JP2023177142A - Method for determining effectiveness of cancer treatment using ret kinase inhibitor - Google Patents

Abstract

To provide a method for determining effectiveness of a cancer treatment using a RET kinase inhibitor.SOLUTION: A method for determining effectiveness of a cancer treatment using a RET kinase inhibitor, comprises: a step for detecting, in a sample isolated from a subject, an amino acid mutation in a calmodulin-like motif of a RET protein; and a step for, if the amino acid mutation is detected in the previous step, determining that the cancer treatment using the RET kinase inhibitor is highly effective for the subject.SELECTED DRAWING: None

Description

The present invention relates to a method for determining the effectiveness of cancer treatment using a RET kinase inhibitor. Furthermore, the present invention relates to a reagent for use in the determination method. The present invention also relates to a RET kinase inhibitor that is administered to a patient based on the results of the determination method.

Identification of cancer gene mutations that drive tumorigenesis is important because these mutations serve as therapeutic targets for inhibitors. To date, several protein kinase inhibitors have improved the prognosis of patients with activating mutations in oncogenic kinase genes. Hotspot mutations affecting single or small amino acid residues associated with human cancer have been intensively studied as oncogenic mutations and therapeutic target mutations due to their cell-transforming activity and drug sensitivity. Ta. This includes mutations in the BRAF (V600E), KRAS (G12C), EGFR (L858R), and RET (M918T) genes (Non-patent Documents 1 and 2).

In particular, hotspot mutations in the RET gene in thyroid cancer have been used as drug discovery targets (Non-Patent Documents 3 and 4), and recently developed RET-specific tyrosine kinase inhibitors (TKIs) are It has shown remarkable therapeutic effects on cancer patients with oncogenic RET mutations (Non-patent Documents 1 and 2). Furthermore, regarding the RET gene, the molecular mechanism of carcinogenicity brought about by hot spot mutations has been elucidated. Specifically, it has been revealed that hot spot mutations in the kinase domain of RET improve kinase activity by enhancing enzyme activity and substrate presentation for autophosphorylation (Non-Patent Document 4). In addition, hotspot mutations in the core cysteine motif (CCM) of RET generate unpaired cysteine residues that form intermolecular disulfide bonds, constituting RET dimerization and enzyme activation. It has also been revealed that this can cause severe symptoms (Non-patent Documents 3 and 5).

On the other hand, genome analysis of tens of thousands of cancer cases using next-generation sequencing has revealed that many cancer genomes contain "non-clustered" mutations, even in well-known cancer genes. It has also been found that there are (non-patent documents 6 and 7). Such mutations, called genetic mutations of unknown clinical significance (VUS), occur infrequently and are therefore unlikely to serve as therapeutic targets, and their role in tumorigenesis and drug sensitivity remains unclear. , has not been extensively studied so far. In cancer precision medicine, predicting such carcinogenic effects of VUS is an urgent task because it is effective for selecting cases for which molecular target therapy using existing inhibitors is effective. In fact, since there are a huge number of VUSs, it is speculated that their functional annotations can be quickly performed using in silico methods such as AlphaFold2 analysis (Non-Patent Document 8), which predicts protein structures from amino acid sequences. There is. However, there is still a gap between structural prediction and functional annotation, making it impossible to distinguish between functionally important "driver" mutations and biologically neutral "passenger" mutations (Non-patent Document 9). ).

In this way, even in the RET gene, where the molecular mechanism of oncogenicity brought about by hotspot mutations has been elucidated, it is still difficult to further identify mutations that are oncogenic or therapeutic targets among non-clustered mutations. If possible, it is expected that the number of cancer patients for whom RET kinase inhibitor treatment is effective will increase. However, such mutations had not yet been identified.

Wirth LJ. et al., N Engl J Med. , 2020, 383: pages 825-835. Subbiah V. et al., The Lancet Diabetes & Endocrinology, 2021, 9:491-501. Mulligan LM. , Nat Rev Cancer, 2014, 14:173-186. Plaza-Menacho I. et al., Mol Cell, 2014, 53:738-751. Romei C. et al., Nat Rev Endocrinol. , 2016, 12: 192-202 pages. Ikemura S. et al., Proceedings of the National Academy of Sciences of the United States of America, 2019, 116:10025-10030. Foley SB. et al., EBioMedicine, 2015; 2: 74-81 pages. Jumper J. et al., Nature, 2021, 596:583-589. Mulligan LM. Nat Rev Cancer 2014 14:173-186 Li J. et al., “Cryo-EM analyzes reveal the common mechanism and diversification in the activation of RET by different ligands.”, Elife , 2019;8

The present invention has been made in view of the above-mentioned problems of the prior art, and it identifies mutations that are carcinogenic or therapeutic target among non-cluster mutations in the RET gene, and uses the mutations as an indicator. The present invention aims to provide a method for determining the effectiveness of cancer treatment using a RET kinase inhibitor.

In order to achieve the above objective, the present inventors first conducted an in silico-driven in vitro functional analysis to identify oncogenic mutations and therapeutic target mutations from among a large number of VUS of the RET gene, which is a therapeutic target. I tried to identify it. More specifically, we performed in silico genome-wide modeling on 71,756 variants in different human cancers to identify mutations in known RET oncogenes above local background levels of 3- or 5-base substitutions. We identified a novel nucleotide motif that induces This group of mutations occurs in the calmodulin-like motif (CaLM, Non-Patent Document 10), which was recently identified by cryo-electron microscopy (EM) analysis of the RET extracellular domain (ECD), and forms a three-dimensional amino acid substitution cluster. Ta. Furthermore, molecular dynamics (MD) simulations using the high-performance supercomputer "Fugaku" showed that mutations in CaLM destabilize the bond between RET and calcium ion complexes. In addition, comprehensive functional analysis (in vitro cell transformation tests, signal transduction assays, etc.) of 110 representative RET mutants revealed that amino acid substitutions in ECD mutants are almost neutral, whereas CaLM mutations was revealed to be a new activating mutation cluster. Furthermore, we found that the CaLM mutation causes homodimerization of RET kinase in a ligand-independent manner through the formation of disulfide bonds, which occurs only under artificial Ca ²⁺ -free conditions in wild-type RET, and completed the present invention. It's arrived.

That is, the present invention provides the following aspects.

[1] A method for determining the effectiveness of cancer treatment with a RET kinase inhibitor, comprising:
Detecting amino acid mutations in the calmodulin-like motif of the RET protein in the sample isolated from the subject;
If the amino acid mutation is detected in the step, determining that the effectiveness of cancer treatment with the RET kinase inhibitor in the subject is high.

[2] The amino acid mutation is such that in the RET protein, at least one amino acid selected from aspartic acid at position 567, glycine at position 568, aspartic acid at position 571, glutamic acid at position 574, and aspartic acid at position 584 is The method according to [1], wherein the mutation is an amino acid substitution.

[3] A reagent for use in the method described in [1] or [2], which contains the oligonucleotide described in (i) or (ii) below, or the antibody described in (iii). ) a pair of oligonucleotides designed to sandwich the nucleotides encoding said amino acid mutation; (ii) an oligonucleotide that hybridizes to the nucleotides encoding said amino acid mutation; and (iii) specifically recognizing said amino acid mutation. antibody.

[4] A RET kinase inhibitor, characterized in that it is ingested by the subject who has been determined to be highly effective in cancer treatment by the method described in [1] or [2].

According to the present invention, by detecting amino acid mutations in the calmodulin-like motif of the RET protein, it is possible to predict the effectiveness of cancer treatment with a RET kinase inhibitor. This makes it possible to avoid administering the drug to cancer patients for whom administration of the drug is considered ineffective, thereby making it possible to achieve efficient cancer treatment.

FIG. 3 shows a mutation model for positive selection. The upper graph shows the number of occurrences of 1,290 RET mutations (circles) and a smoothed representation of the mutation distribution (line). The bar graph below shows the mutation clusters determined by OncodriveCLUSTL along with p-values and domain/motif distributions. CLD, cadherin-like domain; CRD, cysteine-rich domain; TM, transmembrane domain; KD, kinase domain; CaLM, calmodulin-like motif; CCM, core cysteine motif. The pie chart shows the ratio of cancer types and OncoKB significance (Reference 19) for CaLM and CCM mutation clusters. Note that the literature number shown in parentheses (19 here) corresponds to the literature listed in each number shown in <References> below (the same applies hereinafter). It is a bubble plot showing the ability of RET mutations to transform NIH3T3 cells and ERK phosphorylation activity. The horizontal axis shows amino acid residue numbers, and the vertical axis shows ERK phosphorylation compared to wild-type RET in log10 fold change. The size of the plot for each mutation indicates the strength of focus formation in three levels. The color of each plot indicates the grade annotated with OncoKB (Reference 19). Under the color display, blue indicates carcinogenicity, light blue indicates high possibility of carcinogenicity, gray indicates significance is unknown, purple indicates unknown, and green indicates high probability of neutrality. Representative images of Giemsa-stained NIH3T3 cells are shown in the graph area. It is a photograph showing the results of ERK phosphorylation assay. Immunoblot analysis of phosphorylated ERK (pERK), ERK, and RET was performed using HEK293H cells transfected with empty vector or expression plasmids for wild-type RET, 110 RET mutants, and a kinase-dead mutant (K758M). went. Relative pERK levels normalized to ERK are plotted in Figure 1B. FIG. 2 is a diagram showing an overview of a method for selecting RET mutants. The exploration cohort consisted of 71,756 mutation data from 79,720 tumors excluding changes such as fusions and copy number changes, and Oncodrive CLUSTL analysis was performed to output the RET gene modeling results. Among the 860 missense/in-frame insertion deletion RET mutations detected in GENIE samples, 6 CaLM, 25 known activating candidate mutations (References 5, 6, 9, 10, 12, 13), and GENIE Gene-wide cell-based assays were performed on 79 other RET variants observed in three or more cases. FIG. 3 is a diagram showing Oncodrive CLUSTL analysis. A total of 1,290 RET variants obtained from the GENIE cohort were subjected to trinucleotide enrichment analysis using a background mutation rate model based on mutant trinucleotide contexts generated from TCGA (v0.2.8) samples. (Left figure). The TCGA dataset was used as a background model (right panel) because it contains a similar proportion of diverse types of mutations as the GENIE dataset (version 7.0). The table within the graph shows the number of samples, number of mutations, and number of genes for the two datasets. Figure 2 is a pie chart showing RET mutations in ECD. The pie chart shows the proportion of cancer types and the annotation in OncoKB (Reference 22) regarding ECD mutations. Figure 2 is a pie chart showing RET mutations in ICD. The pie chart shows the proportion of cancer types and the annotation in OncoKB (Reference 22) regarding ICD mutations. Figure 2 is a pie chart showing single and combined mutations in CaLM and CCM segments. Both are mostly detected as single mutations in each tumor. FIG. 3 is a three-dimensional diagram showing the structure of CaLM. An overhead view of the extracellular portion of the RET-GDNF-GFRα1 hexamer (PDB: 6Q2N) and an enlarged view of CaLM are shown. The enlarged view illustrates the position of Ca ²⁺ ions (under color display, green sphere) and the amino acid residues that contribute to Ca ²⁺ ion retention. Mutated residues are highlighted in red. In the overhead view, orange: cadherin-like domain (CLD), gray: cysteine-rich domain (CRD), light blue: glial cell-derived neurotrophic factor (GDNF), pink: GDNF family receptor α1 (GFRα1). FIG. 3 is a three-dimensional diagram showing the structure of calmodulin. Represents the Ca ²⁺ binding motif of calmodulin (PDB code: 2BE6). Under the color display, mutated residues found in long QT syndrome patients are highlighted in red. FIG. 3 is a violin plot of binding free energies calculated at 10 nanosecond intervals between Ca ²⁺ ions and RET-CRD monomers. FIG. The width of each strip represents the percentage of time points exhibiting binding free energy values. The black dashed line represents the median value. Each dotted line represents the end of the first quartile and the beginning of the fourth quartile. *: P value <0.05; ****: P value < 0.0001. P values were determined by one-way ANOVA using Tukey's multiple comparison test. FIG. 3 is a violin plot of the RMSD of Cα atoms in CRD monomers. The RMSD of the heavy atom of each amino acid was calculated and plotted with respect to the initial position at 100 picosecond intervals. The left shows the RMSD of wild type (WT) and clinically observed CaLM mutants. The right side shows the results for the artificial alanine mutant. It is a photograph showing the results of analyzing the cancerous ability of artificial CaLM mutants. Left: ERK phosphorylation assay. Immunoblot analysis of pERK, ERK, RET, and β-actin was performed using HEK293H cells transfected with empty vector or WT RET, 110 RET mutants, and kinase dead mutant (K758M) expression plasmids. Right: NIH3T3 focus formation assay. Characterization of NIH3T3 cells using lentiviruses expressing parental cells (uninfected), empty virus infection, D567Y (CaLM mutant), C634R (CCM mutant), M918T (kinase domain mutant) and artificial CaLM mutant proteins. Conversion ability was evaluated. 1 is a graph showing binding free energies calculated every 10 nanoseconds in a 1 microsecond simulation. The three lines represent the binding of wild-type (lower color display, black), D567Y (lower color display, red), and D567N (lower color display, blue) RET-CRD to Ca ²⁺ ions. It shows the change in free energy over time in each time frame of 10 nanoseconds. Amino acid residues of Ca ²⁺ and CaLM in wild type (lower color display, black line), D567Y (lower color display, red line), and D567N (lower color display, blue line) during CRD monomer simulations. Figure 2 is a radar plot showing the average distance between bases. The distance between the carboxyl carbon atom of the negatively charged side chain of the amino acid that contributes to the coordination bond or the oxygen atom of the main chain and the Ca ²⁺ ion was calculated. Calculations for amino acid residue No. 567 were performed for aspartic acid (wild type), asparagine (D567N), and tyrosine (D567Y). The distance from the Cγ atom for the D567N mutant and from the aromatic ring center of the side chain to the Ca ²⁺ ion for the D567Y mutant was calculated. Each number represents the average distance (Å) calculated every 100 ps during the MD simulation time. It is a violin plot diagram showing the distance between Ca ²⁺ ions and amino acid residues (T564, C565, D567, H569, E574, D584) that contribute to coordinate bond formation calculated every 100 picoseconds in MD simulation. The width of each plot is proportional to the frequency of the relative minimum distance. The black dashed line represents the median minimum distance. Each dotted line represents the end of the first quartile and the beginning of the fourth quartile. Asterisks indicate significantly different distributions. n. s. : No significant difference, ***: P<0.0001. P values were determined by one-way ANOVA using Tukey's multiple comparison test. RMSD values for the initial coordinate positions of Ca ²⁺ ions in the wild type and the D567 mutant. In the left panel, the three lines represent wild type (lower color display, black), D567Y (lower color display, red), and D567N (lower color display, blue) RET in each time frame of 100 ps. - Shows the change in CRD over time. The figure on the right is a violin plot showing the RMSD values calculated for each 100 ps time frame. The black dashed line indicates the median RMSD. Each dotted line marks the end of the first quartile and the beginning of the fourth quartile. Asterisks indicate significantly different distributions. n. s. : No significant difference, ***: P<0.0001. P values were determined by one-way ANOVA using Tukey's multiple comparison test. Ca ²⁺ ions (x-axis) and CRD during MD simulation of RET wild type (lower color display, black dots), D567Y (lower color display, red dots), and D567N (lower color display, blue dots) FIG. 2 is a scatter plot of the RMSD of the heavy atoms (y-axis) of each residue of the monomer. Each spot shows the RMSD value calculated every 100 ps during the MD simulation time. This is the RMSD value calculated for the heavy atom of each residue based on the initial coordinate position of wild-type RET and the D567 mutant. The color coding is the same as in FIG. 4D. Interaction energy between Ca ²⁺ ions and CRD calculated from MD simulation of RET-GDNF-GFRα1 extracellular complex (left), RMSD of CRD (middle), cysteine residues in CRD (C565, C570, C581, C585) ) is a violin plot showing the SASA (right). MD simulations were performed three times each for the wild type (WT) and the RET mutant, and structures extracted every 20 picoseconds from a 0.5-1 microsecond trajectory were used as targets for calculation. The black dashed line represents the median value. Each dotted line represents the end of the first quartile and the beginning of the fourth quartile. n. s. : No significant difference, ***: P value <0.0001. P values were determined by one-way ANOVA using Tukey's multiple comparison test. FIG. 2 is a schematic diagram of RET protein. CLD: cadherin-like domain, CRD: cysteine-rich domain, KD: kinase domain, (under color display) green circle: Ca ²⁺ ion binding site, dashed line: cell membrane. 1 is a photograph showing the results of an intermolecular RET dimer formation assay. Flp-In T-Rex 293 cells transfected with/without doxycycline-inducible FLAG-tagged full-length RET cDNA were cultured with doxycycline in calcium-containing (left) or calcium-free (right) medium for 48 hours. Whole cell lysates were prepared under reducing or non-reducing conditions, ie, in the presence or absence of dithiothreitol, separated by SDS-PAGE, and subjected to immunoblot analysis using an anti-FLAG antibody. This is a photograph showing the results of analysis of intermolecular dimer formation in CLD4-CRD protein. GST-tagged RET CRD-CLD4 polypeptide expressed in Sf21 cells was purified under reducing or non-reducing conditions, i.e., in the presence or absence of 2-mercaptoethanol, separated by SDS-PAGE, and purified using an anti-GST antibody. Immunoblot analysis was performed. The left panel shows intermolecular dimerization of WT, C634R, D567N mutant polypeptides. Right shows intermolecular dimerization of purified WT RET polypeptide under calcium chelating conditions, ie, in the presence or absence of EDTA. 2 is a photograph showing the results of analysis of RET autophosphorylation and downstream ERK phosphorylation induced by calcium deficiency. HEK293H cells transfected with WT or mutant full-length RET cDNA were cultured in calcium-containing or calcium-free medium for 48 hours. The RET CaLM mutant was validated along with the K758M (kinase dead) and C634R (CCM) mutants. Protein lysates were subjected to immunoblot analysis for pRET, RET, pERK, and ERK. Interaction energy between RET-ECD and GDNF/GFRα1 (left) or RET-CRD and GDNF/GFRα1 (middle) calculated from MD simulation of RET-GDNF/GFRα1 extracellular complex and contact between CRD and GDNF/GFRα1 Violin plot showing area (right). The black dashed line represents the median value. Each dotted line represents the end of the first quartile and the beginning of the fourth quartile. n. s. : No significant difference, ***: P value <0.0001. P values were determined by one-way ANOVA using Tukey's multiple comparison test. FIG. 3 shows an increase in the contact area between RET-CRD and ligand/co-receptor due to the D567N mutation. The average structure of a WT (left) or D567N (right) RET subunit bound to two GDNF and one GFRα1 molecule is 0.5-1 microns obtained from each of three MD simulations of the RET extracellular complex. Calculated using trajectories for seconds and shown as a protein surface structure model. The above figure shows the CRD binding interface of two GDNF and GFRα1 molecules, and amino acid residues within 5 Å from the CRD domain are highlighted in red under the color display. An enlarged view of the CRD-GFRα1 binding interface is shown below. It is a photograph showing the results of ERK phosphorylation assay. Empty vector or expression plasmids of WT RET, K758M (kinase dead), C634R (CCM), D567N, and D567Y RET mutants were transfected and treated with GDNF/GFRa1 as described. HEK293H cell lysates were subjected to immunoblock analysis of pERK, ERK, RET and β-actin. FIG. 3 is a violin plot showing the RMSD of CRD residues located within 5 Å from Ca ²⁺ ions calculated from MD simulations of the RET-GDNF-GFRα1 extracellular complex. MD simulations were performed three times for the wild type (WT) or each RET mutant, and structures extracted every 20 picoseconds from 0.5-1 microsecond trajectories were used as targets for calculation. The black dashed line represents the median value. Each dotted line represents the end of the first quartile and the beginning of the fourth quartile. ***: P<0.0001. P values were determined by one-way ANOVA using Tukey's multiple comparison test. This is a photograph showing the results of CBB staining analysis of purified GST-tagged RET CLD4-CRD proteins of WT or D567N and C634R mutants expressed in Sf21 cells. It was carried out under reducing or non-reducing conditions, ie, in the presence or absence of 2-ME. This is a photograph showing the results of CBB staining analysis of purified GST-tagged RET CLD4-CRD proteins of D567N, D567Y, and D567A mutants expressed in Sf21 cells. It was carried out under reducing or non-reducing conditions in the presence of calcium, ie, in the presence or absence of 2-ME. 1 is a graph showing the results of a NanoBiT assay for investigating intracellular protein-protein interactions. U2OS cells were transfected using a plasmid expressing LgBiT or SmBiT fused to the RET-ECD C terminus. Twenty-four hours after transfection, cells were incubated with Nano-Glo Live Cell substrate with or without GDNF and GFRα1 at a final concentration of 7.4 μg/mL. The left panel shows RET dimerization evaluated by the increase in luminescence after adding GDNF and GFRα1 for 44 minutes. The solid line and the dotted line indicate the measured values of the WT, D567Y, D567N, and C634R mutants with and without the R77E/R144E mutation, respectively. Points represent mean±SD. The right figure shows the results of comparing the relative maximum increase in luminescence of WT without the R77E/R144E mutation and mutant RET with that with the R77E/R144E mutation. Columns represent mean±SD. It is a photograph showing the results of analyzing the transformation ability of CaLM mutants. The left figure shows NIH3T3 focus formation. The transformation ability when using a lentivirus expressing the CaLM mutant was examined in comparison with empty virus infection and using a lentivirus expressing the C634R (CCM) mutant. Transformation ability was evaluated in the absence and presence of GDNF/GFRα1 (1.0 or 2.5 ng/mL). The figure on the right shows the transformation ability of each mutant graphically. The focus area was calculated using the ImageJ program using the value at the time of empty virus infection as the background. Each value represents the mean ± SD of three independent experiments. It is a photograph showing the results of analysis of suppression of signal transduction by a RET inhibitor. HEK293H cells expressing RET CaLM and C634R (CCM) mutants were treated with selpercatinib (top) or pralsetinib (bottom) for 6 hours, and cell lysates were subjected to immunoblot analysis for pERK, ERK, RET, and β-actin. did. FIG. 3 is a diagram showing the results of analysis of suppression of RET-dependent cell proliferation by a RET inhibitor. IL-3-independent proliferation of Ba/F3 cells expressing RET CaLM, CCM, M918T mutants was verified by treatment with the indicated concentrations of selpercatinib (left) or pralsetinib (right) for 72 hours. Relative survival rate (%) is shown in color. 1 is a graph showing the results of analysis of suppression of allograft tumor growth of RET mutant-expressing NIH3T3 cells by a RET inhibitor. Drug treatment was started when the subcutaneous tumor had grown to a volume of approximately 100-200 mm ³ . All variants received vehicle or 3 mg/kg selpercatinib or pralsetinib twice daily. Each treatment group includes 4 mice. Data are mean±S. D. Indicated by *, P value <0.05; **, P value <0.01; ****, P value <0.001; ****, P value <0.0001. P values were determined by one-way ANOVA using Tukey's multiple comparison test. 1 is a photograph showing the results of analysis of suppression of allograft tumor growth of RET mutant-expressing NIH3T3 cells by a RET inhibitor. 1 is a graph showing the results of analysis of suppression of allograft tumor growth of RET mutant-expressing NIH3T3 cells by a RET inhibitor. FIG. 3 shows that the carcinogenic/tumorigenic activity of RET-CaLM mutants is suppressed by RET-TKI. In "a", GI50 values of selpercatinib and pralsetinib against Ba/F3 cells expressing RET mutants are shown. Data represent the average of 6 replicates. GI50, 50% growth inhibitory concentration. "b" shows the results of tumor observation after treating mice with allografted tumors of RET mutant-expressing NIH3T3 cells with vehicle (top), selpercatinib (middle), or pralsetinib (bottom). D567Y (left) and C634R (right) mutant tumors were excised from mice after the end of treatment.

As shown in the Examples below, among the many VUS of the oncogene RET gene, multiple amino acid substitutions that are oncogenic mutations or therapeutic target mutations are present in the calmodulin-like motif. I discovered that. Therefore, the present invention provides a method for determining the effectiveness of cancer treatment using a RET kinase inhibitor, which comprises the steps of: detecting amino acid mutations in the calmodulin-like motif of the RET protein in a sample isolated from a subject; If the amino acid mutation is detected in the step, the present invention relates to a method including the step of determining that the effectiveness of cancer treatment using a RET kinase inhibitor in the subject is high.

<RET protein etc.>
The "RET (REARRANGED DURING TRANSFECTION) protein" according to the present invention is also referred to as RET tyrosine kinase protein or RET receptor tyrosine kinase protein, and is a protein that is expressed in a gene located at 10q11.2 in humans. It is an encoded protein. In the present invention, "RET protein" typically refers to a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 (RET51, RET isoform a) and a protein set forth in SEQ ID NO: 4, if it is of human origin. (RET9, RET isoform c) consisting of the amino acid sequence. Further, the polynucleotide encoding the protein typically consists of a polynucleotide (RET transcript variant 2) consisting of the base sequence set forth in SEQ ID NO: 1 and a base sequence set forth in SEQ ID NO: 3, respectively. polynucleotide (RET transcript variant 4). The protein (RET51) consisting of the amino acid sequence set forth in SEQ ID NO: 2 and the protein (RET9) consisting of the amino acid sequence set forth in SEQ ID NO: 4 have the same amino acid sequence from methionine at position 1 to glycine at position 1063. They are identical, but differ in the sequence and length of the C-terminal portion.

In addition, as shown in Figure 1A, the RET protein contains, in order from the N-terminus, cadherin-like domain (CLD) 1 (positions 29 to 154), CLD2 (positions 172 to 261), CLD3 (positions 265 to 379), and CLD4 ( extracellular domain (ECD, positions 1 to 635), transmembrane domain (TM, positions 636 to 657), and kinase domain (KD, positions 636 to 657) , 723-1012)) (ICD, amino acids from position 666 onwards to the C-terminus). Furthermore, CRD has a calmodulin-like motif (CaLM, positions 560-586) and a core cysteine motif (CCM, positions 629-643).

The present invention targets amino acid mutations in CaLM, and as shown in the Examples below, mutations that reduce Ca ²⁺ ion binding, mutations that form disulfide bonds even in the presence of Ca ²⁺ ions, and mutations that reduce Ca ²⁺ ion presence. The mutation may be at least one of the following: a mutation that induces homodimerization even in the presence of Ca ²⁺ ions, and a mutation that constitutively activates RET kinase even in the presence of Ca 2+ ions.

More specifically, the amino acid mutation according to the present invention is a mutation in which one or more amino acids are substituted, deleted, added, and/or inserted in the amino acid sequence of CaLM. Here, "plurality" means within 27 amino acids (for example, within 26 amino acids, within 25 amino acids, within 24 amino acids, within 23 amino acids, within 22 amino acids, within 21 amino acids), preferably within 20 amino acids (for example, within 19 amino acids, within 18 amino acids, within 17 amino acids, within 16 amino acids), more preferably within 15 amino acids (for example, within 14 amino acids, within 13 amino acids, within 12 amino acids, within 11 amino acids), and even more preferably within 10 amino acids (for example, within 9 amino acids). within 8 amino acids, within 7 amino acids, within 6 amino acids), more preferably within 5 amino acids (for example, within 4 amino acids, within 3 amino acids, within 2 amino acids).

Furthermore, as described above, the "amino acid sequence of CaLM" into which amino acid mutations are introduced is usually an amino acid sequence consisting of positions 560 to 586, preferably an amino acid sequence consisting of positions 567 to 584, and more preferably an amino acid sequence consisting of positions 567 to 584. , aspartic acid at position 567, glycine at position 568, aspartic acid at position 571, glutamic acid at position 574, and aspartic acid at position 584.

Furthermore, the type of mutation introduced is preferably a substitution with another amino acid. Here, the term "other amino acid" refers to an amino acid different from the wild-type amino acid at each position, and the other amino acid substituted for aspartic acid at position 567 is preferably tyrosine or asparagine. The other amino acid substituted for glycine at position 568 is preferably serine. The other amino acid substituted for aspartic acid at position 571 is preferably asparagine. The other amino acid substituted for glutamic acid at position 574 is preferably aspartic acid. The other amino acid substituted for aspartic acid at position 584 is preferably asparagine.

<Detection of amino acid mutations in RET protein>
In the present invention, the aforementioned amino acid mutations are detected in a sample isolated from a subject.

In the present invention, the "subject" who is the subject of determining the effectiveness of cancer treatment is usually a human being suffering from cancer (cancer patient), but there is a possibility that the cancer will recur. The subject may be a human being, or a human being at risk of developing cancer.

The "sample" isolated from such a subject may be a biological sample (e.g., cells, tissues, organs, body fluids (blood, lymph, etc.), digestive fluids, sputum, alveolar/bronchial lavage fluid, urine, stool). The sample may contain cancer cells or may not contain cancer cells. Furthermore, samples according to the present invention include nucleic acid extracts (genomic DNA extracts, mRNA extracts, cDNA preparations and cRNA preparations prepared from mRNA extracts, etc.) and protein extracts obtained from these biological samples. Also included. Further, the sample may be one that has been subjected to formalin fixation, alcohol fixation, freezing, or paraffin embedding. In addition, those skilled in the art can select and prepare known methods suitable for the type and condition of the sample, etc., by those skilled in the art.

Furthermore, detection of amino acid mutations in such samples can be carried out by those skilled in the art using known detection methods. Such an amino acid mutation can be detected by isolating DNA encoding the mutation site (hereinafter also simply referred to as "DNA mutation site") and determining the sequence of the isolated DNA. The DNA can be isolated, for example, by PCR using genomic DNA as a template, using a pair of oligonucleotides designed to sandwich the DNA encoding the mutation site. The isolated DNA sequence can be determined by methods known to those skilled in the art, such as the Maxam-Gilbert method and the Sanger method. Furthermore, the sequence can also be determined by subjecting it to Next Generation Sequencing (NGS). Then, based on the sequence information determined in this way, it becomes possible to detect the amino acid mutations according to the present invention.

There are no particular restrictions on next-generation sequencing methods, but synthetic sequencing methods (sequencing-by-synthesis, for example, sequencing using Illumina's Solexa genome analyzer, Hiseq or Miseq), pyrosequencing methods (for example, Roche Sequencing using the sequencer GSLX or FLX manufactured by Diagnostics (454) (so-called 454 sequencing), ligase reaction sequencing method (for example, sequencing using SoliD or 5500xl manufactured by Life Technologies), and the like.

Furthermore, amino acid mutations can be detected without determining the DNA sequence as described above. Such methods include the PCR-SSP (PCR-sequence specific primer) method. When detecting an amino acid mutation according to the present invention by the PCR-SSP method, the 3' end of one of the pair of oligonucleotides constituting the primer is complementary to a specific base type at the DNA mutation site. Designed to be a base species. PCR using a pair of oligonucleotides designed in this way can only be amplified when genomic DNA having a specific base type at the site is used as a template, and when the site is a different base type. If a certain genomic DNA is used as a template, it will not be amplified. Therefore, by using such a pair of oligonucleotides, the amino acid mutation according to the present invention can be detected.

Yet another method for detecting amino acid mutations includes the PCR-SSCP (PCR-single strand conformation polymorphism) method. That is, double-stranded DNA amplified by PCR using a pair of oligonucleotides designed to sandwich a DNA mutation site is denatured by treatment with heat, alkali, etc. to become single-stranded DNA, and then When subjected to polyacrylamide gel electrophoresis that does not contain a denaturing agent, single-stranded DNA is folded in the gel due to intramolecular interactions, forming a higher-order structure. Since the interaction of the folded structure changes depending on the base type, the separated single-stranded DNA is detected by silver staining or radioisotope, and the mobility of the single-stranded DNA on the gel is measured. Amino acid mutations according to the present invention can be detected by comparison with a control.

Another method for detecting amino acid mutations according to the present invention includes a method using an intercalator. In this method, first, genomic DNA is prepared from a sample. Next, a region containing the DNA mutation site is amplified using the genomic DNA as a template in a reaction system containing an intercalator that emits fluorescence when inserted between DNA double strands. Then, the temperature of the reaction system is changed, the fluctuation in the intensity of the fluorescence emitted by the intercalator is detected, and the fluctuation in the intensity of the fluorescence accompanying the detected temperature change is compared with a control. Such a method includes a high resolution melting (HRM) method.

Still another method for detecting amino acid mutations according to the present invention includes a method using an oligonucleotide probe that hybridizes to a region containing a DNA mutation site. In this method, first, genomic DNA is prepared from a sample. On the other hand, an oligonucleotide probe that specifically hybridizes to a region containing a DNA mutation site and is labeled with a reporter fluorescent dye and a quencher fluorescent dye is prepared. Then, the oligonucleotide probe is hybridized to the genomic DNA, and the genomic DNA hybridized with the oligonucleotide probe is used as a template to amplify the DNA containing the polynucleotide. Then, fluorescence emitted by the reporter fluorescent dye is detected due to decomposition of the oligonucleotide probe accompanying the amplification. Such a method includes a double die probe method, so-called TaqMan (registered trademark) probe method.

Furthermore, the present invention is not limited to the above method. For example, RFLP (Restriction Fragment Length Polymorphism), CAPS method (PCR-RFLP method), denaturing gradient gel electrophoresis method (DGGE method), Invader method, single nucleotide primer extension ( Other known techniques for detecting amino acid mutations (DNA mutations) are also utilized in the present invention, such as the SNuPE method, allele-specific oligonucleotide (ASO) hybridization method, ribonuclease A mismatch cleavage method, and DNA array method. obtain.

Note that the above-mentioned method does not target only genomic DNA, but may also target its transcription product (cDAN). Furthermore, when the sample is a transcription product (mRNA, cDNA), in addition to the above-mentioned detection methods, RT-PCR, direct sequencing, Northern blotting, dot blotting, cDNA microarray analysis, etc. can also be used.

In addition, in the present invention, when the sample is a translation product (RET protein), methods for detecting amino acid mutations thereof include, for example, immunostaining, Western blotting, ELISA, flow cytometry, and immunoprecipitation. methods and antibody array analysis. In these immunological detection methods, an antibody that specifically recognizes the amino acid mutation according to the present invention (an antibody against RET protein whose epitope is a region containing the amino acid mutation site according to the present invention) is used.

Such antibodies against RET protein can be prepared by those skilled in the art by appropriately selecting known techniques. Such a known method involves inoculating an immunized animal with a polypeptide consisting of a region containing an amino acid mutation site according to the present invention, activating the animal's immune system, and then injecting the animal's serum (polyclonal antibody) into the immunized animal. Examples include recovery methods, and monoclonal antibody production methods such as hybridoma methods, recombinant DNA methods, and phage display methods.

Further, in detecting translation products, if an antibody to which a labeling substance is bound is used, it is possible to directly detect the RET protein by detecting the label. The labeling substance is not particularly limited as long as it can bind to the antibody and can be detected, such as peroxidase, β-D-galactosidase, microperoxidase, horseradish peroxidase (HRP), fluorescein Examples include thiocyanate (FITC), rhodamine isothiocyanate (RITC), alkaline phosphatase, biotin and radioactive substances. Furthermore, in addition to the method of directly detecting the RET protein using an antibody bound to a labeling substance, there is also a method of indirectly detecting the RET protein using a secondary antibody, protein G, protein A, etc. bound to a labeling substance. You can also use

<Determination of effectiveness of cancer treatment>
As shown in the Examples below, the amino acid mutations according to the present invention are thought to cause activation of RET protein and contribute to malignant progression of cancer. On the other hand, RET kinase inhibitors such as selpercatinib and pralsetinib are highly effective against cancers having such mutations. Therefore, there is a high probability that treatment with a RET kinase inhibitor will be effective in subjects in whom such amino acid mutations according to the present invention are detected.

In the present invention, the "RET kinase inhibitor" to be evaluated for the effectiveness of cancer treatment is not particularly limited as long as it is a substance that can directly or indirectly inhibit the function of RET protein. Known RET kinase inhibitors that can be applied to the present invention include, for example, 6-(2-hydroxy-2-methylpropoxy)-4-(6-{6-[(6-methoxypyridin-3-yl)methyl ]-3,6-diazabicyclo[3.1.1]heptan-3-yl}pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile (generic name: Selpercatinib), cis-N-{(1S)-1-[6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl]ethyl}-1-methoxy-4-{4-methyl-6-[ (5-Methyl-1H-pyrazol-3-yl)amino]pyrimidin-2-yl}cyclohexane-1-carboxamide (generic name: pralsetinib (BLU-667)), 4-(4-bromo-2-fluoro Anilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline (generic name: Vandetanib), 4-[4-[3-[4-chloro-3-(trifluoromethyl) ) phenyl]ureido]phenoxy]-N-methylpyridine-2-carboxamide (generic name: Sorafenib), N-[2-(diethylamino)ethyl]-5-[(Z)-(5-fluoro-2 -oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide mono[(2S)-2-hydroxysuccinate] (common name: Sunitinib), N-(3,3-dimethylindolin-6-yl)-2-(pyridin-4-ylmethylamino)nicotinamide (generic name: Motesanib), N-(4-( 6,7-dimethoxyquinolin-4-yloxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (common name; XL184).

Further, the "cancer" to be treated with such a RET kinase inhibitor is not particularly limited, but may be a solid tumor or a liquid tumor. Examples of solid tumors include lung cancer (non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, bronchiolopulmonary cell carcinoma, mesothelioma, etc.), thyroid cancer (medullary thyroid cancer, papillary thyroid cancer, etc.) ), colorectal cancer, and central nervous system cancer. Examples of liquid tumors include blood cancer and leukemia. Furthermore, the condition may be progressive, metastatic, or recurrent.

<Drugs for determining the effectiveness of cancer treatment>
The present invention also provides a reagent for use in the above-mentioned determination method, which includes the oligonucleotide described in (i) or (ii) below.
(i) A pair of oligonucleotides designed to sandwich nucleotides encoding the amino acid mutations according to the present invention. (ii) Oligonucleotides that hybridize to the nucleotides encoding the amino acid mutations according to the present invention.

These polynucleotides have nucleotide sequences that are complementary to specific nucleotide sequences of the RET gene. Here, "complementary" does not need to be completely complementary as long as they hybridize. These polynucleotides usually have 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 100% homology to the specific nucleotide sequence.

(i) Regarding a pair of oligonucleotides (primer set) designed to sandwich nucleotides encoding amino acid mutations according to the present invention, the length of the oligonucleotides is usually 15 to 100 nucleotides, preferably 17 to 100 nucleotides. 30 nucleotides, more preferably 17 to 22 nucleotides. Note that, depending on the above-mentioned detection method, one of the pair of oligonucleotides may contain a nucleotide sequence complementary to the nucleotide encoding the amino acid mutation according to the present invention.

(ii) An oligonucleotide that hybridizes to a nucleotide encoding an amino acid mutation according to the invention. Regarding the (oligonucleotide probe), the oligonucleotide probe is preferably one that specifically hybridizes to the region containing the above DNA mutation under normal hybridization conditions, preferably stringent hybridization conditions.

Furthermore, the oligonucleotide of the present invention may be used after being appropriately labeled with an isotope, a fluorescent dye, biotin, or the like. Labeling methods include labeling by phosphorylating the 5' end of the oligonucleotide with ^32P using T4 polynucleotide kinase, and labeling random hexamer oligonucleotides using DNA polymerase such as Klenow enzyme. Examples include a method (random prime method, etc.) in which a substrate base labeled with an isotope such as ³² P, a fluorescent dye, or biotin is incorporated as a primer.

The oligonucleotide of the present invention can be produced using, for example, a commercially available oligonucleotide synthesizer. Oligonucleotide probes can also be produced as double-stranded DNA fragments obtained by restriction enzyme treatment or the like. Furthermore, the oligonucleotide of the present invention does not need to be composed only of natural nucleotides (deoxyribonucleotides (DNA) and ribonucleotides (RNA)), and may be partially or entirely composed of non-natural nucleotides. It's okay. The non-natural nucleotide used in the present invention is not particularly limited as long as it has the same function as a natural nucleotide, but it increases the efficiency of hybridization to regions containing DNA mutations, and is useful for oligonucleotide primers and From the viewpoint of being able to shorten the chain length of oligonucleotide probes, PNA (polyamide nucleic acid), LNA (registered trademark, locked nuclear acid), ENA (registered trademark, 2'-O,4'-C-Ethylene-) bridged nuclear acids), and complexes thereof are preferred. Note that PNA is a DNA or RNA whose main chain consisting of phosphoric acid and pentose is replaced with a polyamide chain. LNA is also referred to as BNA (Bridged Nucleic Acid), and is RNA in which the 2' oxygen and 4' carbon of a nucleotide are bridged.

In the above reagent, in addition to the oligonucleotide as an active ingredient, for example, sterile water, physiological saline, vegetable oil, surfactant, lipid, solubilizing agent, buffer, preservative, etc. are mixed as necessary. It's okay.

Furthermore, the present invention can also provide a kit for determining the effectiveness of cancer treatment with a RET kinase inhibitor, which includes the oligonucleotide. The kit of the present invention can contain preparations other than the oligonucleotides described above. Examples of such preparations include reagents for extracting DNA and the like from biological samples, and reagents necessary for PCR reactions (eg, deoxyribonucleotides, thermostable DNA polymerase, etc.). The kit can also include instructions for use that also describe the determination method and the like.

Further, as described above, an antibody against RET protein whose epitope is a region containing an amino acid mutation according to the present invention can also be used in the determination method of the present invention. Therefore, like the oligonucleotide described above, the present invention can also provide an embodiment of a drug or kit containing the antibody for determining the effectiveness of cancer treatment with a RET kinase inhibitor.

<Methods for treating cancer, therapeutic agents for cancer>
As mentioned above, it is thought that cancer treatment using a RET kinase inhibitor will be highly effective for subjects in whom the amino acid mutation according to the present invention has been detected. Therefore, by selectively administering a RET kinase inhibitor to patients who carry the amino acid mutation according to the present invention among subjects, it is possible to efficiently treat cancer. Therefore, the present invention provides a method for treating cancer, in which the RET kinase inhibitor is administered to a subject who has been determined to have high efficacy in cancer treatment with a RET kinase inhibitor by the method of the present invention. A method comprising the step of administering. This is what we provide.

The present invention also provides a therapeutic agent for cancer containing a RET kinase inhibitor as an active ingredient, which is applicable to subjects who have been determined to have high efficacy in cancer treatment with the RET kinase inhibitor by the method of the present invention. The present invention provides therapeutic agents to be administered to patients.

The "RET kinase inhibitor" is as described above. The method of administering the drug to the subject is appropriately selected depending on the type of drug and the type of cancer, but examples include oral, intravenous, intraperitoneal, transdermal, intramuscular, and intratracheal ( Administrative forms such as aerosol), intrarectal, intravaginal, etc. can be adopted.

EXAMPLES Hereinafter, the present invention will be described in more detail based on Examples, but the present invention is not limited to the following Examples. Further, this example was carried out using the materials and methods shown below.

(Selection of RET mutations)
In the exploratory cohort, we collected global information such as gene fusions and copy number changes from 79,720 tumors from the American Cancer Society's Project GENIE (version 7.0) (https://genie.cbioportal.org/) website. We downloaded the mutation data for 71,756 items excluding the major changes (Figure 2A). Mutations were analyzed using OncodriveCLUSTL (Reference 14), a nucleotide sequence-based clustering algorithm that detects cancer driver genes based on a local background model derived from trinucleotide mutation probabilities in the study cohort (i.e., GENIE). (Left of Figure 2B). Processing was performed using GENIE mutation data as mutation profile data. Since the background dataset (training cohort) has a similar proportion of each mutation type to the GENIE dataset, we used the whole exome sequence Mutation Annotation Format (version 0.2.8) provided by the TCGA MC3 working group. ) (Reference 41) (Fig. 2B right). Note that the literature numbers shown in parentheses (herein, 14, 41) correspond to the literature described in each number shown in <References> below (the same applies hereinafter).

(Cell lines and reagents)
HEK293H and Flp-in T-REx ^™ 293 cells were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Ba/F3 cells were provided by Dr. Hiroyuki Yasuda of Keio University School of Medicine. 293FT cells were obtained from Invitrogen (Carlsbad, CA, USA) and WEHI-3B cells were obtained from RIKEN BioResource Center (Ibaraki, Japan). U2OS cells were obtained from American Type Culture Collection (Manassas, VA, USA). 293H, Flp-in T-rex 293, and 293FT cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) purchased from Thermo Fisher Scientific. WEHI-3B cells were cultured in RPMI medium containing 10% FBS. Ba/F3 cells were cultured in RPMI medium containing 10% FBS and 10% WEHI-3B conditioned medium (source of IL-3). All cells were incubated at 37°C, 5% _CO2 . Primary antibodies against RET (Cat. No. 14556), phosphorylated Tyr905 RET (Cat. No. 3221), ERK (Cat. No. 9102), phosphorylated Thr202/Tyr204 ERK (Cat. No. 4370) and rabbit IgG (Cat. No. 7074) and mouse IgG. (Cat. No. 7076) was purchased from Cell Signaling Technology (Danvers, Mass., USA). Antibody against β-actin (catalog number ab8227) was purchased from Abcam (Cambridge, UK). Antibody against FLAG (catalog number F1804) was purchased from Sigma-Aldrich (Saint Louis, MO, USA). Antibody against GST (catalog number 27-4577-01) was purchased from Cytiva (Tokyo, Japan). Human GDNF recombinant protein (catalog number 212-GD) and human GFRα1 recombinant protein (catalog number 714-GR) were purchased from R&D Systems (Minneapolis, MN, USA). Selpercatinib (catalog number S8781) and pralsetinib (catalog number S8716) were purchased from Selleck (Houston, TX, USA). These compounds were dissolved in DMSO to make a 10mM stock and stored at -20°C.

(Construction of lentiviral vectors expressing wild type and mutant RET cDNA)
Full-length cDNAs of wild-type and mutant RET were synthesized by Invitrogen and ligated into pLenti-6/V5-DEST plasmid (Invitrogen). The final construct was confirmed by Sanger sequencing. Expression of the cDNA product was confirmed by immunoblotting of transiently transfected cells. Lentivirus was generated in 293FT cells (6×10 ⁶ cells per 10 cm plate) transfected with pLenti-6/V5-DEST plasmid and ViraPower packaging mix (Invitrogen) using Lipofectamine 3000 (Invitrogen). Viral supernatants were collected 24-48 hours after medium change and used for infection.

(phosphorylation assay)
HEK293H cells seeded in a 6-well plate were transiently transfected with 0.25 μg of plasmid DNA using Lipofectamine 3000. After transfection, the cells were cultured under serum-starved conditions for 48 hours and incubated in RIPA buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na EDTA, 1 mM EGTA] containing protease/phosphatase inhibitor cocktail ( _Cell Signaling Technology). . _It was _. The cell lysate was centrifuged at 14,000 rpm for 15 minutes and the supernatant was collected. The supernatant was subjected to SDS-PAGE and then immunoblotted onto a polyvinylidene fluoride (PVDF) membrane. PVDF membranes were blocked with TBS containing 0.1% Tween20 and 1.0% bovine serum albumin (TBST) for 1 hour and then probed with the following primary antibodies: anti-RET, anti-phospho-Thy202/Tyr204 ERK, and Anti-ERK. After washing the PVDF membrane with TBST, it was incubated with HRP-labeled anti-rabbit secondary antibody and visualized with chemiluminescence reagent (Perkin Elmer, Waltham, Mass., USA). Signal intensity was quantified using a LAS3000 imaging system (Quansys Biosciences, West Logan, UT, USA) and Multigauge software (Fujifilm, Tokyo, Japan). For experiments with calcium-containing/non-calcium media conditions, cells were cultured in calcium-containing/non-calcium media for 48 hours after transfection. For phosphorylation assays, GDNF and GFRα1 were added 48 hours post-transfection and incubated for an additional 6 hours. Inhibitors were added 48 hours post-transfection and incubated for an additional 6 hours. The assay was repeated three times independently.

(Ba/F3 cell assay)
Ba/F3 cells (4.0×10 ⁵ ) were centrifuged at 3000×g for 150 minutes at 32° C. in the presence of 10 μg/mL polybrene (Sigma-Aldrich) and infected with lentivirus. After overnight culture at 37° C. and 5% CO ₂ , cells were seeded in 24-well plates and selected for 10 days in medium containing IL-3 and 8 μg/mL Blasticidin (Invitrogen). Blasticidin-resistant cells were cultured in IL-3 free medium for 2 weeks. Expression of exogenous RET protein was confirmed by immunoblotting. For drug treatment, ^5x10 Ba/F3 cells were plated in six replicates in a 96-well plate, and serially diluted inhibitors were added to the wells. Cell viability 72 hours after drug treatment was measured using CellTiter-Glo luminescent cell viability reagent (Promega, Madison, Wis., USA) using EnVision (Perkin Elmer). Cell viability was calculated as the number of cells in the drug-treated sample relative to the number of cells in the untreated sample (n=6). Data were displayed graphically using GraphPad Prism version 9.0 (GraphPad Software Inc., San Diego, CA, USA).

(NIH3T3 transformation assay)
NIH3T3 cells were infected with lentivirus for 24 hours in the presence of 8 μg/mL polybrene (Sigma-Aldrich), and cultured for 2 weeks in DMEM-F12 supplemented with 5% calf serum as previously described (Reference 26). For focus formation assays performed with GDNF and GFRα1, infected NIH3T3 cells were cultured with 0, 1, or 2.5 ng/mL GDNF and GFRα1 in the presence of 5% calf serum for 14 days. Cell transformation was assessed by Giemsa staining. Reproducibility was confirmed by performing the same experiment three times. Focus areas were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

(RET dimerization assay)
Using the Flp-In ^™ T-REx ^™ -293 system, T-REx ^™ 293 cells expressing wild type, D567N, D567Y, C634R full-length RET cDNA and empty vector were established according to the manufacturer's protocol. The cloned cells were cultured for 6 hours in DMEM with/without calcium supplemented with 0.1 μg/mL doxycycline (Fuji Film, Osaka, Japan). In this assay, lysates were prepared in RIPA buffer with/without 40 mM dithiothreitol (Cell Signaling Technology). In immunoblot analysis, anti-FLAG antibody was used as the primary antibody.

In the Sf21 cell assay, pFastBac ^™ (Thermo Fisher Scientific) baculovirus plasmid containing cDNA encoding the CLD4-CRD (amino acid number 382-635) region of RET was transfected with Cellfectin ^™ II Reagent (Thermo Fisher Scientific). her Scientific) into Sf21 cells. It was effected. Transfected cells were lysed with extraction buffer (50mM HEPES, 0.3M NaCl, 0.2% NP-40, pH 7.5) with/without 5mM EDTA. After centrifugation (10,000×g), GST-Sepharose beads were added to the supernatant and mixed with rotation at 4° C. for 30 minutes. The beads were thoroughly washed with extraction buffer and loaded with/without 1% 2-mercaptoethanol (2-ME) (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.1% bromophenol). blue) and incubated at 95°C for 5 minutes. Samples were electrophoresed and dimer formation was detected by immunoblotting using an anti-GST antibody as the primary antibody or Coomassie brilliant blue (CBB) staining.

(NanoBiT assay)
Small BiT (SmBit) or Large BiT (LgBit) luciferase subunit (Promega) fused via a peptide linker to the C-terminus of the RET-ECD domain (amino acid number 1-662) of WT, C634R, D567N, D567Y RET. The expressing plasmid was transfected into U2OS cells. The transfected cells were then seeded into 96-well plates. Cells (1.13×10 ⁴ per well) were then transfected with 5 ng of a mixture of SmBit and LgBiT plasmid DNA at a mass ratio of 1:1. After 24 hours, cells were serum starved with Opti-MEM for 5 hours. Fifteen minutes after adding the NanoGlo live cell luciferase substrate, GDNF and GFRα1 were added to a final concentration of 7.4 μg/mL, and the luminescence kinetics was measured for 45 minutes using a Syngy HTX plate reader (BioTek, Winooski, VT, US). It was measured using

(In vivo tumor formation assay)
NIH3T3 cells expressing RET C634R, D567N, D567Y mutants were selected by exposure to 8 μg/mL blasticidin for 20 days after lentiviral infection. Cell numbers were counted and resuspended on ice in a 1:1 mixture of 100 μL culture medium and 100 μL Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). NIH3T3 cells (5×10 ⁶ cells/mouse) were injected subcutaneously into the flank of 6-week-old female BALB/c-nu/nu mice (Charles River, Wilmington, MA, US). Mice were divided into three groups for each mutant, and treatment was started when the tumor size increased from approximately 100 ^mm to 200 ^mm . In all mutant experiments, mice received vehicle or 3 mg/kg selpercatinib or pralsetinib twice daily. Tumor growth was measured at regular intervals using calipers, and tumor volume was calculated using the following formula: tumor volume (mm ³ ) = 0.5 x length (mm) x width (mm) x width (mm). . At the end of the experiment, subcutaneous tumor weights were measured and mice were euthanized according to standard protocols. This experiment was approved by the National Cancer Center (NCC) Animal Ethics Committee (approval number: A277bM1-21).

(MD simulation of monomer)
Initial cryo-electron microscopy structural data of RET CRD was obtained from Protein Data Bank (PDB code: 6Q2N). A monomer model for MD simulations was constructed using residues 508-622 of the E chain, and other residues were deleted. Bond order assignment and hydrogen atom addition were performed using the Protein Preparation module of Maestro 2020-2 (Schrodinger, Inc., New York, USA). The disordered loop structure (residues 508-514 and 547-548) was modeled using Prime (42, 43). The N-terminus and C-terminus of all protein models were capped with acetyl and N-methyl groups, respectively. A RET mutation model was created based on the 6Q2N structure using the "Sequence Viewer" of Maestro (Schrodinger, Inc.) according to the same protocol. Hydrogen bond optimization and energy minimization were performed using PROPKA (Reference 44) and OPLS3e force field (Reference 45).

Desmond (Schrodinger, Inc.) was used for MD simulation for interaction analysis and binding free energy evaluation, and Maestro's Molecular Dynamics System Setup module was used for creating the hydrate model. All energy minimized models were placed in an orthorhombic box with a buffer distance of 10 Å to create a hydration model. The SPC water model (Reference 46) was used as the hydration model. Sodium chloride (0.15M) was included as a counterion to neutralize the system. The cutoff radius of van der Waals interaction was 9 Å, and the time step, initial temperature of the system, and pressure were 2.0 fs, 300 K, and 1.01325 bar, respectively. The sampling interval of trajectory frames was set to 100 picoseconds. The final simulation was performed in 1 microsecond using the NPT ensemble.

All trajectories of the MD simulations were aligned to the first frame of protein Cα. RMSD and distance analysis was performed using Maestro's Simulation Interactions Diagram module based on the coordinates of the first frame. The MM/PB(GB)SA method was adopted to predict the binding free energy of Ca ions to CRD (Reference 47). Binding free energy analysis was performed for each trajectory at 100 frame intervals, and Prime (References 42, 43) was used for the analysis.

(MD simulation of RET-GDNF-GFRA1 extracellular complex)
Initial structural data for the RET/GFR1/GDNF extracellular complex was obtained from the Protein Data Bank (PDB code: 6Q2N). The disordered loop structure was modeled using the Structure Preparation module of the MOE program (Chemical Computing Group Inc., Montreal, QC, Canada). The N-terminus and C-terminus of all protein models were capped with acetyl and N-methyl groups, respectively. For titratable residues, the predominant protonation state at pH 7.0 was assigned. The D567Y or D567N mutation was introduced into the wild-type RET structure using MOE.

All MD simulations were performed using the GROMACS 2021 program (Reference 48). Amber ff99SB-ILDN force field (Reference 49) was used for proteins and ions, and TIP3P (Reference 50) was used for the water molecule model. Approximately 280,000 water molecules were placed around each complex model with a surrounding distance of 10 Å, and sodium and chloride ions (150 mM each) were introduced into the simulation box to neutralize the system. The electrostatic interaction was calculated by the Particle Mesh Ewald method (Reference 51), and the cutoff radius was set to 10 Å. The P-LINCS algorithm was used to constrain all bond lengths to their equilibrium values (ref. 53). After energy minimization, each system was equilibrated for 100 ps in a constant particle number, volume, and temperature (NVT) ensemble, positional restraints were applied to protein heavy atoms, and equilibrated for 100 ps in a constant particle number, pressure, and temperature (NPT) ensemble. Got it working. The temperature was maintained at 310 K by stochastic rate rescaling (ref. 54) and the pressure at 1 bar by a Parrinello-Rahman barostat (ref. 55). The temperature and pressure time constants were set to 0.1 ps and 2 ps, respectively. Three independent 1 μsec production runs were performed at different speeds for wild-type RET and its D567Y and D567N mutants. The contact area between CRD and GDNF/GFRα1 was calculated using High Throughput Molecular Dynamics (HTMD) environment 1.14.0 (Reference 56) with a probe radius of 1.4 Å.

Below we show the results obtained using the above materials and methods.

(Example 1) CaLM is a hidden oncogenic mutation cluster In order to discover hidden clusters of oncogenic mutations among a large number of VUS, the OncodriveCLUSTL program (Reference 17) was used to analyze the entire genome. Three base enrichment analysis was performed. A total of 71,756 variants obtained from the GENIE cohort (Reference 18) (version 7; 79,720 cases) based on the context of mutant trinucleotides created from 9,104 samples of The Cancer Genome Atlas (TCGA). This was analyzed in comparison to a background mutation rate model (Figures 2A and 2B). In the VUS study, RET had many types (860) of missense and in-frame insertion deletion mutations in 1,290 (1.6%) tumors, most of which were classified as “significant” by OncoKB (Reference 19). Since this cancer gene was annotated as "unknown," it was selected as a target cancer gene for therapeutic target expansion research (Figures 2C and 2D). There are two mutational cluster elements in the RET kinase CRD: a known CCM cluster containing the C634R hotspot mutation and a novel cluster at residues 557-580 consisting only of VUS (Fig. 1A). Three other clusters containing hotspot mutations V804M, R886W, and M918T (Reference 12) were confirmed in the intracellular domain (ICD), demonstrating the reliability of the mutation model. Amino acid residues at positions 557 to 580 corresponded to CaLM of ECD, which was recently identified by cryo-electron microscopy analysis (Reference 15). Although CaLM mutations were observed in various types of tumors, CCM mutations were mainly observed in thyroid cancer (Figure 1A). Most of these mutations were observed as independent mutations, and similar to CCM, they were rarely observed as compound mutations (Fig. 2E). This is consistent with the positive selectivity in in silico analysis.

Next, 110 RET variants including 6 CaLM mutations, 25 known mutations (References 5, 6, 11, 12, 20, 21), and 79 other mutations found in 3 or more GENIE cases. Gene-comprehensive cell-based assays were performed. The effects of introduction of RET mutant cDNA on transforming activity in NIH3T3 cells and ERK phosphorylation downstream of RET in HEK293H cells were investigated (FIGS. 1B and 1C). The results confirmed that CaLM is a cluster of oncogenic mutants with similar potency to hotspot mutants in CCM and ICD, whereas most mutants, especially in ECD, are functionally It was neutral.

(Example 2) Structural destabilization of CRD by CaLM mutation Similar to calmodulin (Figure 3B), RET CaLM forms coordinate bonds with the negatively charged side chains and carbonyl skeletons of contributing amino acids. (Fig. ^3A ) (Reference 15). The RET-CaLM mutations form a three-dimensional cluster due to substitution of amino acids involved in binding to Ca ²⁺ ions (ie, D567Y and D567N, G568S, D571N, E574D, and D584N). This is similar to germline mutations in the D95 and N97 residues of the CALM1 gene encoding calmodulin, which causes long QT syndrome. Since these CALM1 mutations are associated with destabilization of Ca ²⁺ ion retention (References 22, 23), MD simulations were performed to predict the effect of CaLM mutations on the conformational retention of the RET-Ca ²⁺ ion complex. was carried out.

First, a 1 microsecond simulation was performed using RET-CRD monomer structures with or without CaLM mutations to investigate the effect on local structure maintenance. The binding free energy (ΔGbind) of Ca ²⁺ ions to CRD of D567Y and D567N mutants was higher than that of wild-type RET (Figure 3C and Figure 4A), suggesting that the CaLM mutation destabilizes the CRD-Ca ²⁺ complex. . In the simulation, the position of the Ca ²⁺ ion was shifted toward the opposite side of the mutated residue at position 567 (FIGS. 4B and 4C). The instability of Ca ²⁺ ion positions due to CaLM mutations was indicated by an increase in the root mean square deviation (RMSD) of Ca ²⁺ ions from their initial positions (Fig. 4D). In parallel, the structural fluctuations of the CRD associated with the D567 residue mutation were indicated by the RMSD of the backbone Cα atoms (FIG. 4D left, FIGS. 4E and 4F). Similar results were obtained with other CaLM mutants (Fig. 3D left), strongly suggesting that destabilization of the CaLM-Ca ²⁺ complex is behind the carcinogenicity. Furthermore, in order to verify the simulation results, the present inventors investigated the characteristics of an artificial mutant (mutant substituted with alanine) in which the influence of the side chain that contributes to Ca ²⁺ ion retention was removed (Fig. 3D, right). ). As a result, the alanine mutant showed a high RMSD value similar to the clinically recognized CaLM mutant. The tumorigenicity of this alanine mutant was confirmed by ERK activation downstream of RET and transformation of NIH3T3 cells (Fig. 3E).

(Example 3) CaLM mutation induces abnormal intermolecular disulfide bond formation Under physiological conditions, RET protein co-recepts glial cell-derived neurotrophic factor (GDNF) ligands and GDNF family receptor α1 (GFRα1). and homodimerizes to form a hexameric complex (Fig. 3A) (Reference 15). To investigate the effects of CaLM mutations on the structure of RET and its interactions with ligands/co-receptors, we constructed a cryo-electron microscopy structure (PDB: 6Q2N) consisting of dimerized extracellular RET, two molecules of GFRα1 and GDNF. A microsecond-scale MD simulation was carried out using this method. Compared to the wild-type RET complex, the CaLM mutant lost the interaction between Ca ²⁺ ions and surrounding residues, and the CRD structure was distorted (Fig. 5A left and middle, and Supplementary Fig. 6A). Furthermore, distortion of the CRD structure of the CaLM mutant increased the solvent exposed surface area (SASA) of cysteine residues C565, C570, and C585 (Fig. 5A right). These cysteine residues are physiologically buried within the CRD domain and form intradomain disulfide bonds in the wild-type RET structure (Reference 15). Based on these simulation results, we investigated whether CaLM mutations induce the formation of intermolecular disulfide bonds.

When the full-length RET cDNA was exogenously expressed in HEK293H cells, the CaLM mutant dimerized through an abnormal intermolecular disulfide bond, similar to the C634R CCM mutant (Fig. 5B, C left). Mimicking the attenuation of Ca ²⁺ ion retention in the CaLM mutant, depleting Ca ²⁺ ions from the culture medium also caused dimerization through intermolecular disulfide bond formation in wild-type RET (Fig. 5C, right); It was suggested that it suppresses abnormal dimerization of RET. RET has three Ca ²⁺ ion binding sites in the region spanning cadherin-like domains (CLD) 2 and 3 (Fig. 5B) (Reference 15). Therefore, a dimerization assay using purified CLD4-CRD protein was performed to verify the inhibitory role of CaLM on dimerization. The CaLM mutant promoted dimerization via disulfide bonds in the presence of Ca ²⁺ ions, and depletion of Ca ²⁺ ions also promoted dimerization via disulfide bonds in wild-type RET (Figure 5D, Figures 6B and 6C). ). Similarly, RET and downstream ERK phosphorylation upon Ca2 ⁺ ion depletion was more pronounced in wild-type RET than in the CaLM mutant (Fig. 5E).

Coreceptors/ligands contribute to dimerization of RET molecules (24). A time-course bimolecular interaction assay of the RET-ECD domain using the NanoBiT system (Reference 25) revealed that not only wild-type RET but also the dimerization of CaLM (D567N, D567Y) and CCM (C634R) mutants were detected as ligands. /coreceptor addition (Fig. 6D, left). Introducing the R77E and R144E double mutations, which suppress inactive dimerization, did not abolish dimerization reactions, suggesting that these reactions are caused by active dimerization containing a ligand/co-receptor. It was shown that there was no intervening inactive dimerization that did not involve these molecules (Fig. 6D, right). In the present MD simulations, the D567N RET mutant forms a more stable RET-GDNF-GFRα1 hexameric complex than wild-type RET or the D567Y mutant due to enhanced RET-ligand/co-receptor contact. (Figures 5F and 5G). Furthermore, the addition of GDNF and GFRα clearly increased the transformation ability of the D567N mutant in NIH3T3 cells and the activation of ERK in HEK293H cells compared to the D567Y and C634R mutants (Figure 5H, Figure 6E). . These data suggest that the oncogenic properties of the D567N mutant are increased in the presence of ligand/coreceptor, consistent with the enhanced protein-protein interactions observed in MD simulations.

(Example 4) RET-CaLM mutants are targets of RET-TKIs Finally, it was verified whether RET-CaLM mutants are targets of existing RET-TKIs. Enhanced ERK phosphorylation caused by exogenously expressed CaLM mutants in HEK293H cells was suppressed by selpercatinib and pralsetinib, two RET-specific TKIs approved by the US Food and Drug Administration (Figure 7A) ( Reference 20). Stable expression of the RET-CaLM mutant enabled proliferation of Ba/F3 cells in an interleukin-3 (IL-3)-independent manner, similar to the C634R and M918T hotspot mutants, and the oncogenic activity of the CaLM mutant. was confirmed. Proliferation of Ba/F3 cells expressing the CaLM mutant was inhibited by selpercatinib and pralsetinib at concentrations comparable to those effective for the C634R and M918T mutants (FIGS. 7B and 8a). NIH3T3 cells stably expressing the D567N and D567Y mutants formed tumors in nude mice similar to cells expressing the C634R mutant. Both selpercatinib and pralsetinib suppressed the proliferation of NIH3T3 cells expressing D567N and D567Y, similar to their effects on NIH3T3 cells expressing the C634R mutant (FIGS. 7C-7E and FIG. 8b). These results indicate that RET-CaLM mutants can be therapeutic targets with existing RET-TKIs.

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As explained above, according to the present invention, it is possible to predict the effectiveness of cancer treatment using a RET kinase inhibitor by detecting amino acid mutations in the calmodulin-like motif of the RET protein. This makes it possible to avoid administering the drug to cancer patients for whom administration of the drug is considered ineffective. Therefore, the present invention is extremely useful in increasing the efficiency of cancer treatment.

Claims (4)

A method for determining the effectiveness of cancer treatment with a RET kinase inhibitor, the method comprising:
Detecting amino acid mutations in the calmodulin-like motif of the RET protein in the sample isolated from the subject;
If the amino acid mutation is detected in the step, determining that the effectiveness of cancer treatment with the RET kinase inhibitor in the subject is high. The amino acid mutation is a substitution of at least one amino acid selected from aspartic acid at position 567, glycine at position 568, aspartic acid at position 571, glutamic acid at position 574, and aspartic acid at position 584 with another amino acid in the RET protein. 2. The method according to claim 1, wherein the mutation is A reagent for use in the method according to claim 1 or 2, comprising the oligonucleotide according to (i) or (ii) below, or the antibody according to (iii). a pair of oligonucleotides designed to sandwich the encoding nucleotides; (ii) an oligonucleotide that hybridizes to the nucleotide encoding the amino acid mutation; and (iii) an antibody that specifically recognizes the amino acid mutation. A RET kinase inhibitor, characterized in that it is ingested by the subject who has been determined to have high effectiveness in cancer treatment by the method according to claim 1 or 2.