A secondary RET mutation in the activation loop conferring resistance to vandetanib

Abstract

Resistance to vandetanib, a type I RET kinase inhibitor, developed in a patient with metastatic lung adenocarcinoma harboring a CCDC6-RET fusion that initially exhibited a response to treatment. The resistant tumor acquired a secondary mutation resulting in a serine-to-phenylalanine substitution at codon 904 in the activation loop of the RET kinase domain. The S904F mutation confers resistance to vandetanib by increasing the ATP affinity and autophosphorylation activity of RET kinase. A reduced interaction with the drug is also observed in vitro for the S904F mutant by thermal shift assay. A crystal structure of the S904F mutant reveals a small hydrophobic core around F904 likely to enhance basal kinase activity by stabilizing an active conformer. Our findings indicate that missense mutations in the activation loop of the kinase domain are able to increase kinase activity and confer drug resistance through allosteric effects.

Fig. 1

Identification of a RET-S904F mutation conferring resistance to vandetanib. a Clinical course of the patient and axial chest computed tomographic (CT) scan. (Upper) The blue line indicates the serum CEA level, and the orange line indicates the size of the target lesion (the right metastatic cervical lymph node). The time points of the biopsy of metastatic lymph nodes are indicated by an arrowhead in Biopsy #1 and an arrow in Biopsy #2 (the details of the clinical course are shown in Supplementary Fig. 1). (Lower) CT scan images of the metastatic lymph node as a target lesion. b Sanger sequencing results of RT-PCR products from pretreatment specimens (Biopsy #1, pre) and specimens obtained at disease progression (Biopsy #2, pro). The same CCDC6-RET fusion transcript in which exon 1 of CCCDC6 is joined to exon 15 of RET was expressed. c Histological findings of hematoxylin/eosin-stained lymph node biopsy specimens obtained before treatment (Biopsy #1) and after disease progression (Biopsy #2). The identical pathological features are shown. d Sanger sequencing of genomic-PCR and RT-PCR products from peripheral blood, pretreatment specimens (pre), and specimens obtained at disease progression (pro). A mutation of cytosine to thymine at residue 2902 was detected only in the resistant tumor specimen. Genomic and RT-PCR analysis was performed using a primer in CCDC6-exon 1 and a reverse primer in RET-exon 15. The detection of the substitution, which causes an amino acid substitution of serine-to phenylalanine at codon 904 (in magenta), in genomic DNA and in the fusion transcript suggested that the mutation occurred on the rearranged RET allele in the resistant tumor

Fig. 2

Resistance to vandetanib by RET-S904F mutation. a Immunoblot analysis of the wild type and S904F mutant CCDC6-RET fusion proteins. (Left) An expression vector encoding full-length wild type or S904F mutant CCDC6-RET cDNA was transiently introduced into H1299 lung cancer cells. After exposure to the indicated concentrations of vandetanib for 6 h, phosphorylation at tyrosines 905 and 1015 were detected. The signal intensities, calculated as the ratio of phosphorylated to total CCDC6-RET fusion proteins, are shown at the bottom. (Right) The experiment (shown on the left) was performed separately three times (Supplementary Fig. 8). The graph shows the mean ratios of phospho-Ret (pTyr905) to total RET from three separate experiments with standard deviations (shown as error bars). Concentrations showing statistical significance (p < 0.05 by t-test) are marked with an asterisk. ACTB: beta-actin. b Cell growth assay using IL3-independent Ba/F3 cells carrying lentivirally transduced CCDC6-RET cDNA with or without the S904F mutation. Ba/F3 cells (2000 cells) were plated in quadruplicate in 96-well plates and treated with serially diluted vandetanib. After incubation for 72 h, cell viability was measured using the CellTiter-Glo luminescent reagent with EnVision and the viability curves with standard deviations (shown as error bars) were calculated with GraphPad Prism version 6.0. Reproducibility was confirmed by performing the same experiment three times. c In vitro kinase assays. Recombinant RET kinase domain (KD) (amino acids 658–1072) with or without the S904F mutation was expressed by baculovirus in Sf9 insect cells using an N-terminal GST tag (gene accession number: NM_020630). The assay was initiated by addition of ³²P-ATP to the reaction mixture and serially diluted vandetanib, and the IGF1Rtide synthetic peptide was added as the substrate (KKKSPGEYVNIEFG). After incubation at 30 °C for 20 min, the radioactivity was measured in a TriLux scintillation counter after transferring the products onto a phosphocellulose P81 plate. IC₅₀ values with 95% confidence interval were calculated using GraphPad Prism version 6.0. Error bars show standard deviations. Reproducibility was confirmed by performing the same experiment three times

Fig. 3

Kinetic and structural properties of the RET-S904F mutant. a Enzyme kinetics parameters of wild type and S904F mutant RET KD proteins. The kinase assay was performed in triplicate at 25 °C for different incubation times (0, 10, 20, 30, 40, and 50 min) using purified RET KDs, increasing concentrations of ATP (3.125–400 nM), and serially diluted vandetanib. The assay was initiated by addition of ³²P-ATP, and the reaction mixture was incubated at 30 °C. These experiments were performed independently three times. The data were analyzed using GraphPad Prism version 6.0 to calculate kinetic parameters, K_i, and IC₅₀ values. b Saturation curve graphed by Michaelis–Menten equation. The graph with standard deviations (shown as error bars) was generated using GraphPad Prism. c Western blotting showing the autophosphorylation time course in the wild type and S904F mutant RET KDs. Phosphorylation of the recombinant purified RET KDs treated with ATP (5 mM) and MgCl₂ (10 mM) for 0–80 min was detected with the indicated antibodies. d Comparison of S904F and wild-type RET structures. (Upper) Detail of side chain contacts close to the F904 mutation site of mutant RET kinase domain (PDB code 6FEK). Selected sidechains are labeled. Bound waters are shown as red spheres and hydrogen-bonds drawn in gray as defined by Pymol Molecular Graphics System (Schrödinger, LLC, New York, NY). (Middle) Detail of side chain contacts close to the S904 of wild-type RET kinase domain (PDB code 2IVT), colored as per panel (upper). (Lower) Overlay of the RET core kinase domain wild type and S904F mutant structures omitting bound waters for clarity

Fig. 4

Decreased thermal stability of the RET kinase-vandetanib complex induced by the S904F mutation. a A thermal shift assay was performed to determine the drug-induced changes in the melting temperature (∆T_m) of purified RET KD, which reflect the stability of the complex. Recombinant wild type or S904F mutant RET KD was generated using previously published methods. Each protein was dephosphorylated using CIP-phosphatase and then either used directly (unphosphorylated) or phosphorylated by addition of Mg-ATP, followed by incubation with DMSO or 1 µM vandetanib. Wild type and S904F mutant RET KDs without drug showed T_m values of 43.00 ± 0.06 °C and 44.16 ± 0.04 °C, respectively (Supplementary Table 1). Addition of vandetanib increased the ∆T_m of wild-type RET KD by 6.11 ± 0.19 °C, whereas it increased the ∆T_m of phosphorylated S904F RET KD only by 3.76 ± 0.17 °C. Unphosphorylated RET KDs showed little or no (∆T_m) increase irrespective of the mutation status (Supplementary Table 1). b  Geometry of the hydrogen-bond network consisting of E734, D771, and R912, which regulates the accessibility of ATP to the nucleotide-binding pocket and active site. The mean structures of E734, F735, R770, D771, S/F904, pY905, K907, R912, and vandetanib, generated by molecular dynamics simulations of 1 μs × 3 times, are represented by thick sticks (gray, carbon; blue, nitrogen; orange, phosphorus; red, oxygen; light blue, fluorine; and light pink, bromine). Mean geometry of the hydrogen-bond network formed by E734, D771, and R912 is depicted by dashed green lines, showing the formation of an E734-R912 hydrogen bond at an irregular position in the S904F mutant, and the intermediate conformer is likely to be stabilized by this aberrant hydrogen-bond network