Dataset Information

Selective inhibition of RET mediated cell proliferation in vitro by the kinase inhibitor SPP86.

ABSTRACT:

Background

The RET tyrosine kinase receptor has emerged as a target in thyroid and endocrine resistant breast cancer. We previously reported the synthesis of kinase inhibitors with potent activity against RET. Herein, we have further investigated the effect of the lead compound SPP86 on RET mediated signaling and proliferation. Based on these observations, we hypothesized that SPP86 may be useful for studying the cellular activity of RET.

Methods

We compared the effects of SPP86 on RET-induced signaling and proliferation in thyroid cancer cell lines expressing RET-PTC1 (TPC1), or the activating mutations BRAFV600E (8505C) and RASG13R (C643). The effect of SPP86 on RET- induced phosphatidylinositide 3-kinases (PI3K)/Akt and MAPK pathway signaling and cell proliferation in MCF7 breast cancer cells was also investigated.

Results

SPP86 inhibited MAPK signaling and proliferation in RET/PTC1 expressing TPC1 but not 8505C or C643 cells. In TPC1 cells, the inhibition of RET phosphorylation required co-exposure to SPP86 and the focal adhesion kinase (FAK) inhibitor PF573228. In MCF7 cells, SPP86 inhibited RET- induced phosphatidylinositide 3-kinases (PI3K)/Akt and MAPK signaling and estrogen receptor? (ER?) phosphorylation, and inhibited proliferation to a similar degree as tamoxifen. Interestingly, SPP86 and PF573228 inhibited RET/PTC1 and GDNF- RET induced activation of Akt and MAPK signaling to a similar degree.

Conclusion

SPP86 selectively inhibits RET downstream signaling in RET/PTC1 but not BRAFV600E or RASG13R expressing cells, indicating that downstream kinases were not affected. SPP86 also inhibited RET signaling in MCF7 breast cancer cells. Additionally, RET- FAK crosstalk may play a key role in facilitating PTC1/RET and GDNF- RET induced activation of Akt and MAPK signaling in TPC1 and MCF7 cells.

SUBMITTER: Alao JP

PROVIDER: S-EPMC4252022 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Publications

Selective inhibition of RET mediated cell proliferation in vitro by the kinase inhibitor SPP86.

Alao John P JP Michlikova Sona S Dinér Peter P Grøtli Morten M Sunnerhagen Per P

BMC cancer 20141120

<h4>Background</h4>The RET tyrosine kinase receptor has emerged as a target in thyroid and endocrine resistant breast cancer. We previously reported the synthesis of kinase inhibitors with potent activity against RET. Herein, we have further investigated the effect of the lead compound SPP86 on RET mediated signaling and proliferation. Based on these observations, we hypothesized that SPP86 may be useful for studying the cellular activity of RET.<h4>Methods</h4>We compared the effects of SPP86 o ...[more]

PMID: 25409876

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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.

Project description:IntroductionThe structural and dynamic determinants that confer highly selective RET kinase inhibition are poorly understood.ObjectivesTo explore the druggability landscape of the RET active site in order to uncover structural and dynamic vulnerabilities that can be therapeutically exploited.MethodsWe apply an integrated structural, computational and biochemical approach in order to explore the druggability landscape of the RET active site.ResultsWe demonstrate that the that the druggability landscape of the RET active site is determined by the conformational setting of the ATP-binding (P-) loop and its coordination with the αC helix. Open and intermediate P-loop structures display additional druggable vulnerabilities within the active site that were not exploited by first generation RET inhibitors. We identify a cryptic pocket adjacent to the catalytic lysine formed by K758, L760, E768 and L772, that we name the post-lysine pocket, with higher druggability potential than the adenine-binding site and with important implications in the regulation of the phospho-tyrosine kinase activity. Crystal structure and simulation data show that the binding mode of highly-selective RET kinase inhibitors LOXO-292 and BLU-667 is controlled by a synchronous open P-loop and αC-in configuration that allows accessibility to the post-lysine pocket. Molecular dynamics simulations show that these inhibitors efficiently occupy the post-lysine pocket with high stability through the simulation time-scale (300 ns), with both inhibitors forming hydrophobic contacts further stabilized by pi-cation interactions with the catalytic K758. Engineered mutants targeting the post-lysine pocket impact on inhibitor binding and sensitivity, as well as RET tyrosine kinase activity.ConclusionsThe identification of the post-lysine pocket as a new druggable vulnerability in the RET kinase and its exploitation by second generation RET inhibitors have important implications for future drug design and the development of personalized therapies for patients with RET-driven cancers.

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:BackgroundRearranged during transfection (RET) gene fusions are a validated target in non-small-cell lung cancer (NSCLC). RET-selective inhibitors selpercatinib (LOXO-292) and pralsetinib (BLU-667) recently demonstrated favorable antitumor activity and safety profiles in advanced RET fusion-positive NSCLC, and both have received approval by the US Food and Drug Administration for this indication. Insights into mechanisms of resistance to selective RET inhibitors remain limited.Patients and methodsThis study was performed at five institutions. Tissue and/or cell-free DNA was obtained from patients with RET fusion-positive NSCLC after treatment with selpercatinib or pralsetinib and assessed by next-generation sequencing (NGS) or MET FISH.ResultsWe analyzed a total of 23 post-treatment tissue and/or plasma biopsies from 18 RET fusion-positive patients who received an RET-selective inhibitor (selpercatinib, n = 10; pralsetinib, n = 7; pralsetinib followed by selpercatinib, n = 1, with biopsy after each inhibitor). Three cases had paired tissue and plasma samples, of which one also had two serial resistant tissue specimens. The median progression-free survival on RET inhibitors was 6.3 months [95% confidence interval 3.6-10.8 months]. Acquired RET mutations were identified in two cases (10%), both affecting the RET G810 residue in the kinase solvent front. Three resistant cases (15%) harbored acquired MET amplification without concurrent RET resistance mutations, and one specimen had acquired KRAS amplification. No other canonical driver alterations were identified by NGS. Among 16 resistant tumor specimens, none had evidence of squamous or small-cell histologic transformation.ConclusionsRET solvent front mutations are a recurrent mechanism of RET inhibitor resistance, although they occurred at a relatively low frequency. The majority of resistance to selective RET inhibition may be driven by RET-independent resistance such as acquired MET or KRAS amplification. Next-generation RET inhibitors with potency against RET resistance mutations and combination strategies are needed to effectively overcome resistance in these patients.

Dataset Information

Selective inhibition of RET mediated cell proliferation in vitro by the kinase inhibitor SPP86.

Background

Methods

Results

Conclusion

Publications

Selective inhibition of RET mediated cell proliferation in vitro by the kinase inhibitor SPP86.

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