs to inhibit RET kinase domain mutants. Besides changes in the drug-interacting residues, mutations at distant sites could exert long-range effects resulting in TKI resistance. Among the four TKIs analysed here, nintedanib remained unaffected by mutations at the three distant sites.

SUBMITTER: Liu X

PROVIDER: S-EPMC6086982 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Drug resistance profiles of mutations in the RET kinase domain.

Liu Xuan X Shen Tao T Mooers Blaine H M BHM Hilberg Frank F Wu Jie J

British journal of pharmacology 20180719 17

<h4>Background and purpose</h4>Alterations in the tyrosine kinase enzyme RET are found in thyroid and lung cancer. While RET TK inhibitors (TKIs) are used to treat thyroid cancer and are in clinical trials for RET fusion-positive non-small cell lung cancer, the impact of mutations in the RET kinase domain on drug sensitivity is largely uncharacterized.<h4>Experimental approach</h4>We identified and analysed mutations in the RET kinase domain that conferred resistance to the TKIs cabozantinib, le ...[more]

PMID: 29908090

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Project description:INTRODUCTION:Novel rearranged in transfection (RET)-specific tyrosine kinase inhibitors (TKIs) such as selpercatinib (LOXO-292) have shown unprecedented efficacy in tumors positive for RET fusions or mutations, notably RET fusion-positive NSCLC and RET-mutated medullary thyroid cancer (MTC). However, the mechanisms of resistance to these agents have not yet been described. METHODS:Analysis was performed of circulating tumor DNA and tissue in patients with RET fusion-positive NSCLC and RET-mutation positive MTC who developed disease progression after an initial response to selpercatinib. Acquired resistance was modeled preclinically using a CCDC6-RET fusion-positive NSCLC patient-derived xenograft. The inhibitory activity of anti-RET multikinase inhibitors and selective RET TKIs was evaluated in enzyme and cell-based assays. RESULTS:After a dramatic initial response to selpercatinib in a patient with KIF5B-RET NSCLC, analysis of circulating tumor DNA revealed emergence of RET G810R, G810S, and G810C mutations in the RET solvent front before the emergence of clinical resistance. Postmortem biopsy studies reported intratumor and intertumor heterogeneity with distinct disease subclones containing G810S, G810R, and G810C mutations in multiple disease sites indicative of convergent evolution on the G810 residue resulting in a common mechanism of resistance. Acquired mutations in RET G810 were identified in tumor tissue from a second patient with CCDC6-RET fusion-positive NSCLC and in plasma from patients with additional RET fusion-positive NSCLC and RET-mutant MTC progressing on an ongoing phase 1 and 2 trial of selpercatinib. Preclinical studies reported the presence of RET G810R mutations in a CCDC6-RET patient-derived xenograft (from a patient with NSCLC) model of acquired resistance to selpercatinib. Structural modeling predicted that these mutations sterically hinder the binding of selpercatinib, and in vitro assays confirmed loss of activity for both anti-RET multikinase inhibitors and selective RET TKIs. CONCLUSIONS:RET G810 solvent front mutations represent the first described recurrent mechanism of resistance to selective RET inhibition with selpercatinib. Development of potent inhibitor of these mutations and maintaining activity against RET gatekeeper mutations could be an effective strategy to target resistance to selective RET inhibitors.

Project description:Background:Alterations involving the RET kinase are implicated in the pathogenesis of lung, thyroid and other cancers. However, the clinical activity of multikinase inhibitors (MKIs) with anti-RET activity in RET-altered patients appears limited, calling into question the therapeutic potential of targeting RET. LOXO-292 is a selective RET inhibitor designed to inhibit diverse RET fusions, activating mutations and acquired resistance mutations. Patients and methods:Potent anti-RET activity, high selectivity, and central nervous system coverage were confirmed preclinically using a variety of in vitro and in vivo RET-dependent tumor models. Due to clinical urgency, two patients with RET-altered, MKI-resistant cancers were treated with LOXO-292, utilizing rapid dose-titration guided by real-time pharmacokinetic assessments to achieve meaningful clinical exposures safely and rapidly. Results:LOXO-292 demonstrated potent and selective anti-RET activity preclinically against human cancer cell lines harboring endogenous RET gene alterations; cells engineered to express a KIF5B-RET fusion protein -/+ the RET V804M gatekeeper resistance mutation or the common RET activating mutation M918T; and RET-altered human cancer cell line and patient-derived xenografts, including a patient-derived RET fusion-positive xenograft injected orthotopically into the brain. A patient with RET M918T-mutant medullary thyroid cancer metastatic to the liver and an acquired RET V804M gatekeeper resistance mutation, previously treated with six MKI regimens, experienced rapid reductions in tumor calcitonin, CEA and cell-free DNA, resolution of painful hepatomegaly and tumor-related diarrhea and a confirmed tumor response. A second patient with KIF5B-RET fusion-positive lung cancer, acquired resistance to alectinib and symptomatic brain metastases experienced a dramatic response in the brain, and her symptoms resolved. Conclusions:These results provide proof-of-concept of the clinical actionability of RET alterations, and identify selective RET inhibition by LOXO-292 as a promising treatment in heavily pretreated, multikinase inhibitor-experienced patients with diverse RET-altered tumors.

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Drug resistance profiles of mutations in the RET kinase domain.

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Drug resistance profiles of mutations in the RET kinase domain.

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