Method for predicting sensitivity of cancer cells to helicase inhibitors

Abstract

To develop a therapeutic strategy capable of specifically targeting cancer cells with loss-of-function mutations, more specifically, to develop a therapeutic strategy that specifically targets cancer cells with TTK mutations and / or Rad50 mutations.SOLUTION: Provided is a method for predicting the sensitivity of cancer cells to a helicase inhibitor, comprising: predicting that a cancer cell in which at least one mutation selected from a first group consisting of a TTK mutation and a RAD50 mutation is detected is sensitive to a helicase inhibitor; and predicting that a cancer cell in which at least one mutation selected from a second group consisting of a RAD50 mutation, an MRE11 mutation, an NBN mutation, a DNA2 mutation, and an RBBP8 mutation is detected is sensitive to a helicase inhibitor.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for predicting the sensitivity of cancer cells to a helicase inhibitor. The present invention also relates to a method for predicting the sensitivity of cancer patients to treatment with a helicase inhibitor, a method for selecting cancer patients to be treated with a helicase inhibitor, a method for treating cancer, a method for screening a compound for use in the treatment of cancer, and a cancer therapeutic agent.

Background Art

[0002] In recent years, rapid progress in genomic sequencing technology has made it possible to decode genomic information including unique gene mutations in cancer cells. Among them, in the development of anticancer drugs, drugs have been created that specifically inhibit the functions of cancer cells with gain-of-function gene mutations, such as EGFR gene mutations, BRAF gene mutations, and ALK fusion genes (Non-Patent Documents 1 to 3). A treatment method specific to such cancer cells targeting cancer cells having these gene mutations is a highly selective and effective treatment method for cancer.

[0003] Also, for example, in cancer cells showing MSI-H (high-frequency microsatellite instability), it has been reported that their survival depends on WRN (Werner syndrome protein) (Non-Patent Documents 4 to 7), and it is considered that treatment that inhibits WRN can specifically target such cancer cells showing MSI-H.

[0004] On the other hand, gene mutations found in human cancer cells include not only the above-described gain-of-function type but also, conversely, loss-of-function gene mutations. Loss-of-function gene mutations are difficult to develop drugs specific to the gene mutations, and a treatment strategy different from that for treating cancer cells having gain-of-function gene mutations is required.

[0005] As one of the few successful examples of specifically targeting cancer cells with loss-of-function mutations, PARP inhibitors for BRCA1 / 2-deficient tumors can be cited (Non-Patent Document 8). However, treatment strategies for specifically targeting cancer cells with other loss-of-function mutations have not yet been developed at present.

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Summary of the Invention

Problems to be Solved by the Invention

[0007] The present invention has been made in view of the above circumstances. The object of the present invention is to develop a treatment strategy capable of specifically targeting cancer cells in which a loss-of-function mutation has occurred. More specifically, at least one mutation selected from the first group consisting of mutations in TTK and mutations in RAD50, and / or at least one mutation selected from the second group consisting of mutations in RAD50, mutations in MRE11, mutations in NBN, mutations in DNA2, and mutations in RBBP8 is detected. The purpose is to develop a treatment strategy that specifically targets cancer cells.

Means for Solving the Problems

[0008] The present inventors have conducted intensive studies to solve the above problems. First, the inventors comprehensively suppressed the expression of WRN by siRNA in approximately 70 cancer cell lines, and analyzed the mutations common to the cell lines in which growth suppression was confirmed. As a result, in cancer cells in which at least one mutation (preferably a loss-of-function mutation) selected from the first group consisting of mutations in TTK and mutations in RAD50 has occurred, when the expression or function of helicases such as WRN is suppressed, the growth of the cancer cells is significantly suppressed. On the other hand, it was found that such growth suppression does not occur in cells in which neither mutation in TTK nor mutation in RAD50 has occurred.

[0009] Furthermore, the present inventors added the target cancer cell line and comprehensively analyzed approximately 200 cancer cell lines including the above approximately 70 cancer cell lines. As a result, in addition to the mutations selected from the first group, at least one mutation (preferably a loss-of-function mutation) selected from the second group consisting of mutations in RAD50, mutations in MRE11, mutations in NBN, mutations in DNA2, and mutations in RBBP8 is common in cancer cells in which the mutation has occurred. It was found that when the expression or function of the helicase is suppressed, the growth of the cancer cells is suppressed.

[0010] In addition, the above-mentioned mutations are also part of the mutations frequently identified in cancer cells showing MSI-H. As described above, MSI-H is considered to be an indicator for the selection of cancer cells that are targets for treatment that inhibits WRN, a helicase (for example, Non-Patent Documents 4 to 7). However, the inventors have found that the above-mentioned growth inhibition does not occur in cancer cells that show MSI-H but do not have any of the above-mentioned mutations, particularly the mutations selected from the above-mentioned second group. Therefore, the newly discovered mutations can be used as an indicator for the selection of cancer cells that are targets for treatment that inhibits helicase, regardless of whether they show MSI-H, and can be a more specific indicator for the selection of the above-mentioned cancer cells than MSI-H.

[0011] Therefore, the inventors have found that treatment that inhibits helicase is a promising approach for treatment targeting cancer cells in which at least one mutation selected from the first group consisting of mutations in TTK and mutations in RAD50, and / or at least one mutation selected from the second group consisting of mutations in RAD50, mutations in MRE11, mutations in NBN, mutations in DNA2, and mutations in RBBP8 has occurred. In addition, in this treatment strategy, it has also been clarified that, based on companion diagnostics, efficient treatment is possible because cancer patients can be selected based on the above-mentioned mutations and then a helicase inhibitor can be administered.

[0012] Furthermore, the inventors have also found that screening for drugs useful for the treatment of cancer in which the above-mentioned mutations have occurred can be performed using as an indicator whether or not helicase is inhibited, and have thus completed the present invention.

[0013] Accordingly, the present invention also relates to a treatment method for specifically targeting cancer cells in which at least one mutation selected from the first group consisting of mutations of TTK and mutations of RAD50, and / or at least one mutation selected from the second group consisting of mutations of RAD50, mutations of MRE11, mutations of NBN, mutations of DNA2, and mutations of RBBP8 has occurred, and more specifically, provides the following inventions. [1] A method for predicting the sensitivity of cancer cells to a helicase inhibitor, comprising the step of predicting that cancer cells in which at least one mutation selected from the first group consisting of mutations of TTK and mutations of RAD50 is detected are sensitive to a helicase inhibitor. A method as described above. [2] A method for predicting the sensitivity of cancer cells to a helicase inhibitor, (a) a step of detecting the presence or absence of at least one mutation selected from the first group consisting of mutations of TTK and mutations of RAD50 in cancer cells; (b) a step of predicting that the cancer cells in which the mutation is detected are sensitive to a helicase inhibitor. A method as described above. [3] A method for predicting the sensitivity of a cancer patient to treatment with a helicase inhibitor, comprising the step of predicting that a cancer patient in which at least one mutation selected from the first group consisting of mutations of TTK and mutations of RAD50 is detected in cancer cells contained in a sample derived from the cancer patient is sensitive to treatment with a helicase inhibitor. A method as described above. [4] A method for predicting the sensitivity of a cancer patient to treatment with a helicase inhibitor, (a) a step of detecting the presence or absence of at least one mutation selected from the first group consisting of mutations of TTK and mutations of RAD50 in cancer cells contained in a sample derived from the cancer patient; (b) Predicting that a cancer patient in whom the mutation has been detected in the cancer cells is sensitive to treatment with a helicase inhibitor; A method comprising: [5] A method for selecting a cancer patient to be treated with a helicase inhibitor, selecting, as a subject for cancer treatment with a helicase inhibitor, a cancer patient in whom at least one mutation selected from the first group consisting of a TTK mutation and a RAD50 mutation is detected in cancer cells contained in a sample derived from the cancer patient; A method comprising: [6] A method for selecting a cancer patient to be treated with a helicase inhibitor, (a) Detecting the presence or absence of at least one mutation selected from the first group consisting of a TTK mutation and a RAD50 mutation in cancer cells contained in a sample derived from a cancer patient; (b) Selecting, as a subject for cancer treatment with a helicase inhibitor, a cancer patient in whom the mutation has been detected in the cancer cells; A method comprising: [7] A method for treating cancer, administering a helicase inhibitor to a cancer patient in whom at least one mutation selected from the first group consisting of a TTK mutation and a RAD50 mutation is detected in cancer cells contained in a sample derived from the cancer patient; A method comprising: [8] A method for treating cancer, (a) Detecting the presence or absence of at least one mutation selected from the first group consisting of a TTK mutation and a RAD50 mutation in cancer cells contained in a sample derived from a cancer patient; (b) Administering a helicase inhibitor to a cancer patient in whom the mutation has been detected in the cancer cells; A method comprising: [9] The method according to any one of [1] to [8], wherein the helicase inhibitor is a WRN inhibitor.

[10] The method according to any one of [1] to [9], wherein the cancer cells are cancer cells in which a mutation in MSH3 is further detected.

[11] A method for screening a compound for use in the treatment of cancer, comprising cancer cells in which at least one mutation selected from the first group consisting of a mutation in TTK and a mutation in RAD50 is detected, and a step of selecting a compound using as an index whether or not it inhibits helicase The method comprising the above.

[12] A cancer therapeutic agent comprising, as an active ingredient, a compound that inhibits helicase and comprising cancer cells in which at least one mutation selected from the group consisting of a mutation in TTK and a mutation in RAD50 is detected.

[13] The method according to

[11] , wherein the helicase is WRN.

[14] The cancer therapeutic agent according to

[12] , wherein the helicase is WRN.

[15] A method for predicting the sensitivity of cancer cells to a helicase inhibitor, and a step of predicting that cancer cells in which at least one mutation selected from the second group consisting of a mutation in RAD50, a mutation in MRE11, a mutation in NBN, a mutation in DNA2, and a mutation in RBBP8 is detected are sensitive to a helicase inhibitor. The method comprising the above.

[16] A method for predicting the sensitivity of cancer cells to a helicase inhibitor, (a) A step of detecting the presence or absence of at least one mutation selected from the second group consisting of a mutation in RAD50, a mutation in MRE11, a mutation in NBN, a mutation in DNA2, and a mutation in RBBP8 in cancer cells; and (b) A step of predicting that the cancer cells in which the mutation is detected are sensitive to a helicase inhibitor. The method comprising the above.

[17] A method for predicting the sensitivity of a cancer patient to treatment with a helicase inhibitor, Predicting that a cancer patient in which at least one mutation selected from the second group consisting of mutations of RAD50, mutations of MRE11, mutations of NBN, mutations of DNA2, and mutations of RBBP8 is detected in cancer cells contained in a sample derived from the cancer patient is sensitive to treatment with a helicase inhibitor; A method comprising the same.

[18] A method for predicting the sensitivity of a cancer patient to treatment with a helicase inhibitor, (a) Detecting the presence or absence of at least one mutation selected from the second group consisting of mutations of RAD50, mutations of MRE11, mutations of NBN, mutations of DNA2, and mutations of RBBP8 in cancer cells contained in a sample derived from the cancer patient; (b) Predicting that a cancer patient in which the mutation is detected in the cancer cells is sensitive to treatment with a helicase inhibitor; A method comprising the same.

[19] A method for selecting a cancer patient to be treated with a helicase inhibitor, Selecting, as a subject for cancer treatment with a helicase inhibitor, a cancer patient in which at least one mutation selected from the second group consisting of mutations of RAD50, mutations of MRE11, mutations of NBN, mutations of DNA2, and mutations of RBBP8 is detected in cancer cells contained in a sample derived from the cancer patient; A method comprising the same.

[20] A method for selecting a cancer patient to be treated with a helicase inhibitor, (a) Detecting the presence or absence of at least one mutation selected from the first group consisting of mutations of RAD50, mutations of MRE11, mutations of NBN, mutations of DNA2, and mutations of RBBP8 in cancer cells contained in a sample derived from the cancer patient; (b) Selecting, as a subject for cancer treatment with a helicase inhibitor, a cancer patient in which the mutation is detected in the cancer cells; A method comprising the same.

[21] A method for treating cancer, For a cancer patient in whom at least one mutation selected from the second group consisting of mutations of RAD50, mutations of MRE11, mutations of NBN, mutations of DNA2, and mutations of RBBP8 is detected in cancer cells contained in a sample derived from the cancer patient, the step of administering a helicase inhibitor A method comprising

[22] A method for treating cancer, (a) The step of detecting the presence or absence of at least one mutation selected from the second group consisting of mutations of RAD50, mutations of MRE11, mutations of NBN, mutations of DNA2, and mutations of RBBP8 in cancer cells contained in a sample derived from a cancer patient; (b) The step of administering a helicase inhibitor to a cancer patient in whom the mutation is detected in the cancer cells; A method comprising

[23] The method according to any one of claims

[15] to

[22] , wherein the helicase inhibitor is a WRN inhibitor.

[24] The method according to any one of

[15] to

[23] , wherein the cancer cells are cancer cells in which at least one mutation selected from the third group consisting of mutations of EXO1, mutations of RPA1, mutations of RPA2, and mutations of RPA3 is further detected.

[25] A method for screening a compound for use in the treatment of cancer, the method comprising cancer cells in which at least one mutation selected from the second group consisting of mutations of RAD50, mutations of MRE11, mutations of NBN, mutations of DNA2, and mutations of RBBP8 is detected, The step of selecting a compound using the inhibition of helicase as an indicator A method comprising

[26] A cancer therapeutic agent, which contains a compound that inhibits helicase as an active ingredient and is for the treatment of cancer, the cancer comprising cancer cells in which at least one mutation selected from the second group consisting of mutations of RAD50, mutations of MRE11, mutations of NBN, mutations of DNA2, and mutations of RBBP8 is detected.

[27] The method according to

[25] , wherein the helicase is WRN.

[28] The cancer therapeutic agent according to

[26] , wherein the helicase is WRN. [Advantages of the Invention]

[0014] According to the present invention, at least one mutation selected from the first group consisting of mutations of TTK and mutations of RAD50, and / or at least one mutation selected from the second group consisting of mutations of RAD50, mutations of MRE11, mutations of NBN, mutations of DNA2, and mutations of RBBP8 are used as indices, and it becomes possible to efficiently predict the sensitivity to cancer treatment with a helicase inhibitor. Further, according to the present invention, the presence or absence of the above mutations in a sample derived from a cancer patient is detected, and after selecting a patient in whom the mutation is detected, the patient can be treated with a helicase inhibitor for cancer. Therefore, it becomes possible to greatly improve the treatment results of cancer. Further, by using an oligonucleotide probe or primer for at least one gene selected from the first group consisting of TTK and RAD50, and / or at least one gene selected from the second group consisting of RAD50, MRE11, NBN (gene encoding NBS1), DNA2, and RBBP8 (gene encoding CtIP), and an antibody against at least one protein selected from the first group consisting of TTK protein and RAD50 protein, and / or at least one protein selected from the second group consisting of RAD50 protein, MRE11 protein, NBS1 protein, DNA2 protein, and CtIP protein, it becomes possible to efficiently perform companion diagnosis by detecting the presence or absence of the above mutations. [Brief Description of the Drawings]

[0015]

Figure 1

Figure 2

Figure 3

Mode for Carrying Out the Invention

[0016] Hereinafter, the present invention will be described in detail according to its preferred embodiments.

[0017] <Method for predicting the sensitivity of cancer cells to a helicase inhibitor, method for predicting the sensitivity of a cancer patient to treatment with a helicase inhibitor, method for selecting a cancer patient to be treated with a helicase inhibitor> In the present invention, at least one mutation selected from the first group consisting of a mutation of TTK and a mutation of RAD50 (hereinafter sometimes referred to as "TTK mutation and / or RAD50 mutation" or "mutation of the first group" in this specification), preferably a loss-of-function mutation, has been found to be able to suppress the growth of cancer cells when a helicase is inhibited in cancer cells in which the mutation has occurred. Further, in addition, at least one mutation selected from the second group consisting of a mutation of RAD50, a mutation of MRE11, a mutation of NBN, a mutation of DNA2, and a mutation of RBBP8 (hereinafter sometimes referred to as "mutation of the second group" in this specification), preferably a loss-of-function mutation, has also been found to be able to suppress the growth of cancer cells when a helicase is inhibited in cancer cells in which the mutation has occurred.

[0018] Based on these findings, the sensitivity of cancer cells to a helicase inhibitor can be predicted using the mutation of the first group and / or the mutation of the second group as an index. Therefore, the present invention provides (a) a step of detecting the presence or absence of at least one mutation selected from the first group consisting of a mutation of TTK and a mutation of RAD50 in cancer cells; (b) a step of predicting that the cancer cells in which the mutation has been detected are sensitive to a helicase inhibitor; A method for predicting the sensitivity of cancer cells to a helicase inhibitor, comprising: The step of detecting the presence or absence of at least one mutation selected from the second group consisting of mutations of RAD50, mutations of MRE11, mutations of NBN, mutations of DNA2, and mutations of RBBP8 in cancer cells; The step of predicting that the cancer cells in which the mutation is detected are sensitive to a helicase inhibitor; Provided is a method for predicting the sensitivity of cancer cells to a helicase inhibitor (hereinafter, these are collectively referred to as, optionally, the "cancer cell sensitivity prediction method").

[0019] Also, based on the above findings, the sensitivity to cancer treatment with a helicase inhibitor can be predicted using the mutations in the first group and / or the mutations in the second group as indicators. Therefore, the present invention The step of detecting the presence or absence of at least one mutation selected from the first group consisting of mutations of TTK and mutations of RAD50 in cancer cells contained in a sample derived from a cancer patient; The step of predicting that the cancer patient in which the mutation is detected in the cancer cells is sensitive to treatment with a helicase inhibitor; A method for predicting the sensitivity of a cancer patient to treatment with a helicase inhibitor, comprising: The step of detecting the presence or absence of at least one mutation selected from the second group consisting of mutations of RAD50, mutations of MRE11, mutations of NBN, mutations of DNA2, and mutations of RBBP8 in cancer cells contained in a sample derived from a cancer patient; The step of predicting that the cancer patient in which the mutation is detected in the cancer cells is sensitive to treatment with a helicase inhibitor; Provided is a method for predicting the sensitivity of a cancer patient to treatment with a helicase inhibitor (hereinafter, these are collectively referred to as, optionally, the "cancer patient sensitivity prediction method").

[0020] Furthermore, since patients in whom the mutations of the first group and / or the mutations of the second group are detected in this way can be said to be suitable for the treatment of cancer with a helicase inhibitor, patients who are effective and those who are not effective in cancer treatment with a helicase inhibitor can be selected using the mutations of the first group and / or the mutations of the second group as indicators, and efficient treatment can be performed. Therefore, the present invention provides (a) a step of detecting the presence or absence of at least one mutation selected from the first group consisting of mutations of TTK and mutations of RAD50 in cancer cells contained in a sample derived from a cancer patient; (b) a step of selecting, as a subject for cancer treatment with a helicase inhibitor, a cancer patient in whom the mutation has been detected in the cancer cells; A method for selecting a cancer patient to be treated with a helicase inhibitor, comprising: and (a) a step of detecting the presence or absence of at least one mutation selected from the first group consisting of mutations of RAD50, mutations of MRE11, mutations of NBN, mutations of DNA2, and mutations of RBBP8 in cancer cells contained in a sample derived from a cancer patient; (b) a step of selecting, as a subject for cancer treatment with a helicase inhibitor, a cancer patient in whom the mutation has been detected in the cancer cells; A method for selecting a cancer patient to be treated with a helicase inhibitor (hereinafter, these are collectively referred to as, in some cases, "cancer patient selection method") is provided.

[0021] (Sample, etc.) In the present invention, malignant neoplasms such as cancer (epithelial tumor), leukemia, malignant lymphoma, myeloma, sarcoma, carcinosarcoma, etc. are collectively referred to as "cancer", and the cells constituting the cancer are referred to as "cancer cells". Examples of cancers containing cancer cells capable of detecting the presence or absence of the mutations in the first group and / or the mutations in the second group include, but are not limited to, colorectal cancer, gastric cancer, cervical cancer, endometrial cancer, prostate cancer, esophageal cancer, breast cancer, lung cancer, bladder cancer, head and neck cancer, kidney cancer, ovarian cancer, lymphoma, adenoid cystic cancer, pancreatic cancer. In the present invention, the cancer cells may be cancer cells further having a mutation in MSH3 described later, or cancer cells further having at least one mutation selected from the third group consisting of mutations in EXO1, mutations in RPA1, mutations in RPA2, and mutations in RPA3. When detecting the mutations in the first group, it is preferably cancer cells further having a mutation in MSH3. When detecting the mutations in the second group, it is preferably cancer cells further having a mutation in the third group.

[0022] In the present invention, the "cancer patient" may be not only a human suffering from the cancer but also a human suspected of suffering from the cancer. In the method of the present invention, there is no particular limitation on the cancer patient to be detected for the mutations in the first group and / or the mutations in the second group, and all cancer patients can be targeted.

[0023] The "sample derived from a cancer patient" used in the present invention is not particularly limited as long as it is a biological sample capable of detecting the presence or absence of the mutations in the first group and / or the mutations in the second group, but is preferably a cancer biopsy specimen, blood, urine, body cavity fluid, a specimen such as circulating tumor DNA (ctDNA) derived from tumor cells, etc. Further, it may be a protein extract or a nucleic acid extract (mRNA extract, cDNA preparation or cRNA preparation prepared from the mRNA extract, etc.) obtained from the specimen. In the present specification, the "biological sample" includes a sample derived from a cancer patient and a sample derived from a cancer cell culture.

[0024] (Helicase inhibitor) The "helicase inhibitor" in the present invention refers to a composition containing at least one compound that inhibits helicase, which may consist only of the said compound or its combination, or may further contain the following additive components. The "compound that inhibits helicase" in the present invention includes compounds that inhibit at least either the activity or the expression of helicase.

[0025] The "helicase" targeted by the helicase inhibitor in the present invention is not particularly limited. For example, it includes RecQ helicases (RecQL1, BLM, WRN, RecQL4 / RTS, RecQL5), preferably a RecQ helicase, and more preferably WRN (Werner syndrome protein). The typical nucleotide sequence of genomic DNA encoding human-derived natural WRN is shown in SEQ ID NO: 1, and the typical amino acid sequence of human-derived natural WRN is shown in SEQ ID NO: 2. Note that even for a WRN gene (a gene encoding WRN) that has not undergone mutations such as substitution, deletion, insertion, and addition of amino acid sequences, individual differences may occur in the sequence due to polymorphisms and the like.

[0026] The inhibition of the helicase activity by a compound can be confirmed, for example, in a system containing a fluorescent molecule, double-stranded DNA labeled with quenching molecules on each strand, and the said helicase, where when the helicase activity unwinds the double-stranded DNA, the fluorescent molecule and the quenching molecule separate and emit fluorescence. By adding a test compound to this system and detecting the suppression of the fluorescence generation (Sommers JA et al., A high-throughput screen to identify novel small molecule inhibitors of the Werner Syndrome Helicase-Nuclease (WRN), PLoS One. 2019 Jan 9;14(1):e0210525).

[0027] In addition, the inhibition of the expression of the helicase by the compound can be confirmed, for example, by detecting a decrease in the expression of the helicase (preferably WRN) in cells treated with the test compound. As a method for detecting a decrease in the expression of the helicase, usually, the expression level of the helicase is detected at the transcriptional level or the translational level, and in comparison with a control (for example, the expression level of cells not treated with the test compound), it is confirmed that the expression level is lower than that.

[0028] In the method for detecting the expression level of the helicase at the transcriptional level, first, RNA or cDNA is prepared from cells treated with the test compound. The method for extracting RNA from the cells is not particularly limited, and known methods can be appropriately selected and used. For example, an extraction method using phenol and chaotropic salts (more specifically, an extraction method using a commercially available kit such as TRIzol (manufactured by Invitrogen), Isogen (manufactured by Wako Pure Chemical Industries, Ltd.), etc.), and other commercially available kits (RNAPrep total RNA extraction kit (manufactured by Beckman Coulter), RNeasy Mini (manufactured by QIAGEN), RNA Extraction Kit (manufactured by Pharmacia Biotech), etc.) can be mentioned. Furthermore, the reverse transcriptase used for preparing cDNA from the extracted RNA is not particularly limited, and examples include reverse transcriptases derived from retroviruses such as RAV (Rous associated virus) and AMV (Avian myeloblastosis virus), and reverse transcriptases derived from mouse retroviruses such as MMLV (Moloney murine leukemia virus).

[0029] Next, an oligonucleotide primer or an oligonucleotide probe is used in an amplification reaction or a hybridization reaction, respectively, and the amplification product or the hybrid product is detected. As such methods, for example, RT-PCR method, Northern blot method, dot blot method, DNA array method, in situ hybridization method, RNase protection assay method, mRNA-seq, etc. can be utilized. A person skilled in the art can design an oligonucleotide primer or an oligonucleotide probe suitable for each method by a conventional method based on the nucleotide sequence of the cDNA encoding the helicase.

[0030] In the method for detecting the expression level of the helicase at the translation level, first, a protein sample is prepared from cells treated with a test compound. Next, an antigen-antibody reaction is performed using an antibody specific to the helicase to detect the helicase. In such a method for detecting a protein using an antibody, for example, an antibody specific to the helicase is added to the protein sample to perform an antigen-antibody reaction, and the binding of the antibody to the helicase is detected. When the antibody specific to the helicase is labeled, the helicase can be directly detected. However, when it is not labeled, a labeled molecule (for example, a secondary antibody or protein A) that recognizes the antibody is further allowed to act, and the helicase can be indirectly detected using the label of the molecule. As such methods, for example, immunohistochemistry (immunostaining) method, Western blotting method, ELISA method, flow cytometry, imaging cytometry, radioimmunoassay, immunoprecipitation method, analysis method using an antibody array, etc. can be utilized.

[0031] There are no particular restrictions on the type and origin of the antibody to be used, but monoclonal antibodies are preferred. As long as the helicase can be detected with sufficient specificity, oligoclonal antibodies (a mixture of several to several dozen antibodies) or polyclonal antibodies can also be used. In addition, functional fragments of antibodies such as Fab, Fab’, F(ab’)2, Fv, scFv, sc(Fv)2, dsFv, and diabodies, and their multimers (e.g., dimers, trimers, tetramers, polymers) can also be used. The anti-helicase antibody may be a commercially available product.

[0032] The detection of the helicase can also be carried out using mass spectrometry (MS). In particular, analysis by a mass spectrometer (LC / MS) coupled with liquid chromatography is advantageous because it is sensitive. Detection by mass spectrometry can be performed, for example, by labeling the protein in the protein sample, fractionating the labeled protein, subjecting the fractionated protein to mass spectrometry, and identifying the helicase from the mass spectrometry values. As the label, an isotope-labeled reagent known in the art can be used, and a suitable labeled reagent can be obtained as a commercially available product. The fractionation can also be carried out by a method known in the art, for example, using a commercially available strong cation column or the like.

[0033] The "compound that inhibits helicase" in the present invention is not particularly limited, and it may be a known compound or a compound identified by the screening described below. However, it is preferably at least one selected from the group consisting of compounds, polypeptides, and polynucleotides.

[0034] Examples of the compound include low molecular weight compounds (molecular weight less than 900) and medium molecular weight compounds (molecular weight 900 to 2000). Examples of the polypeptide include full-length polypeptides encoded by genes, as well as fragments thereof, synthetic polypeptides, cyclic polypeptides, glycopeptides, and non-natural polypeptides. The polypeptide also includes antibodies and antigenic peptides, and the antibody may be a polyclonal antibody or a monoclonal antibody. The antibody includes, in addition to a complete antibody, antibody fragments (e.g., Fab, Fab’, F(ab’)2, Fv, scFv, sc(Fv)2, dsFv, and diabody, etc.), multimers thereof, and low-molecular-weight antibodies in which the variable region of the antibody is bound. Examples of the polynucleotide include DNA, RNA, and siRNA, and in addition to full-length polynucleotides, fragments thereof and synthetic polynucleotides are also included.

[0035] In the present invention, the "helicase inhibitor" can be made into various dosage forms such as tablets, pills, powders, granules, capsules, and solutions according to its properties. Further, according to the dosage form, it may further contain pharmacologically acceptable additive components such as sterilized water, physiological saline, vegetable oil, solvent, base, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, fragrance, excipient, vehicle, preservative, binder, diluent, isotonic agent, soothing agent, bulking agent, disintegrant, buffer, coating agent, lubricant, coloring agent, sweetening agent, thickening agent, taste and odor correcting agent, solubilizing agent, etc., and can be produced by known pharmaceutical methods using these.

[0036] Further, in the helicase inhibitor according to the present invention, the content of the compound that inhibits the helicase (when there are two or more of the compounds, the total content thereof) can be appropriately adjusted according to the dosage form and the purpose of use.

[0037] (Mutation of Group 1, Mutation of Group 2) [[TTK]] "TTK" in the present invention is a gene encoding a serine-threonine kinase (hereinafter sometimes referred to as "TTK protein") involved in the control of centrosome replication and the mitotic checkpoint response. The typical nucleotide sequence of natural-type TTK genomic DNA derived from humans is shown in SEQ ID NO: 3, and the typical amino acid sequence of natural-type TTK protein derived from humans is shown in SEQ ID NO: 4. Note that even for TTK that has not undergone mutations such as substitution, deletion, insertion, and addition of amino acid sequences, individual differences may occur in the sequence due to polymorphisms and the like.

[0038] "Mutations of TTK" in the present invention include substitution, deletion, insertion, and addition of amino acids in the amino acid sequence of the TTK protein. Such mutations of TTK are not particularly limited as long as the activity originally possessed by the TTK protein is changed, but mutations that cause a decrease in TTK protein activity (including complete loss (inactivation) of TTK protein activity), that is, loss-of-function type mutations, are preferred. A decrease in TTK protein activity can occur, for example, due to missense mutations in TTK, nonsense mutations throughout the entire region, or changes in gene structure such as deletion of all or part of TTK, or changes in gene expression levels, but is not limited thereto.

[0039] Whether the gene structure and gene expression level originally possessed by TTK have changed can be confirmed and determined, for example, by the following methods: obtaining the nucleotide sequence of TTK by sequencing genomic DNA; fluorescence detection using an oligonucleotide probe that specifically binds to the nucleotide sequence of TTK; detection by PCR using an oligonucleotide primer that specifically binds to the nucleotide of TTK, etc., by comparing with a control (for example, a healthy person or non-cancerous tissue of the same patient) to determine whether there is a change (preferably whether a loss-of-function type mutation has occurred, and in the case of mRNA expression level, whether it has decreased).

[0040] Whether the activity (functional activity) originally possessed by the TTK protein has changed can be confirmed and determined, for example, by the following methods: detection by immunostaining or Western blotting using an antibody that specifically binds to the TTK protein; and a method for determining whether the intracellular TTK protein purified by immunoprecipitation or the like phosphorylates a substrate peptide by Western blotting or the like. By comparing with a control (for example, a healthy subject or non-cancerous tissue of the same patient), it can be determined whether there is a change (preferably a decrease, that is, in the case of the expression level of the protein detected by immunostaining or Western blotting, whether it is lower than that of the control; in the case of the molecular weight of the protein detected by Western blotting, whether it has changed compared to the control; in the case of phosphorylation activity, whether the activity is lower than that of the control).

[0041] Examples of specific TTK mutations that cause such changes in TTK protein activity include, for example, p.L84*(COSMIC Legacy Mutation ID: COSM1643150), p.S162Vfs*9(COSMIC Legacy Mutation ID: COSM3176137), p.K192Sfs*18(COSMIC Legacy Mutation ID: COSM1446079), p.Q193Afs*33(COSMIC Legacy Mutation ID: COSM3176143), p.R232Sfs*26(COSMIC Legacy Mutation ID: COSM5895418), p.Q480Hfs*30(COSMIC Legacy Mutation ID: COSM150902), p.N606Kfs*3(COSMIC Legacy Mutation ID: COSM7741406), p.S618Ifs*3(COSMIC Legacy Mutation ID: COSM6811382), p.E851Kfs*42(COSMIC Legacy Mutation ID: COSM3176214), p.R854Gfs*10(COSMIC Legacy Mutation ID: COSM1446097), p.R854Gfs*10(COSMIC Legacy Mutation ID: COSM3176218), p.R854Gfs*39(COSMIC Legacy Mutation ID: COSM252896), p.R854Kfs*11(COSMIC Legacy Mutation ID: COSM253159), p.K857Nfs*36(COSMIC Legacy Mutation ID: COSM273397), and the like.

[0042] 〔RAD50〕 "RAD50" in the present invention is a gene that encodes a protein (hereinafter sometimes referred to as "RAD50 protein") that forms a complex (MRN complex) together with the following MRE11 protein and NBS1 protein and is involved in a DNA repair mechanism (particularly, a double-stranded DNA homologous end repair mechanism). A typical nucleotide sequence of a natural RAD50 genomic DNA derived from a human is shown in SEQ ID NO: 5, and a typical amino acid sequence of a natural RAD50 protein derived from a human is shown in SEQ ID NO: 6. Note that even RAD50 that has not undergone mutations such as substitution, deletion, insertion, and addition of amino acid sequences may have individual differences in the sequence due to polymorphisms and the like.

[0043] "Mutations of RAD50" in the present invention include substitution, deletion, insertion, and addition of amino acids in the amino acid sequence of the RAD50 protein. Such mutations of RAD50 are not particularly limited as long as the activity originally possessed by the RAD50 protein is changed, but mutations that cause a decrease in RAD50 protein activity (including complete loss (inactivation) of RAD50 protein activity), that is, loss-of-function type mutations, are preferred. A decrease in RAD50 protein activity can occur, for example, due to missense mutations in RAD50, nonsense mutations throughout the entire region, or changes in gene structure such as deletion of all or part of RAD50, or changes in gene expression levels, but is not limited thereto.

[0044] Whether the gene structure and gene expression level originally possessed by RAD50 have changed can be confirmed and determined by, for example, the following methods: obtaining the nucleotide sequence of RAD50 by sequencing genomic DNA; fluorescence detection using an oligonucleotide probe that specifically binds to the nucleotide sequence of RAD50; detection by PCR using an oligonucleotide primer that specifically binds to the nucleotide of RAD50, etc., by comparing with a control (for example, a healthy person or non-cancerous tissue of the same patient) to determine whether there is a change (preferably whether a loss-of-function type mutation has occurred, and in the case of the expression level of mRNA, whether it has decreased).

[0045] Whether the activity (functional activity) originally possessed by the RAD50 protein has changed can be determined, for example, by the following methods: detection by immunostaining or Western blotting using an antibody that specifically binds to the RAD50 protein; and a method for determining whether the intracellular RAD50 protein purified by immunoprecipitation or the like binds to the MRE11 protein or NBS1 protein, which are components of the MRN complex, by Western blotting or the like; and a method for determining whether the intracellular RAD50 protein purified by immunoprecipitation or the like has ATPase activity by an ATPase activity measurement method. Whether there is a change (preferably a decrease) compared to a control (for example, a healthy person or non-cancerous tissue of the same patient) can be confirmed and determined by whether there is a change (that is, in the case of the expression level of the RAD50 protein detected by immunostaining or Western blotting, whether it is decreased compared to the control; in the case of the molecular weight of the protein detected by Western blotting, whether it has changed compared to the control; in the case of the amount of the MRE11 protein or NBS1 protein, which are components of the MRN complex, that binds to the RAD50 protein detected by Western blotting, whether it is decreased compared to the control; in the case of ATPase activity, whether the activity is decreased compared to the control).

[0046] Examples of specific RAD50 mutations that cause such changes in RAD50 protein activity include, for example, p.A149Gfs*10 (COSMIC Legacy Mutation ID: COSM7087398), p.C157Lfs*7 (COSMIC Legacy Mutation ID: COSM9001198), p.S181Ffs*7 (COSMIC Legacy Mutation ID: COSM9008445), p.K279Efs*7 (COSMIC Legacy Mutation ID: COSM5016099), p.T410Lfs*5 (COSMIC Legacy Mutation ID: COSM6941414), p.K425Tfs*4 (COSMIC Legacy Mutation ID: COSM9494174), p.L439Kfs*4 (COSMIC Legacy Mutation ID: COSM1158978), p.N459Mfs*2 (COSMIC Legacy Mutation ID: COSM8515312), p.L541Afs*7 (COSMIC Legacy Mutation ID: COSM6971853), p.R617Efs*26 (COSMIC Legacy Mutation ID: COSM4747889), p.D675Tfs*45 (COSMIC Legacy Mutation ID: COSM5016101), p.Q689Rfs*31 (COSMIC Legacy Mutation ID: COSM6761894), p.K722Gfs*5 (COSMIC Legacy Mutation ID: COSM6048265), p.K722Rfs*14 (COSMIC Legacy Mutation ID: COSM1433045), p.E723Gfs*5 (COSMIC Legacy Mutation ID: COSM4611459), p.K722Nfs*6, p.L929Sfs*10 (COSMIC Legacy Mutation ID: COSM1740881), p.N934Ifs*6 (COSMIC Legacy Mutation ID: COSM1433049), p.N934Kfs*10 (COSMIC Legacy Mutation ID: COSM1287518), p.Examples include E995Rfs*2 (COSMIC Legacy Mutation ID: COSM6962279), p.L1042Ffs*15 (COSMIC Legacy Mutation ID: COSM5617248), p.Y1182Lfs*2 (COSMIC Legacy Mutation ID: COSM1633926), etc.

[0047] [[MRE11, NBN]] "MRE11" (also known as "MRE11A") in the present invention is a gene encoding a protein (sometimes referred to as the "MRE11 protein" in this specification) that forms a complex (MRN complex) with the above RAD50 protein. The MRE11 protein is a nuclease that can degrade DNA as an exonuclease at the ends of DNA and as an endonuclease at internal sites. Also, "NBN" in the present invention is a gene encoding NBS1 (sometimes referred to as the "NBS1 protein" in this specification), which is a protein that forms a complex (MRN complex) with the above RAD50 protein. These proteins are related to each other and are involved in the DNA repair mechanism (especially the double-stranded DNA homologous end-joining repair mechanism) (for example, Lei Bian et al., Molecular Cancer, (2019) 18:169, DOI: https: / / doi.org / 10.1186 / s12943-019-1100-5; Kwi H Koh et al., Laboratory Investigation, (2005) 85, p. 1130-1138, etc.). If the function of any one of these is deleted, the function of the MRN complex itself decreases and the above DNA repair mechanism does not function properly. Therefore, among the second group of mutations, for RAD50, MRE11, and NBN that encode proteins forming such an MRN complex, it is particularly preferable to detect at least one mutation among them, and it is more preferable to detect at least one mutation among the mutations of RAD50 and NBN.

[0048] The typical nucleotide sequence of human-derived natural MRE11 genomic DNA is shown in SEQ ID NO:9, and the typical amino acid sequence of human-derived natural MRE11 protein is shown in SEQ ID NO:10. Also, the typical nucleotide sequence of human-derived natural NBN genomic DNA (genomic DNA encoding NBS1) is shown in SEQ ID NO:11, and the typical amino acid sequence of human-derived natural NBS1 protein is shown in SEQ ID NO:12. Note that even MRE11 and NBN without mutations such as substitution, deletion, insertion, and addition of amino acids in the amino acid sequence may have individual differences in the sequence due to polymorphisms and the like.

[0049] In the present invention, the "mutation of MRE11" and "mutation of NBN" include, respectively, substitution, deletion, insertion, and addition of amino acids in the amino acid sequence of MRE11 protein and the amino acid sequence of NBS1 protein. Such mutations of MRE11 and NBN are not particularly limited as long as the activities originally possessed by MRE11 protein and NBS1 protein are respectively changed. However, mutations that cause a decrease in MRE11 protein activity (including complete loss (inactivation) of MRE11 protein activity) and a decrease in NBS1 protein activity (including complete loss (inactivation) of NBS1 protein activity), that is, loss-of-function type mutations are preferred. A decrease in MRE11 protein activity can occur, for example, due to missense mutations in MRE11, nonsense mutations throughout the entire region, or changes in gene structure such as deletion of the whole or part of MRE11, or changes in gene expression levels, but is not limited thereto. A decrease in NBS1 protein activity can occur, for example, due to missense mutations in NBN, nonsense mutations throughout the entire region, or changes in gene structure such as deletion of the whole or part of NBN, or changes in gene expression levels, but is not limited thereto.

[0050] Whether the gene structure and gene expression level originally possessed by MRE11, and whether the gene structure and gene expression level originally possessed by NBN have changed, respectively, and whether the activity (functional activity) originally possessed by the MRE11 protein and the activity (functional activity) originally possessed by the NBS1 protein have changed, respectively, can be confirmed and determined by, for example, the same methods as those for confirming and determining whether the gene structure and gene expression level originally possessed by RAD50 have changed, and whether the activity (functional activity) originally possessed by the RAD50 protein has changed. For the MRE11 protein, it can also be confirmed and determined by, for example, a method of measuring whether the intracellular MRE11 protein purified by immunoprecipitation or the like cleaves substrate DNA, and comparing it with a control (for example, a healthy person or non-cancerous tissue of the same patient) to determine whether there is a change (preferably whether there is a decrease, that is, whether the activity is lower than that of the control).

[0051] Examples of specific MRE11 mutations that cause changes in MRE11 protein activity include, for example, p.I93Ffs*17 (COSMIC Legacy Mutation ID: COSM6975993), p.R188Kfs*9 (COSMIC Legacy Mutation ID: COSM8923701), p.V198* (COSMIC Legacy Mutation ID: COSM6938551), p.F321Lfs*8 (COSMIC Legacy Mutation ID: COSM6927045), p.N322* (COSMIC Legacy Mutation ID: COSM5176176), p.F399Sfs*29 (COSMIC Legacy Mutation ID: COSM2061331), p.T481Hfs*43 (COSMIC Legacy Mutation ID: COSM2061313), p.Q482Afs*4 (COSMIC Legacy Mutation ID: COSM6909136), p.N511Ifs*13 (COSMIC Legacy Mutation ID: COSM1357925), p.A526Gfs*16 (COSMIC Legacy Mutation ID: COSM7513337), p.Q629Afs*9 (COSMIC Legacy Mutation ID: COSM6920246), p.D647Yfs*28 (COSMIC Legacy Mutation ID: COSM6962317), and the like.

[0052] In addition, as examples of specific NBN mutations that cause changes in NBS1 protein activity, for example, p.N30Tfs*5 (COSMIC Legacy Mutation ID: COSM6978819), p.D61* (COSMIC Legacy Mutation ID: COSM7449862), p.S72Lfs*20 (COSMIC Legacy Mutation ID: COSM8559558), p.M83Cfs*9 (COSMIC Legacy Mutation ID: COSM6722467), p.G103Efs*6 (COSMIC Legacy Mutation ID: COSM391695), p.K125Rfs*34 (COSMIC Legacy Mutation ID: COSM28402), p.V153Kfs*17 (COSMIC Legacy Mutation ID: COSM6959155), p.G206Lfs*26 (COSMIC Legacy Mutation ID: COSM6981006), p.K219Nfs*16 (COSMIC Legacy Mutation ID: COSM1740923), p.K233Sfs*5 (COSMIC Legacy Mutation ID: COSM9494223), p.S240Cfs*8 (COSMIC Legacy Mutation ID: COSM6924583), p.F316Sfs*2 (COSMIC Legacy Mutation ID: COSM7450031), p.N440Kfs*2 (COSMIC Legacy Mutation ID: COSM7513565), p.R466Gfs*18 (COSMIC Legacy Mutation ID: COSM1458550), p.R466Kfs*5 (COSMIC Legacy Mutation ID: COSM8498945), p.L490* (COSMIC Legacy Mutation ID: COSM2790257), p.N503Kfs*2 (COSMIC Legacy Mutation ID: COSM9061551), p.E505Gfs*6 (COSMIC Legacy Mutation ID: COSM7513880), p.R551Gfs*8 (COSMIC Legacy Mutation ID: COSM1458549), p.R551Kfs*5 (COSMIC Legacy Mutation ID: COSM6918499), p.M553Wfs*6 (COSMIC Legacy Mutation ID: COSM6955358), p.L654Afs*5 (COSMIC Legacy Mutation ID: COSM1458548), p.A713Gfs*29 (COSMIC Legacy Mutation ID: COSM6983893), p.N731Ifs*20 (COSMIC Legacy Mutation ID: COSM30401), etc. are included.

[0053] 〔DNA2〕 "DNA2" in the present invention is a gene that encodes a protein (hereinafter sometimes referred to as "DNA2 protein") that is involved in the DNA repair mechanism (particularly, the double-stranded DNA homologous end-joining repair mechanism) together with the MRN complex. The DNA2 protein is a nuclease that can degrade DNA as an exonuclease at the ends of DNA and as an endonuclease at internal sites, and is also a helicase that can unwind double-stranded DNA into single strands. The typical nucleotide sequence of the human-derived native DNA2 genomic DNA is shown in SEQ ID NO: 13, and the typical amino acid sequence of the human-derived native DNA2 protein is shown in SEQ ID NO: 14. Note that even DNA2 that has not undergone mutations involving substitutions, deletions, insertions, and additions of amino acid sequences may have individual differences in the sequence due to polymorphisms, etc.

[0054] Examples of the "mutation of DNA2" in the present invention include substitution, deletion, insertion, and addition of amino acids in the amino acid sequence of the DNA2 protein. Such mutations of DNA2 are not particularly limited as long as the activities originally possessed by the DNA2 protein are each changed, but mutations that cause a decrease in DNA2 protein activity (including complete loss (inactivation) of DNA2 protein activity), that is, loss-of-function mutations, are preferred. A decrease in DNA2 protein activity can be caused by, for example, but not limited to, missense mutations in DNA2, nonsense mutations spanning the entire region, or changes in gene structure such as deletion of all or part of DNA2, or changes in gene expression levels.

[0055] Whether the gene structure and gene expression level originally possessed by DNA2 have changed, and whether the activity (functional activity) originally possessed by the DNA2 protein has changed can be confirmed and determined by the same methods as those for confirming and determining whether the gene structure and gene expression level originally possessed by RAD50 have changed, and whether the activity (functional activity) originally possessed by the RAD50 protein has changed.

[0056] Examples of specific mutations in DNA2 that cause such changes in DNA2 protein activity include, for example, p.K590Nfs*5 (COSMIC Legacy Mutation ID: COSM8485077), p.L697Ffs*28 (COSMIC Legacy Mutation ID: COSM1348694), p.L776Ffs*9 (COSMIC Legacy Mutation ID: COSM5081629), p.L776Pfs*24 (COSMIC Legacy Mutation ID: COSM7631030), p.S779Tfs*20 (COSMIC Legacy Mutation ID: COSM4747435), p.S779Ffs*7 (COSMIC Legacy Mutation ID: COSM5092627), p.S779Hfs*6 (COSMIC Legacy Mutation ID: COSM295321), p.S779Ffs*21 (COSMIC Legacy Mutation ID: COSM2159320), p.V825Cfs*5 (COSMIC Legacy Mutation ID: COSM6645872), p.I940Lfs*8 (COSMIC Legacy Mutation ID: COSM5423531), p.S975Vfs*4 (COSMIC Legacy Mutation ID: COSM1727571), and the like.

[0057] [[RBBP8]] "RBBP8" in the present invention is a gene that encodes CtIP (hereinafter sometimes referred to as "CtIP protein"), which is a protein involved in the DNA repair mechanism (particularly, the double-stranded DNA homologous end-joining repair mechanism) together with the MRN complex. The CtIP protein is a nuclease that can degrade DNA as an endonuclease at internal sites of DNA. The typical nucleotide sequence of the natural RBBP8 genomic DNA (genomic DNA encoding CtIP) derived from human is shown in SEQ ID NO: 15, and the typical amino acid sequence of the natural CtIP protein derived from human is shown in SEQ ID NO: 16. Note that even for RBBP8 that has not undergone mutations such as substitution, deletion, insertion, and addition of amino acid sequences, individual differences may occur in the sequence due to polymorphisms and the like.

[0058] Examples of the "mutation of RBBP8" in the present invention include substitution, deletion, insertion, and addition of amino acids in the amino acid sequence of the CtIP protein. Such mutations of RBBP8 are not particularly limited as long as the activities originally possessed by the CtIP protein are each changed, but it is preferable that they are mutations that cause a decrease in CtIP protein activity (including complete loss (inactivation) of CtIP protein activity), that is, loss-of-function type mutations. A decrease in CtIP protein activity can occur, for example, due to missense mutations, nonsense mutations spanning the entire region, or changes in gene structure such as deletion of the whole or part of RBBP8, or changes in gene expression levels, but is not limited thereto.

[0059] Whether the gene structure and gene expression level originally possessed by RBBP8 have changed, and whether the activity (functional activity) originally possessed by the CtIP protein has changed can be confirmed and determined by the same methods as those described as methods for confirming and determining whether the gene structure and gene expression level originally possessed by RAD50 have changed, and whether the activity (functional activity) originally possessed by the RAD50 protein has changed.

[0060] Examples of specific RBBP8 mutations that cause such changes in CtIP protein activity include, for example, p.R100Pfs*8 (COSMIC Legacy Mutation ID: COSM1744943), p.H183Pfs*11 (COSMIC Legacy Mutation ID: COSM9138797), p.S231 M235del (COSMIC Legacy Mutation ID: COSM7203765), p.L286Tfs*24 (COSMIC Legacy Mutation ID: COSM1745525), p.K357Nfs*3 (COSMIC Legacy Mutation ID: COSM8188329), p.H358Tfs*8 (COSMIC Legacy Mutation ID: COSM1744941), p.T375Nfs*2 (COSMIC Legacy Mutation ID: COSM8188329), p.E455Tfs*2 (COSMIC Legacy Mutation ID: COSM4720593), p.F479Efs*4 (COSMIC Legacy Mutation ID: COSM1263883), p.F650Kfs*16 (COSMIC Legacy Mutation ID: COSM4189100), p.V672Efs*2 (COSMIC Legacy Mutation ID: COSM1190926), p.K801Efs*14 (COSMIC Legacy Mutation ID: COSM7088614), p.E803Rfs*12 (COSMIC Legacy Mutation ID: COSM2885323), p.L808Tfs*7 (COSMIC Legacy Mutation ID: COSM7513270), and the like.

[0061] 〔Detection of mutation〕 Each of the methods for "detection of TTK mutation", "detection of RAD50 mutation", "detection of MRE11 mutation", "detection of NBN mutation", "detection of DNA2 mutation", and "detection of RBBP8 mutation" in the present invention is not particularly limited, and each independently, for example, the following methods can be mentioned.

[0062] In the present invention, "mutation detection" means detecting mutations in each gene on genomic DNA. When the mutations on the genomic DNA are reflected in changes in bases in the transcription products or changes in amino acids in the translation products, the detection of mutations in the gene includes detecting such changes in these transcription products or translation products (i.e., indirect detection).

[0063] A preferred embodiment of the method of the present invention is a method for detecting mutations by directly determining the base sequences of gene regions (gene regions for detecting mutations in cancer cells: at least one selected from the TTK gene region and the RAD50 gene region for detecting mutations in the first group (hereinafter sometimes referred to as "gene regions of the first group" in this specification); gene regions for detecting mutations in the second group: at least one selected from the RAD50 gene region, the MRE11 gene region, the NBN gene region, the DNA2 gene region, and the RBBP8 gene region (hereinafter sometimes referred to as "gene regions of the second group" in this specification)). In the present invention, "TTK gene region", "RAD50 gene region", "MRE11 gene region", "NBN gene region", "DNA2 gene region", and "RBBP8 gene region" respectively mean a certain region on genomic DNA containing TTK, a certain region on genomic DNA containing RAD50, a certain region on genomic DNA containing MRE11, a certain region on genomic DNA containing NBN, a certain region on genomic DNA containing DNA2, and a certain region on genomic DNA containing RBBP8. Each of these regions independently includes an expression control region (e.g., promoter region, enhancer region) of each gene and the 3'-terminal untranslated region of each gene.

[0064] In this method, first, a DNA sample is prepared from a biological sample. Examples of the DNA sample include a genomic DNA sample and a cDNA sample prepared by reverse transcription from RNA.

[0065] There are no particular restrictions on the method for extracting genomic DNA or RNA from a biological sample, and known methods can be appropriately selected and used. For example, as a method for extracting genomic DNA, the SDS-phenol method (a method in which tissues stored in a solution containing urea or ethanol are treated with a proteolytic enzyme (proteinase K), a surfactant (SDS), and phenol to denature the proteins of the tissue, and DNA is precipitated from the tissue with ethanol for extraction), Clean Columns (registered trademark, manufactured by NexTec), AquaPure (registered trademark, manufactured by Bio-Rad), ZR Plant / Seed DNA Kit (manufactured by Zymo Research), AquaGenomicSolution (registered trademark, manufactured by Mo Bi Tec), prepGEM (registered trademark, manufactured by ZyGEM), BuccalQuick (registered trademark, manufactured by TrimGen) can be mentioned as DNA extraction methods.

[0066] In addition, as a method for extracting RNA from a biological sample and preparing cDNA from the extracted RNA, the same methods as those mentioned in the method for detecting the expression level of helicase at the transcriptional level can be mentioned.

[0067] In this embodiment, next, DNA containing the gene region of the first group or the gene region of the second group is isolated, and the nucleotide sequence of the isolated DNA is determined. The isolation of the DNA can be carried out, for example, by PCR or the like using genomic DNA or RNA as a template with a pair of oligonucleotide primers designed to sandwich all or part of the gene region of the first group or the gene region of the second group. The determination of the nucleotide sequence of the isolated DNA can be carried out by methods known to those skilled in the art such as the Maxam-Gilbert method or the Sanger method.

[0068] By comparing the determined nucleotide sequence of the DNA or cDNA with a control (for example, when the biological sample is a sample derived from a cancer patient, the nucleotide sequence of DNA or cDNA derived from a non-cancerous tissue of the same patient), it is possible to determine the presence or absence of mutations in the gene region of the first group or the gene region of the second group in the cancer cells of the biological sample.

[0069] The method for detecting mutations in the gene region of Group 1 or the gene region of Group 2 can be carried out by various methods capable of detecting mutations, in addition to the method of directly determining the nucleotide sequences of DNA or cDNA.

[0070] For example, the detection of mutations in the present invention can also be carried out by the following method. First, a DNA or cDNA sample is prepared from a biological sample. Next, an oligonucleotide probe having a nucleotide sequence complementary to the nucleotide sequence containing the mutation site in the gene region of Group 1 or the gene region of Group 2 and labeled with a reporter fluorescent dye and a quencher fluorescent dye is prepared. Then, the oligonucleotide probe is hybridized to the DNA or cDNA sample, and further, using the DNA or cDNA sample to which the oligonucleotide probe has hybridized as a template, the nucleotide sequence containing the mutation site in the gene region of Group 1 or the gene region of Group 2 is amplified. Then, the fluorescence emitted by the reporter fluorescent dye is detected due to the degradation of the oligonucleotide probe accompanying the amplification, and then the detected fluorescence is compared with a control. Examples of such methods include the double-dye probe method and the so-called TaqMan (registered trademark) probe method.

[0071] In yet another method, a DNA or cDNA sample is prepared from a biological sample. Next, in a reaction system containing an intercalator that emits fluorescence when inserted between DNA double strands, using the DNA or cDNA sample as a template, the nucleotide sequence containing the mutation site in the gene region of Group 1 or the gene region of Group 2 is amplified. 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 change in temperature is compared with a control. Examples of such methods include the HRM (high resolution melting) method.

[0072] In yet another method, first, a DNA or cDNA sample is prepared from a biological sample. Next, DNA containing all or part of the gene regions of the first group or the second group is amplified. Further, the amplified DNA is cleaved with a restriction enzyme. Next, the DNA fragments are separated according to their sizes. Then, the sizes of the detected DNA fragments are compared with a control. Such methods include, for example, methods using restriction fragment length polymorphism (RFLP) and PCR-RFLP methods.

[0073] In yet another method, first, a DNA or cDNA sample is prepared from a biological sample. Next, DNA containing all or part of the gene regions of the first group or the second group is amplified. Further, the amplified DNA is dissociated into single-stranded DNA. Next, the dissociated single-stranded DNA is separated on a non-denaturing gel. The mobility of the separated single-stranded DNA on the gel is compared with a control. Such methods include, for example, the PCR-SSCP (single-strand conformation polymorphism) method.

[0074] In yet another method, first, a DNA or cDNA sample is prepared from a biological sample. Next, DNA containing all or part of the gene regions of the first group or the second group is amplified. Further, the amplified DNA is separated on a gel with a gradually increasing concentration of a DNA denaturant. Then, the mobility of the separated DNA on the gel is compared with a control. Such methods include, for example, the denaturant gradient gel electrophoresis (DGGE) method.

[0075] In yet another method, there is a method using DNA containing a mutation site in the gene regions of the first group or the second group prepared from a biological sample and a substrate on which an oligonucleotide probe hybridizing to the DNA is immobilized. Such methods include, for example, the DNA array method.

[0076] In yet another method, first, a DNA or cDNA sample is prepared from a biological sample. Also, an oligonucleotide primer having a base sequence complementary to the base on the 3'-side of one base of all or part of the base of the first group of gene regions or the second group of gene regions and the base sequence on the 3'-side thereof is prepared. Next, using the DNA as a template and the primer, a fluorescence-labeled ddNTP primer extension reaction is carried out. Next, the primer extension reaction product is subjected to a DNA sequencer, and nucleotide sequencing is performed based on the length and fluorescence of the extension reaction product. Next, the genotype is determined from the results of the DNA sequencer. Next, the determined genotype is compared with a control. Such a method includes, for example, the Sanger method.

[0077] In yet another method, first, a DNA or cDNA sample is prepared from a biological sample. Next, an oligonucleotide probe consisting of 5'- "all or part of the bases of the first group of gene regions or the second group of gene regions and the base sequence complementary to the base sequence on the 5'-side thereof" - "the base on the 3'-side of one base of all or part of the first group of gene regions or the second group of gene regions and the base sequence that does not hybridize with the base sequence on the 3'-side thereof" - 3' (flap) is prepared. Also, an oligonucleotide probe having a base sequence complementary to all or part of the bases of the first group of gene regions or the second group of gene regions and the base sequence on the 3'-side thereof is prepared. Next, the two types of oligonucleotide probes are hybridized to the prepared DNA or cDNA sample. Next, the hybridized DNA is cleaved with a single-stranded DNA cleavage enzyme to release the flap. The single-stranded DNA cleavage enzyme is not particularly limited, and for example, cleavase can be mentioned. In this method, next, an oligonucleotide probe having a sequence complementary to the flap and labeled with a reporter fluorescence and a quencher fluorescence is hybridized to the flap. Next, the intensity of the generated fluorescence is measured. Next, the measured fluorescence intensity is compared with a control. Such a method includes, for example, the Invader method.

[0078] In yet another method, first, a DNA or cDNA sample is prepared from a biological sample. Next, DNA containing all or part of the first group of gene regions or the second group of gene regions is amplified. Then, the amplified DNA is dissociated into single strands, and only the single-stranded DNA among the dissociated single-stranded DNAs is separated. Next, an extension reaction is performed one base at a time starting from the vicinity of the bases of all or part of the first group of gene regions or the second group of gene regions, and the pyrophosphate generated during this process is enzymatically luminesced, and the intensity of the luminescence is measured. Then, the measured fluorescence intensity is compared with a control. Examples of such a method include the Pyrosequencing method.

[0079] In yet another method, first, a DNA or cDNA sample is prepared from a biological sample. Next, DNA containing all or part of the first group of gene regions or the second group of gene regions is amplified. Next, an "oligonucleotide primer having a base sequence complementary to the base on the 3'-side of the base of all or part of the first group of gene regions or the second group of gene regions and the base sequence on its 3'-side" is prepared. Next, in the presence of a fluorescence polarization dye-labeled nucleotide, using the amplified DNA as a template and the prepared primer, a single-base extension reaction is performed. Then, the degree of fluorescence polarization is measured. Next, the measured degree of fluorescence polarization is compared with a control. Examples of such a method include the AcycloPrime method.

[0080] In yet another method, first, a DNA or cDNA sample is prepared from a biological sample. Next, DNA containing all or part of the first group of gene regions or the second group of gene regions is amplified. Next, an oligonucleotide primer having a base sequence complementary to the base on the 3'-side of the base of all or part of the first group of gene regions or the second group of gene regions and the base sequence on its 3'-side is prepared. Next, in the presence of a fluorescently labeled nucleotide, a single-base extension reaction is carried out using the amplified DNA as a template and the prepared primer. Next, the base species used in the single-base extension reaction is determined. Next, the determined base species is compared with a control. As such a method, for example, the SNuPE method can be mentioned.

[0081] In addition, if the mutation is accompanied by a change in amino acid (e.g., substitution, deletion, insertion, addition) in each protein (when detecting mutations in the first group: at least one selected from TTK protein and RAD50 protein (hereinafter sometimes referred to as "the first group of proteins" in this specification); when detecting mutations in the second group: at least one selected from RAD50 protein, MRE11 protein, NBS1 protein, DNA2 protein, and CtIP protein (hereinafter sometimes referred to as "the second group of proteins" in this specification)), the sample prepared from the biological sample may be a protein. In such a case, to detect the mutation, a method using a molecule (e.g., an antibody) that specifically binds to the site where the amino acid change has occurred due to the above mutation can be utilized.

[0082] For example, in a method for detecting a protein using an antibody, first, a protein sample is prepared from a biological sample. Next, an antigen-antibody reaction is performed using an antibody specific to the protein in the first group or the protein in the second group to detect the protein in the first group or the protein in the second group. As such a method for detecting a protein using an antibody, in a method for detecting the expression level of helicase at the translation level, a method similar to the method listed as the method for detecting a protein using an antibody can be adjusted according to the protein in the first group or the protein in the second group and appropriately employed. In this method, according to immunohistochemistry, there is also an advantage that additional information such as the morphology and distribution state of cancer cells in a tissue can be obtained simultaneously.

[0083] There are no particular restrictions on the type and origin of the antibody to be used, but monoclonal antibodies are preferably used. As long as the protein in the first group or the protein in the second group can be detected with sufficient specificity, oligoclonal antibodies (a mixture of several to several dozen antibodies) or polyclonal antibodies can also be used. In addition, functional fragments of antibodies such as Fab, Fab’, F(ab’)2, Fv, scFv, sc(Fv)2, dsFv, and diabodies, and multimers thereof (for example, dimers, trimers, tetramers, polymers) can also be used. As the anti-TTK protein antibody, anti-RAD50 protein antibody, anti-MRE11 protein antibody, anti-NBS1 protein antibody, anti-DNA2 protein antibody, and anti-CtIP protein antibody, each may be a commercially available product.

[0084] The detection of the protein in the first group or the protein in the second group can also be performed using a mass spectrometry (MS). In particular, analysis by a mass spectrometer (LC / MS) coupled with liquid chromatography is advantageous because it is sensitive. As a detection method by mass spectrometry, in a method for detecting the expression level of helicase at the translation level, a method similar to the method listed as the detection method by mass spectrometry can be adjusted according to the protein in the first group or the protein in the second group and appropriately employed.

[0085] The detection of the protein in the first group or the protein in the second group can also be carried out by measuring the activity of each protein. For these activities, known methods or methods analogous thereto can be adopted as appropriate. For example, the measurement of the phosphorylation activity of the TTK protein can be carried out by detecting a phosphate group with a fluorescent substance or the like. Also, the measurement of the ATPase activity of the RAD50 protein can be carried out by using a luminescent ADP assay. For example, the ATPase activity can be measured by using ADP-Glo (manufactured by Promega). Furthermore, the measurement of the nuclease activity of the MRE11 protein can be carried out by methods such as the HeLa S3 assay method, the activity gel method, and the plasmid assay method.

[0086] 〔Prediction of sensitivity and selection of cancer patients〕 When the detection of the mutation in the first group and / or the detection of the mutation in the second group is thus carried out from a biological sample, if the biological sample is tentatively determined to be cancer cells, it can be predicted that the cells are sensitive to a helicase inhibitor. Also, when the biological sample is cancer cells contained in a sample derived from a cancer patient, it can be predicted that the cancer patient in whom the mutation has been detected in the cancer cells is sensitive to treatment with a helicase inhibitor. Furthermore, the cancer patient can be selected as a subject for cancer treatment with a helicase inhibitor.

[0087] Here, "sensitivity to a helicase inhibitor" and "sensitivity to treatment with a helicase inhibitor" are indices indicating whether a helicase inhibitor can exert a therapeutic effect on cancer cells. The sensitivity includes the promotion of the death of the cancer cells and the suppression of the proliferation by the helicase inhibitor. The prediction of the sensitivity may include not only the determination of the presence or absence of sensitivity but also evaluations such as whether sensitivity can be expected / cannot be expected, and the evaluation of the degree in the case of sensitivity (for example, evaluations such as high sensitivity can be expected, medium sensitivity can be expected, etc.). Therefore, depending on the type and degree of the mutation in the first group and / or the second group, for example, patients to be subjects for cancer treatment may be selected at a level where medium sensitivity can be expected.

[0088] On the other hand, when no mutation in the first group and / or no mutation in the second group is detected in a sample derived from a cancer patient, the patient can be excluded from the target of cancer treatment with a helicase inhibitor. Thereby, the efficacy rate of the treatment can be improved.

[0089] (MSH3) In addition, in the present invention, when cancer cells targeted by a helicase inhibitor have mutations in the first group and / or the second group, more preferably mutations in the first group (TTK mutations and / or RAD50 mutations), and further have mutations in MSH3, it has been found that the helicase inhibitor can suppress the growth of the cancer cells equally or more. Therefore, in the cancer cell sensitivity prediction method, the cancer patient sensitivity prediction method, and the cancer patient selection method, the presence or absence of the detection of mutations in MSH3 can be added as an index independently, and the present invention is A method for predicting the sensitivity of cancer cells to a helicase inhibitor, (a) detecting the presence or absence of at least one mutation selected from the first group consisting of mutations in TTK and mutations in RAD50 in cancer cells; (c) detecting the presence or absence of mutations in MSH3 in cancer cells; (d) predicting that cancer cells in which mutations in MSH3 are also detected in addition to TTK mutations and / or RAD50 mutations are sensitive to a helicase inhibitor; A method (cancer cell sensitivity prediction method) comprising: A method for predicting the sensitivity of a cancer patient to treatment with a helicase inhibitor, (a) detecting the presence or absence of at least one mutation selected from the first group consisting of mutations in TTK and mutations in RAD50 in cancer cells contained in a sample derived from a cancer patient; (c) detecting the presence or absence of mutations in MSH3 in cancer cells contained in a sample derived from a cancer patient; (d) predicting that a cancer patient in whom a mutation of MSH3 is also detected in addition to a TTK mutation and / or a RAD50 mutation in the cancer cells is sensitive to treatment with a helicase inhibitor; A method (cancer patient sensitivity prediction method) comprising: A method for selecting a cancer patient to be treated with a helicase inhibitor, (a) detecting the presence or absence of at least one mutation selected from a first group consisting of a TTK mutation and a RAD50 mutation in cancer cells contained in a sample derived from a cancer patient; (c) detecting the presence or absence of a mutation of MSH3 in cancer cells contained in a sample derived from a cancer patient; (d) selecting, as a subject for cancer treatment with a helicase inhibitor, a cancer patient in whom a mutation of MSH3 is also detected in addition to a TTK mutation and / or a RAD50 mutation in the cancer cells; A method (cancer patient selection method) comprising: is also provided.

[0090] "MSH3" in the present invention is a gene encoding a mismatch repair protein (hereinafter sometimes referred to as "MSH3 protein"). A typical nucleotide sequence of human-derived natural MSH3 genomic DNA is shown in SEQ ID NO: 7, and a typical amino acid sequence of human-derived natural MSH3 protein is shown in SEQ ID NO: 8. Note that even MSH3 that has not undergone mutations involving substitution, deletion, insertion, addition, etc. of the amino acid sequence may have individual differences in the sequence due to polymorphisms and the like.

[0091] The "mutation of MSH3" in the present invention includes substitution, deletion, insertion, and addition of amino acids in the amino acid sequence of the MSH3 protein. Such mutations of MSH3 are not particularly limited as long as the activity originally possessed by the MSH3 protein is changed, but mutations that cause a decrease in MSH3 protein activity (including complete loss (inactivation) of MSH3 protein activity), that is, loss-of-function mutations, are preferred. A decrease in MSH3 protein activity can occur, for example, due to missense mutations in MSH3, nonsense mutations throughout the entire region, or changes in gene structure such as the whole or partial deletion of MSH3, or changes in gene expression levels, but is not limited thereto.

[0092] Whether the gene structure and gene expression level originally possessed by MSH3 have changed can be confirmed and determined, for example, by the following methods: obtaining the base sequence of MSH3 by genome DNA sequencing; fluorescence detection using an oligonucleotide probe that specifically binds to the nucleotide sequence of MSH3; detection by PCR using an oligonucleotide primer that specifically binds to the nucleotide of MSH3, etc., by comparing with a control (for example, a healthy person or non-cancerous tissue of the same patient) to determine whether there is a change (preferably whether a loss-of-function mutation has occurred, or whether the expression level of mRNA has decreased in the case of the expression level of mRNA).

[0093] Whether the activity (functional activity) originally possessed by the MSH3 protein has changed can be determined, for example, by the following methods: detection by immunostaining or Western blotting using an antibody that specifically binds to the MSH3 protein; a method for determining whether the intracellular MSH3 protein purified by immunoprecipitation or the like binds to MSH2, which is a constituent protein of the MutSβ complex, by Western blotting or the like; and a method for determining whether the intracellular MSH3 protein purified by immunoprecipitation or the like has ATPase activity by an ATPase activity measurement method. Whether there is a change (preferably a decrease, that is, whether the expression level of the MSH3 protein detected by immunostaining or Western blotting is lower than that of the control, whether the molecular weight of the protein detected by Western blotting has changed compared to the control, whether the amount of MSH2, which is a constituent protein of the MutSβ complex that binds to the MSH3 protein detected by Western blotting, is lower than that of the control, and whether the activity is lower than that of the control in the case of ATPase activity) can be confirmed and determined by comparison with a control (for example, a healthy subject or non-cancerous tissue of the same patient).

[0094] Examples of specific MSH3 mutations that cause such changes in MSH3 protein activity include, for example, p.A22Rfs*3 (COSMIC Legacy Mutation ID: COSM7212418), p.P67Qfs*13 (COSMIC Legacy Mutation ID: COSM5989630), p.P67Qfs*13 (COSMIC Legacy Mutation ID: COSM5356342), p.V292Mfs*15 (COSMIC Legacy Mutation ID: COSM9494178), p.K383Gfs*20 (COSMIC Legacy Mutation ID: COSM1568178), p.K383Rfs*32 (COSMIC Legacy Mutation ID: COSM1438888), p.L503Wfs*5 (COSMIC Legacy Mutation ID: COSM5835081), p.P783Ffs*19 (COSMIC Legacy Mutation ID: COSM4188468), p.E797Sfs*3 (COSMIC Legacy Mutation ID: COSM8468896), p.N861Mfs*6 (COSMIC Legacy Mutation ID: COSM1438891), p.L1006Vfs*10 (COSMIC Legacy Mutation ID: COSM3139259), p.N1020IMfs*40 (COSMIC Legacy Mutation ID: COSM1438892), p.G1062Nfs*12 (COSMIC Legacy Mutation ID: COSM9358418), p.N212Sfs*2 (COSMIC Legacy Mutation ID: COSM9178646), p.E261Gfs*43 (COSMIC Legacy Mutation ID: COSM5868883), p.N385Qfs*19 (COSMIC Legacy Mutation ID: COSM1735453), p.Q406Pfs*42 (COSMIC Legacy Mutation ID: COSM9494259), p.P740Afs*28 (COSMIC Legacy Mutation ID: COSM5701238), p.I785Yfs*18 (COSMIC Legacy Mutation ID: COSM7513821), p.L821Ffs*3 (COSMIC Legacy Mutation ID: COSM4603915), p.N861Kfs*8 (COSMIC Legacy Mutation ID: COSM8183447), p.N1020Kfs*17 (COSMIC Legacy Mutation ID: COSM8565481), p.E1092Rfs*24 (COSMIC Legacy Mutation ID: COSM8851685), etc. can be mentioned.

[0095] As a method for detecting mutations in MSH3, the same methods as those listed as methods for "detecting mutations in TTK" etc. in the above [Detection of mutations] can be adjusted according to the mutations in MSH3 and appropriately adopted.

[0096] (Mutations in the third group) Furthermore, in the present invention, in addition to the cancer cells targeted by the helicase inhibitor having mutations in the first group and / or mutations in the second group, more preferably mutations in the second group, when the cancer cells further have at least one mutation selected from the third group consisting of mutations in EXO1, mutations in RPA1, mutations in RPA2, and mutations in RPA3 (hereinafter sometimes referred to as "mutations in the third group" in this specification), it has been found that the helicase inhibitor can suppress the growth of the cancer cells equally or more. Therefore, in the cancer cell sensitivity prediction method, the cancer patient sensitivity prediction method, and the cancer patient selection method, independently, the presence or absence of detection of mutations in the third group can also be added as an index, and the present invention is a method for predicting the sensitivity of cancer cells to a helicase inhibitor, (a) a step of detecting the presence or absence of at least one mutation selected from the second group consisting of mutations in RAD50, mutations in MRE11, mutations in NBN, mutations in DNA2, and mutations in RBBP8 in cancer cells; (c) a step of detecting the presence or absence of the third group consisting of mutations in EXO1, mutations in RPA1, mutations in RPA2, and mutations in RPA3 in cancer cells; (d) A step of predicting that cancer cells in which mutations in the third group are detected in addition to the mutations in the second group are sensitive to a helicase inhibitor; A method (cancer cell sensitivity prediction method) comprising: A method for predicting the sensitivity of a cancer patient to treatment with a helicase inhibitor, (a) A step of detecting the presence or absence of at least one mutation selected from the second group consisting of mutations in RAD50, MRE11, NBN, DNA2, and RBBP8 in cancer cells contained in a sample derived from a cancer patient; (c) A step of detecting the presence or absence of mutations in the third group consisting of mutations in EXO1, RPA1, RPA2, and RPA3 in cancer cells contained in a sample derived from a cancer patient; (d) A step of predicting that a cancer patient in whom mutations in the third group are detected in addition to the mutations in the second group in the cancer cells is sensitive to treatment with a helicase inhibitor; A method (cancer patient sensitivity prediction method) comprising: A method for selecting a cancer patient to be treated with a helicase inhibitor, (a) A step of detecting the presence or absence of at least one mutation selected from the second group consisting of mutations in RAD50, MRE11, NBN, DNA2, and RBBP8 in cancer cells contained in a sample derived from a cancer patient; (c) A step of detecting the presence or absence of mutations in the third group consisting of mutations in EXO1, RPA1, RPA2, and RPA3 in cancer cells contained in a sample derived from a cancer patient; (d) A step of selecting, as a subject for cancer treatment with a helicase inhibitor, a cancer patient in whom mutations in the third group are detected in addition to the mutations in the second group in the cancer cells; A method (cancer patient selection method) comprising: is also provided.

[0097] [EXO1] "EXO1" in the present invention is a 3'→5' exonuclease that releases 5'-mononucleotides from the 3'-OH end of single-stranded DNA by catalyzing the hydrolysis of phosphoryl ester bonds in DNA, and is also a gene encoding a protein having RNase activity (hereinafter sometimes referred to as "EXO1 protein" in this specification). The exonuclease activity of the EXO1 protein has been reported to be involved in DNA repair mechanisms (particularly, double-stranded DNA homologous end-joining repair mechanisms) in cooperation with the endonuclease activity of the MRE11 protein. The typical nucleotide sequence of the native EXO1 genomic DNA derived from human is shown in SEQ ID NO: 17, and the typical amino acid sequence of the native EXO1 protein derived from human is shown in SEQ ID NO: 18. Note that even for EXO1 that has not undergone mutations such as substitution, deletion, insertion, and addition of amino acid sequences, individual differences may occur in the sequence due to polymorphisms and the like.

[0098] "Mutations of EXO1" in the present invention include substitution, deletion, insertion, and addition of amino acids in the amino acid sequence of the EXO1 protein. Such mutations of EXO1 are not particularly limited as long as the activity originally possessed by the EXO1 protein is changed, but mutations that cause a decrease in EXO1 protein activity (including complete loss (inactivation) of EXO1 protein activity), that is, loss-of-function type mutations, are preferred. A decrease in EXO1 protein activity can occur, for example, due to missense mutations in EXO1, nonsense mutations throughout the entire region, or changes in gene structure such as deletion of the whole or part of EXO1, or changes in gene expression levels, but is not limited thereto.

[0099] Whether the gene structure and gene expression level originally possessed by EXO1 have changed can be confirmed and determined by, for example, the following methods: obtaining the nucleotide sequence of EXO1 by sequencing genomic DNA; fluorescence detection using an oligonucleotide probe that specifically binds to the nucleotide sequence of EXO1; detection by PCR using an oligonucleotide primer that specifically binds to the nucleotide of EXO1, etc., and comparing with a control (for example, a healthy person or non-cancerous tissue of the same patient) to check whether there is a change (preferably whether a loss-of-function mutation has occurred, and in the case of the mRNA expression level, whether it has decreased).

[0100] Whether the activity (functional activity) originally possessed by the EXO1 protein has changed can be confirmed and determined by, for example, the following methods: detection by immunostaining or Western blotting using an antibody that specifically binds to the EXO1 protein; and a method of measuring whether the intracellular EXO1 protein purified by immunoprecipitation etc. cleaves the 3' end of the substrate single-stranded DNA, etc., and comparing with a control (for example, a healthy person or non-cancerous tissue of the same patient) to check whether there is a change (preferably whether it has decreased, that is, in the case of the expression level of the protein detected by immunostaining or Western blotting, whether it has decreased compared to the control, in the case of the molecular weight of the protein detected by Western blotting, whether it has changed compared to the control, and in the case of exonuclease activity, whether the activity has decreased compared to the control).

[0101] Examples of specific EXO1 mutations that cause such changes in EXO1 protein activity include, for example, p.E89Dfs*44 (COSMIC Legacy Mutation ID: COSM5832542), p.V142* (COSMIC Legacy Mutation ID: COSM6657048), p.N159Tfs*9 (COSMIC Legacy Mutation ID: COSM5661778), p.G190Wfs*5 (COSMIC Legacy Mutation ID: COSM392303), p.F215Lfs*9 (COSMIC Legacy Mutation ID: COSM166045), p.C508Afs*13 (COSMIC Legacy Mutation ID: COSM1340675), p.C508Lfs*7 (COSMIC Legacy Mutation ID: COSM5207739), p.R723Ffs*20 (COSMIC Legacy Mutation ID: COSM9226806), p.D731Tfs*4 (COSMIC Legacy Mutation ID: COSM6657053), and the like.

[0102] As a method for detecting EXO1 mutations, the same methods as those listed as methods such as "detection of TTK mutations" in the above [detection of mutations] can be adjusted according to EXO1 mutations and appropriately adopted.

[0103] [[RPA1, RPA2, RPA3]] "RPA1", "RPA2", and "RPA3" in the present invention are genes that encode proteins (hereinafter sometimes referred to as "RPA1 protein", "RPA2 protein", and "RPA1 protein", respectively, and collectively referred to as "RPA1-3 proteins" as the case may be) that are reported to form a replication protein RPA by interacting with each other and interact with the MRN complex (for example, Greg Oakley et al., Biochemistry., August 11, 2009, 48(31), pp. 7473-7481; Ting Liu et al., Acta Biochim Biophys Sin, 2016, 48(7), pp. 665-670, etc.). If the function of any one of these proteins is deleted, the function of the complex itself is reduced and the above DNA repair mechanism does not function properly. Therefore, among the third group of mutations, in particular, it is preferable to detect at least one mutation among RPA1, RPA2, and RPA3 (hereinafter sometimes collectively referred to as "RPA1-3") that encode the proteins forming such a complex.

[0104] The typical base sequences of human-derived natural RPA1-3 genomic DNAs are shown in SEQ ID NOs: 19, 21, and 23, respectively, and the typical amino acid sequences of human-derived natural RPA1-3 proteins are shown in SEQ ID NOs: 20, 22, and 24, respectively. Even for RPA1, RPA2, or RPA3 that has not undergone mutations involving substitutions, deletions, insertions, additions, etc. of the amino acid sequence, individual differences may occur in the sequence due to polymorphisms, etc.

[0105] In the present invention, the "mutation of RPA1", "mutation of RPA2", and "mutation of RPA3" (hereinafter referred to as "mutations of RPA1-3") include amino acid substitutions, deletions, insertions, and additions in the amino acid sequences of RPA1-3 proteins, respectively. Such mutations of RPA1-3 are not particularly limited as long as the activities originally possessed by the RPA1-3 proteins are changed, but mutations that cause a decrease in the activities of RPA1-3 proteins (including complete loss (inactivation) of the activities of RPA1-3 proteins), that is, loss-of-function mutations, are preferred. The decrease in the activities of RPA1-3 proteins can be caused by, for example, missense mutations in RPA1-3, nonsense mutations throughout the entire region, or changes in gene structure such as the whole or partial deletion of RPA1-3, or changes in gene expression levels, but is not limited thereto.

[0106] Whether the gene structure and gene expression levels originally possessed by RPA1-3 have changed can be confirmed and determined by, for example, the following methods: obtaining the nucleotide sequences of RPA1-3 by sequencing genomic DNA; fluorescence detection using oligonucleotide probes that specifically bind to the nucleotide sequences of RPA1-3; detection by PCR using oligonucleotide primers that specifically bind to the nucleotides of RPA1-3, etc., by comparing with a control (for example, a healthy person or non-cancerous tissue of the same patient) to determine whether there is a change (preferably whether a loss-of-function mutation has occurred, or whether the expression level of mRNA has decreased in the case of mRNA expression level).

[0107] Whether the activities (functional activities) originally possessed by the RPA1-3 proteins have changed can be confirmed and determined, for example, by the following methods: detection by immunostaining or Western blotting using an antibody that specifically binds to the RPA1-3 proteins; and discrimination by Western blotting or the like as to whether the intracellular RPA1-3 proteins purified by immunoprecipitation or the like are bound to other RPA1-3 proteins that are constituent proteins of the complex. This is done by comparing with a control (e.g., a healthy subject or non-cancerous tissue of the same patient) to see if there is a change (preferably a decrease, that is, in the case of the expression level of the RPA1-3 proteins detected by immunostaining or Western blotting, whether it is lower than that of the control; in the case of the molecular weight of the protein detected by Western blotting, whether it has changed compared to the control; in the case of the amount of other RPA1-3 proteins that are constituent proteins of the complex detected by Western blotting and are bound, whether it is lower than that of the control).

[0108] Examples of specific RPA1 mutations that cause such changes in RPA1-3 protein activity include, for example, p.N274Mfs*5 (COSMIC Legacy Mutation ID: COSM4722502), p.N338Kfs*28 (COSMIC Legacy Mutation ID: COSM1745322), p.E363Gfs*4 (COSMIC Legacy Mutation ID: COSM6048715), p.E418Kfs*5 (COSMIC Legacy Mutation ID: COSM111541), p.E601Vfs*53 (COSMIC Legacy Mutation ID: COSM8515054), p.S609Rfs*46 (COSMIC Legacy Mutation ID: COSM112025), etc. Examples of RPA2 mutations include, for example, p.G34Afs*69 (COSMIC Legacy Mutation ID: COSM8537429), p.V127Gfs*26 (COSMIC Legacy Mutation ID: COSM907939), p.E158Gfs*5 (COSMIC Legacy Mutation ID: COSM8220461), etc. Further, examples of RPA3 mutations include, for example, p.N50Mfs*6 (COSMIC Legacy Mutation ID: COSM3029082), p.S64Nfs*26 (COSMIC Legacy Mutation ID: COSM6848181), etc.

[0109] As a method for detecting mutations in RPA1-3, the same methods as those listed as methods for "detecting mutations in TTK" etc. in the above [Detection of Mutations] can be adjusted respectively according to the mutations in RPA1-3 and appropriately adopted.

[0110] (Helicase) Furthermore, when the helicase, which is the target of the helicase inhibitor according to the present invention, is not normally expressed and / or not normally functioning, there is a risk that cancer treatment with the helicase inhibitor may not be effectively carried out. Therefore, in the cancer cell sensitivity prediction method, the cancer patient sensitivity prediction method, and the cancer patient selection method, in addition to the above, detection of mutations in the gene encoding helicase and decreased expression of helicase can also be added as indicators.

[0111] As a method for detecting mutations in the gene encoding helicase, the same methods as those listed as methods such as "detection of mutations in TTK" in the above [detection of mutations] can be appropriately adopted after being adjusted according to mutations in the gene encoding helicase (for example, the WRN gene). Also, as a method for detecting decreased expression of helicase, it is as described above.

[0112] <Method for treating cancer> The present invention relates to (a) a step of detecting the presence or absence of at least one mutation selected from the first group consisting of mutations in TTK and mutations in RAD50 in cancer cells contained in a sample derived from a cancer patient; (b) a step of administering a helicase inhibitor to a cancer patient in whom the mutation has been detected in the cancer cells; and a method for treating cancer comprising the above; and (a) a step of detecting the presence or absence of at least one mutation selected from the second group consisting of mutations in RAD50, mutations in MRE11, mutations in NBN, mutations in DNA2, and mutations in RBBP8 in cancer cells contained in a sample derived from a cancer patient; (b) a step of administering a helicase inhibitor to a cancer patient in whom the mutation has been detected in the cancer cells; and a method for treating cancer comprising the above (hereinafter, these are collectively referred to as "cancer treatment methods" as the case may be) are also provided.

[0113] In the cancer treatment method of the present invention, the detection of mutations in the first group, the detection of mutations in the second group, and the helicase inhibitor are as described above, respectively.

[0114] The administration of the helicase inhibitor to cancer patients may be oral or parenteral (e.g., intravenous administration, arterial administration, local administration).

[0115] Also, the dosage of the helicase inhibitor for cancer patients may be an amount effective to inhibit helicase and treat cancer, and it cannot be generally stated as it is appropriately selected according to the properties of the compound that inhibits helicase, the age, weight, symptoms, health status, progression of cancer, etc. of the cancer patient. For example, when administered to humans, it is an amount of the compound that inhibits helicase, 0.001 to 100,000 mg per day, preferably 0.01 to 5,000 mg. Similarly, regarding the dosing frequency of the helicase inhibitor for cancer patients, it cannot be generally stated, but for example, it is preferably administered once a day or divided into 2 to 4 times a day and repeated at appropriate intervals. The above dosage and dosing frequency can be appropriately increased or decreased as necessary according to the judgment of the doctor.

[0116] Thereby, in cancer cells having the mutation of the first group and / or the mutation of the second group in cancer patients, helicase is further inhibited, and cancer can be treated by promoting the death and / or suppressing the proliferation of cancer cells.

[0117] Examples of cancers to be treated include, but are not limited to, colorectal cancer, gastric cancer, cervical cancer, endometrial cancer, prostate cancer, breast cancer, lung cancer, bladder cancer, esophageal cancer, head and neck cancer, kidney cancer, ovarian cancer, lymphoma, adenoid cystic carcinoma, and pancreatic cancer.

[0118] <Reagent for detecting the presence or absence of mutation> The present invention also provides a reagent for detecting the presence or absence of the mutation of the first group and / or the mutation of the second group in the above method, (i) an oligonucleotide primer that specifically binds to one gene selected from the first group consisting of TTK and RAD50, (ii) An oligonucleotide probe that specifically binds to one gene selected from the first group consisting of TTK and RAD50, and (iii) An antibody that specifically binds to one protein selected from the first group consisting of TTK protein and RAD50 protein, A reagent comprising at least any one of these molecules as an active ingredient; and (i) An oligonucleotide primer that specifically binds to one gene selected from the second group consisting of RAD50, MRE11, NBN, DNA2, and RBBP8, (ii) An oligonucleotide probe that specifically binds to one gene selected from the second group consisting of RAD50, MRE11, NBN, DNA2, and RBBP8, and (iii) An antibody that specifically binds to one protein selected from the second group consisting of RAD50 protein, MRE11 protein, NBS1 protein, DNA2 protein, and CtIP protein, A reagent comprising at least any one of these molecules as an active ingredient is provided.

[0119] The above oligonucleotide primers can be designed based on the base sequence information of genomic DNA or cDNA of each gene (for example, SEQ ID NOs: 3, 5, 9, 11, 13, 15) so as to be primers suitable for the above-described methods and amplification regions, and to minimize the amplification products of genes other than the target gene. Such oligonucleotide primer design can be carried out by those skilled in the art by conventional methods. The length of the oligonucleotide primer is usually 15 to 50 bases in length, preferably 15 to 30 bases in length, but may be longer or shorter depending on the method and purpose.

[0120] The above oligonucleotide probe can be designed based on the nucleotide sequence information of genomic DNA or cDNA of each gene (for example, SEQ ID NO: 3, 5, 9, 11, 13, 15) to be a probe suitable for the above-described method and the region to be hybridized, and further, to minimize hybridization to genes other than the target gene. Such design of the oligonucleotide probe can be carried out by a person skilled in the art by a conventional method. The length of the oligonucleotide probe is usually 15 to 200 bases in length, preferably 15 to 100 bases in length, more preferably 15 to 50 bases in length, but may be longer or shorter depending on the method and purpose.

[0121] The oligonucleotide probe is preferably used after being appropriately labeled. Examples of the labeling method include a method of labeling by phosphorylating the 5'-end of the oligonucleotide with 32P using T4 polynucleotide kinase, and a method of incorporating a substrate base labeled with an isotope such as 32P, a fluorescent dye, or biotin using a DNA polymerase such as Klenow enzyme and using a random hexamer oligonucleotide or the like as a primer (random primer method, etc.).

[0122] The above oligonucleotide primer and oligonucleotide probe can be prepared, for example, by a commercially available oligonucleotide synthesizer. The oligonucleotide probe can also be prepared as a double-stranded DNA fragment obtained by restriction enzyme treatment or the like. Further, the oligonucleotide primer and oligonucleotide probe of the present invention do not have to be composed only of natural nucleotides (deoxyribonucleotides (DNA) or ribonucleotides (RNA)), and a part or all thereof may be composed of unnatural nucleotides. Examples of the unnatural nucleotides include PNA (polyamide nucleic acid), LNA (registered trademark, locked nucleic acid), ENA (registered trademark, 2'-O,4'-C-Ethylene-bridged nucleic acids), and complexes thereof.

[0123] An antibody that specifically binds to the protein of the first group or the protein of the second group can be obtained by immunizing an animal with an antigen (each protein (e.g., TTK protein, etc.), its partial peptide, or cells expressing these) if it is a polyclonal antibody, and then purifying it from the antiserum by conventional means (e.g., salting out, centrifugation, dialysis, column chromatography, etc.). Monoclonal antibodies can be produced by the hybridoma method or the recombinant DNA method.

[0124] Typical examples of the hybridoma method include the method of Kohler and Milstein (Kohler & Milstein, Nature 1975; 256: 495). The antibody-producing cells used in the cell fusion step in this method are spleen cells, lymph node cells, peripheral blood leukocytes, etc. of an animal (e.g., mouse, rat, hamster, rabbit, monkey, goat) immunized with an antigen (each protein (e.g., TTK protein, etc.), its partial peptide, or cells expressing these). It is also possible to use antibody-producing cells obtained by allowing an antigen to act on the above cells or lymphocytes, etc. isolated in advance from a non-immunized animal in a medium. Various known cell lines can be used as myeloma cells. The antibody-producing cells and myeloma cells may be of different animal species origins as long as they are fusible, but preferably they are of the same animal species origin. Hybridomas are produced, for example, by cell fusion between spleen cells obtained from a mouse immunized with an antigen and mouse myeloma cells, and then through subsequent screening, hybridomas that produce monoclonal antibodies specific to the protein of the first group or the protein of the second group can be obtained. Monoclonal antibodies against the protein of the first group or the protein of the second group can be obtained by culturing the hybridomas or from the ascites of a mammal administered with the hybridomas.

[0125] The recombinant DNA method involves cloning the DNA encoding the above-mentioned antibody from hybridomas, B cells, etc., integrating it into an appropriate vector, introducing this into host cells (such as mammalian cell lines, Escherichia coli, yeast cells, insect cells, plant cells, etc.), and producing the antibody of the present invention as a recombinant antibody (for example, P.J. Delves, Antibody Production: Essential Techniques, 1997 WILEY, P. Shepherd and C. Dean Monoclonal Antibodies, 2000 OXFORD UNIVERSITY PRESS, Vandamme AM et al., Eur. J. Biochem. 1990; 192: 767-775). In the expression of the DNA encoding the antibody, the DNA encoding the heavy chain or the light chain may be separately integrated into an expression vector and the host cells may be transformed, or the DNA encoding the heavy chain and the light chain may be integrated into a single expression vector and the host cells may be transformed (WO94 / 11523 publication, etc.). The antibody can be obtained in a substantially pure and homogeneous form by culturing the above-mentioned host cells and separating and purifying it from within the host cells or from the culture medium. For the separation and purification of the antibody, methods used in the purification of ordinary polypeptides can be used. By using transgenic animal production technology, transgenic animals (such as cows, goats, sheep, or pigs) into which the antibody gene has been integrated can be produced, and a large amount of monoclonal antibodies derived from the antibody gene can also be obtained from the milk of the transgenic animals.

[0126] Based on the antibody or its gene thus obtained, functional fragments of the antibody such as Fab, Fab’, F(ab’)2, Fv, scFv, sc(Fv)2, dsFv, and diabody, and multimers thereof (for example, dimer, trimer, tetramer, polymer) can be prepared.

[0127] When directly detecting the amount of antibody bound to the protein of the first group or the protein of the second group, the obtained anti-TTK protein antibody, anti-RAD50 protein antibody, anti-MRE11 protein antibody, anti-NBS1 protein antibody, anti-DNA2 protein antibody, anti-CtIP protein antibody, etc. are directly labeled with an enzyme, a radioisotope, a fluorescent dye, an avidin-biotin system, etc. and used. On the other hand, when performing an indirect detection method for detecting the amount of antibody bound to the protein of the first group or the protein of the second group using a secondary antibody or the like, the obtained anti-protein antibody (for example, anti-TTK protein antibody, etc.) (primary antibody) does not need to be labeled, and when detecting, a labeled molecule (for example, a secondary antibody or protein A) that recognizes the antibody may be used.

[0128] In the reagent of the present invention, in addition to the above molecules as active ingredients, other components acceptable as reagents, such as sterilized water, physiological saline, buffers, preservatives, etc., can be included as necessary. Furthermore, at least one of each oligonucleotide primer, oligonucleotide probe, and antibody for detecting the mutation of MSH3 or the mutation of the third group may be further included, or reagents containing such molecules may be further combined.

[0129] <Method for screening compounds for cancer treatment · Cancer therapeutic agent> The present invention A step of selecting a compound using whether or not it inhibits helicase as an index A method for screening a compound for treating cancer, comprising cancer cells in which at least one mutation selected from the first group consisting of a mutation of TTK and a mutation of RAD50 is detected; and A step of selecting a compound using whether or not it inhibits helicase as an index A method for screening a compound for treating cancer, comprising cancer cells in which at least one mutation selected from the second group consisting of a mutation of RAD50, a mutation of MRE11, a mutation of NBN, a mutation of DNA2, and a mutation of RBBP8 is detected; (hereinafter, these are collectively referred to as, optionally, "method for screening a compound") is also provided.

[0130] Also, using each of the compounds that inhibit helicase screened by the above-described method for screening compounds, a cancer therapeutic agent containing, as an active ingredient, a compound that inhibits helicase and containing cancer cells in which at least one mutation selected from the first group consisting of a mutation of TTK and a mutation of RAD50 is detected; and a cancer therapeutic agent containing, as an active ingredient, a compound that inhibits helicase and containing cancer cells in which at least one mutation selected from the second group consisting of a mutation of RAD50, a mutation of MRE11, a mutation of NBN, a mutation of DNA2, and a mutation of RBBP8 is detected (hereinafter, these are collectively referred to as, optionally, "cancer therapeutic agents") can also be provided.

[0131] The test compound to be applied to the method for screening the compounds of the present invention is not particularly limited. For example, at least one selected from the group consisting of the compounds, polypeptides, and polynucleotides exemplified as the above-described compounds that inhibit helicase (however, it is not necessary to know whether or not it inhibits helicase) can be mentioned. More specifically, as the test compound, for example, a synthetic low molecular weight compound library, an expression product of a gene library, a peptide library, siRNA, an antibody, a bacterial released substance, an extract and a culture supernatant of cells (microorganisms, plant cells, animal cells), a purified or partially purified polypeptide, an extract derived from a marine organism, a plant or an animal, and a random phage peptide display library can be mentioned. Further, the test compound may be a derivative of a known helicase inhibitor.

[0132] In a method (screening) for selecting a compound using whether or not it inhibits helicase as an index, the test compound may be allowed to act on the above-described confirmation system for inhibition of helicase activity or expression, and then the subsequent helicase activity or expression may be detected. As a result of the detection, if the activity or expression is decreased as compared with the helicase activity or expression in the control (for example, when the test compound is not added), it can be evaluated that the helicase is inhibited.

[0133] In the above screening, the "helicase" whose inhibition by a compound is evaluated is not particularly limited. For example, RecQ helicases (RecQL1, BLM, WRN, RecQL4 / RTS, RecQL5) can be mentioned. It is preferably a RecQ helicase, and more preferably WRN (Werner syndrome protein).

[0134] The compound identified by the screening method of the compound of the present invention can be appropriately mixed with the above-mentioned additive components such as the pharmacologically acceptable helicase inhibitor, and formulated by a known pharmaceutical method to be a cancer therapeutic agent as a pharmaceutical product.

Examples

[0135] Hereinafter, the present invention will be described more specifically based on test examples, but the present invention is not limited to the following examples.

[0136] (Test Example 1) 1. Experimental materials and methods (1) Cell lines First, for about 70 cancer cell lines, the expression of WRN was suppressed by siRNA, and cell lines with confirmed growth inhibition and cell lines without growth inhibition were selected respectively. In the following tests, as part of the selected cell lines, HCT 116, KM12, SW48, CW-2, HT-29, NCI-H716 (colorectal cancer), RL95-2, C-33A, COLO-684 (uterine cancer), SNU-1, GSU (gastric cancer) are shown as examples used as the cancer cell lines to be tested.

[0137] HCT 116 is a cancer cell line with mutations in all of TTK, RAD50, and MSH3. KM12, RL95-2, and C-33A are cancer cell lines with mutations in at least TTK and RAD50. SNU-1 is a cancer cell line with mutations in at least TTK and MSH3. SW48 is a cancer cell line with a mutation in at least TTK. CW-2 is a cancer cell line with a mutation in at least RAD50. COLO-684, GSU, HT-29, and NCI-H716 are cancer cell lines that have no mutations in either TTK or RAD50 and in which no mutations in MSH3 have been reported. These cancer cell lines and their derived tissues, and information on mutations in TTK, RAD50, and MSH3 in each cancer cell line (information on gene mutations that cause loss-of-function mutations) are shown in Table 1 below.

[0138]

Table 1

[0139] In Table 1, "*1" indicates mutations disclosed in the cancer cell line database "Cancer Cell Line Encyclopedia (CCLE)" created by the Broad Institute, "*2" indicates mutations disclosed in Mol Cell. 2018 Jul 19;71(2), p.319-331.e3, and "*3" indicates mutations disclosed in Eur J Cancer. 2000 May;36(7), p.925-931, respectively, and "N.I." indicates that information on the mutations is not known.

[0140] (2) Short interfering (si) RNA To suppress the expression of WRN, ON-TARGETplus individual siRNA (manufactured by Dharmacon) was used. For transfection, Lipofectamine RNAiMAX (manufactured by Invitrogen) was used. In addition, WRN siRNA (WRN siRNA1, Dharmacon product code: J-010378-06, SEQ ID NO: 25) was used as the test substance, and non-targeted siRNA (non-target siRNA1, Dharmacon product code: D-001810-01, SEQ ID NO: 26) was used as the negative control. Each siRNA was dissolved using 1× siRNA buffer, which was prepared by diluting 5× siRNA buffer (manufactured by Dharmacon: B-002000-UB-100) five-fold with nuclease-free water (manufactured by Ambion: AM9932).

[0141] (3) WRN Expression Suppression Test In each cancer cell line, the cytotoxic activity due to WRN expression suppression was evaluated. Each cell line was cultured in a cell culture flask (manufactured by Corning: 430641U) using the culture medium described in Table 2 below. After washing the cell surface with PBS (manufactured by Nacalai tesque: 14249-24), trypsin (manufactured by Nacalai tesque: 35554-64) was added and incubated at 37°C for 5 minutes, and then suspended using each culture medium. After measuring the cell count using an automatic cell counter (manufactured by Chemometec: NC-200) and Via1-Casette (manufactured by Chemometec: 941-0011), each cell line solution was adjusted with each culture medium to the cell concentration shown in Table 3 below. 100 μL / well of each cell line solution was seeded into a 96-well plate (manufactured by Greiner bio-one), and seeded so that the cell count was the seeding number shown in Table 3.

[0142] Next, Lipofectamine RNAiMAX (manufactured by Thermo Fischer Scientific: 13778150) was diluted 50-fold using Opti-MeM (manufactured by Thermo Fischer Scientific: 31985-062), and then mixed in equal amounts with 120 nM of the test substance (WRN siRNA1) or non-targeting siRNA (non-target siRNA1). After adding 20 μL / well of the mixed solution to the plate seeded with the above cells, the plate was shaken, and siRNA with a final concentration of 10 nM was transfected respectively.

[0143] After culturing for 7 days in a 37 °C incubator, 50 μL / well of CellTiter-Glo 2.0 Cell Viability Assay (manufactured by Promega: G9243) was added, incubated at room temperature for 5 minutes, and then luminescence was measured using EnVision (manufactured by PerkinElmer) to measure the amount of intracellular ATP, which is a marker of cell survival. The cell survival rate due to WRN expression suppression was calculated with the amount of intracellular ATP in each cancer cell line after transfection with the negative control and culturing for 7 days set as 100%. The cell survival rate (% survival) due to WRN expression suppression in each cancer cell line is shown in Figure 1.

[0144]

Table 2

[0145]

Table 3

[0146] 2. Results As shown in FIG. 1, in the WRN expression suppression test, a significant decrease in cell viability was observed in all seven cancer cell lines having at least one of the TTK mutation and the RAD50 mutation, with the cell viability being 70% or less (particularly 40% or less in FIG. 1). Similarly, for the other cancer cell lines tested, a significant decrease in cell viability was observed in the cancer cell lines having at least one of the TTK mutation and the RAD50 mutation. From these results, it was shown that the survival of cancer cell lines with at least one mutation selected from the group consisting of the TTK mutation and the RAD50 mutation (Group 1) strongly depends on the function of helicase, and in these cancer cells, suppression of helicase expression significantly suppresses the growth of the cancer cells.

[0147] (Test Example 2) 1. Experimental Materials and Methods (1) Cell Lines Furthermore, for approximately 200 cancer cell lines including the above-mentioned approximately 70 cancer cell lines, WRN expression was suppressed by siRNA, and cell lines with confirmed growth suppression and cell lines without growth suppression were selected respectively. In the following tests, as part of the selected cell lines, HCT 116, KM12, LS411N, SNU-407, SNU-C5, RKO, CW-2, CCK-81, HT-29 (colorectal cancer), RL95-2, AN3 CA, C-33A, SIHA, JHUEM-3 (uterine cancer), IM95, MKN1 (gastric cancer), TOV-21G, PA-1 (ovarian cancer) are shown as examples used as the following test target cancer cell lines.

[0148] In addition to the above-mentioned TTK, RAD50, and MSH3, HCT 116 is a cancer cell line having mutations (truncating mutations) in at least DNA2. KM12 is a cancer cell line having mutations (truncating mutations) in at least DNA2, RBBP8, and EXO1 in addition to the above-mentioned TTK and RAD50. LS411N is a cancer cell line having mutations (truncating mutations) in at least MRE11, DNA2, RBBP8, and EXO1. SNU-407 is a cancer cell line having mutations (truncating mutations) in at least DNA2, RBBP8, and EXO1. SNU-C5 is a cancer cell line having a mutation (truncating mutation) in at least RAD50. RKO is a cancer cell line having mutations (truncating mutations) in at least RAD50 and RPA3. CW-2 is a cancer cell line having a mutation (truncating mutation) in at least RPA1 in addition to the above-mentioned RAD50. CCK-81 is a cancer cell line having a mutation (truncating mutation) in at least RBBP8. RL95-2 is a cancer cell line having a mutation (truncating mutation) in at least DNA2 in addition to the above-mentioned RAD50. AN3 CA is a cancer cell line having a mutation (truncating mutation) in at least DNA2. C-33A is a cancer cell line having mutations (truncating mutations) in at least DNA2 and RPA1 in addition to the above-mentioned RAD50. IM95 is a cancer cell line having mutations (truncating mutations) in at least RAD50, NBN, DNA2, and RBBP8. TOV-21G is a cancer cell line having a mutation (truncating mutation) in at least NBN. HT-29, SIHA, JHUEM-3, MKN1, and PA-1 are cancer cell lines having no mutations in any of TTK, RAD50, MRE11, NBN, DNA2, RBBP8, EXO1, RPA1, RPA2, and RPA3. Information on these cancer cell lines and their derived tissues (Tissue), and the presence or absence of truncating mutations in RAD50, MRE11, NBN, DNA2, RBBP8, EXO1, RPA1, and RPA3 in each cancer cell line is shown in Table 4 below.

[0149] Each mutation described in Table 4 is a mutation disclosed in the cancer cell line database "Cancer Cell Line Encyclopedia (CCLE)" created by the Broad Institute. Also, in Table 4, "N.I." indicates that information regarding Truncating mutations that cause at least loss-of-function mutations is not known.

[0150] [Table 4]

[0151] (2) Short interfering (si) RNA To suppress the expression of WRN, ON-TARGETplus individual siRNA (manufactured by Dharmacon) was used. For transfection, Lipofectamine RNAiMAX (manufactured by Invitrogen) was used. Also, for the test substance, WRN siRNA (WRN siRNA1, Dharmacon product code: J-010378-06, SEQ ID NO: 25) was used, and for the negative control, non-targeting siRNA (non-target siRNA1, Dharmacon product code: D-001810-01, SEQ ID NO: 26) or non-targeting siRNA (non-target siRNA2, Dharmacon product code: D-001810-03, SEQ ID NO: 27) was used. Each siRNA was dissolved using 1×siRNA buffer, which was a 5-fold dilution of 5×siRNA buffer (manufactured by Dharmacon: B-002000-UB-100) with nuclease free water (manufactured by Ambion: AM9932).

[0152] (3) WRN expression suppression test In each cancer cell line, the cytotoxic activity due to WRN expression suppression was evaluated. Each cell line was cultured in a cell culture flask (manufactured by Corning: 430641U) using the culture solution described in Table 5 below. After washing the cell surface with PBS (manufactured by Nacalai tesque: 14249-24), trypsin (manufactured by Nacalai tesque: 35554-64) was added, and after incubation at 37°C for 5 minutes, it was suspended using each culture solution. After measuring the cell count using an automatic cell counter (manufactured by Chemometec: NC-200) and a Via1-Casette (manufactured by Chemometec: 941-0011), 100 μL / well of each was seeded into a 96-well plate (manufactured by Greiner bio-one) so that the cell count would be the seeding number shown in Table 6 below.

[0153] Next, for HCT 116, KM12, LS411N, SNU-407, SNU-C5, RKO, CW-2, CCK-81, HT-29, RL95-2, AN3 CA, C-33A, SIHA, JHUEM-3, IM95, and TOV-21G, Lipofectamine RNAiMAX (manufactured by Thermo Fischer Scienctific: 13778150) was diluted 50-fold using Opti-MeM (manufactured by Thermo Fischer Scientific: 31985-062), and then mixed in equal amounts with 120 nM of the test substance (WRN siRNA1) or non-targeted siRNA (non-target siRNA1). After adding 20 μL / well of the mixture to the plates seeded with the above cells, the plates were shaken, and each was transfected with siRNA at a final concentration of 10 nM. For MKN1 and PA-1, Lipofectamine RNAiMAX (manufactured by Thermo Fischer Scienctific: 13778150) was diluted 50-fold using Opti-MeM (manufactured by Thermo Fischer Scientific: 31985-062), and then mixed in equal amounts with 12 nM of the test substance (WRN siRNA1) or non-targeted siRNA (non-target siRNA2). After adding 20 μL / well of the mixture to the plates seeded with the above cells, the plates were shaken, and each was transfected with siRNA at a final concentration of 1 nM.

[0154] After culturing for 7 days in a 37°C incubator, 20 μL / well of CellTiter-Glo2.0 Cell Viability Assay (manufactured by Promega: G9243) was added, incubated at room temperature for 5 minutes, and then luminescence was measured using EnVision (manufactured by PerkinElmer) to measure the amount of intracellular ATP, which is a marker of cell survival. The cell survival rate due to WRN expression suppression was calculated with the amount of intracellular ATP in each cancer cell line after transfection with the negative control and culturing for 7 days set as 100%. The cell survival rate (%survival) due to WRN expression suppression in each cancer cell line is shown in Figure 2.

[0155]

Table 5

[0156]

Table 6

[0157] 2. Results As shown in Fig. 2, in the WRN expression suppression test, in all 13 cancer cell lines having at least any one of the mutations of RAD50, MRE11, NBN, DNA2, and RBBP8, particularly at least any one of the mutations of RAD50, NBN, DNA2, and RBBP8, a decrease in cell viability was observed with the cell viability being 70% or less. Similarly, for the other cancer cell lines tested, a significant decrease in cell viability was observed in the cancer cell lines having at least any one of the mutations of RAD50, MRE11, NBN, DNA2, and RBBP8. From these results, it was shown that the survival of cancer cell lines in which at least one mutation selected from the group consisting of the mutations of RAD50, MRE11, NBN, DNA2, and RBBP8 (Group 2) has occurred strongly depends on the function of helicase, and in these cancer cells, when the expression of helicase is suppressed, the proliferation of the cancer cells is significantly suppressed.

[0158] (Test Example 3) 1. Experimental Materials and Methods (1) Cell Lines HCT 116 was used as the cancer cell line to be tested. As described above, HCT 116 is a cancer cell line having mutations (truncating mutations) in at least TTK, RAD50, MSH3, and DNA2.

[0159] (2) Short Interfering (si) RNA To suppress the expression of WRN, ON-TARGETplus individual siRNA (manufactured by Dharmacon) was used. For transfection, Lipofectamine RNAiMAX (manufactured by Invitrogen) was used. In addition, as the siRNA, WRN siRNA (WRN siRNA2, Dharmacon product code: J-010378-07, SEQ ID NO: 28) was used. The siRNA was dissolved using 1×siRNA buffer prepared by diluting 5×siRNA buffer (manufactured by Dharmacon: B-002000-UB-100) five-fold with nuclease free water (manufactured by Ambion: AM9932).

[0160] (3) Transfection To overexpress WRN, ViaFect Transfection Reagent (manufactured by Promega) was used. A plasmid in which a siRNA-resistant wild-type WRN gene (SEQ ID NO: 29) was cloned into the pCMV-3Tag-1a vector was used as the control substance, and a plasmid in which a typical nucleotide sequence (SEQ ID NO: 1) of genomic DNA encoding the above human-derived natural-type WRN as a siRNA-non-resistant wild-type WRN gene was cloned into the pCMV-3Tag-1a vector was used as test substance 1, and a plasmid in which a siRNA-resistant K577M WRN gene (K577M WRN, SEQ ID NO: 30) with a deletion of helicase activity into which the K577M mutation was introduced was cloned into the pCMV-3Tag-1a vector was used as test substance 2, respectively. (4) Rescue test of WRN In the HCT 116 cancer cell line, while suppressing WRN expression, the cytotoxic activities were respectively evaluated when overexpressing the siRNA-resistant wild-type WRN gene, or the siRNA-non-resistant wild-type WRN gene, or the siRNA-resistant K577M WRN gene. First, the HCT 116 cell line was cultured in a cell culture flask (manufactured by Corning: 430641U) using the culture solution described in Table 2 above. After washing the cell surface with PBS (manufactured by Nacalai tesque: 14249-24), trypsin (manufactured by Nacalai tesque: 35554-64) was added, incubated at 37 °C for 5 minutes, and then suspended using the above culture solution. After measuring the cell count using an automatic cell counter (manufactured by Chemometec: NC-200) and a Via1-Casette (manufactured by Chemometec: 941-0011), it was adjusted to 7500 cells / 76 μL with each culture solution and seeded at 76 μL / well.

[0161] Next, using Opti-MeM (manufactured by Thermo Fischer Scientific: 31985-062), these were mixed so that the final concentration of siRNA (WRN siRNA2) was 5 nM and Lipofectamine RNAiMAX (manufactured by Thermo Fischer Scienctific: 13778150) was diluted 100-fold. After incubating the mixture at room temperature for 20 minutes, it was added to the plate seeded with the above cells at 19 μL / well, and the plate was shaken to transfect siRNA at a final concentration of 1 nM.

[0162] The next day, 0.1 μg of the control substance (plasmid of siRNA-resistant wild-type WRN gene), or test substance 1 (plasmid of siRNA-non-resistant wild-type WRN gene) or test substance 2 (plasmid of siRNA-resistant K577M WRN gene) was mixed with 0.3 μL of ViaFect Transfection Reagent in 10 μL of Opti-MeM. After incubating the mixture at room temperature for 20 minutes, 10 μL / well of the above siRNA was added to the wells transfected, and the plate was shaken to overexpress the siRNA-resistant wild-type WRN gene, siRNA-non-resistant wild-type WRN gene, or siRNA-resistant K577M WRN gene, respectively.

[0163] After culturing in a 37°C incubator for 6 days, 50 μL / well of CellTiter-Glo 2.0 Cell Viability Assay (manufactured by Promega: G9243) was added, and after incubating at room temperature for 5 minutes, luminescence was measured using EnVision (manufactured by PerkinElmer), the amount of intracellular ATP, which is a marker of cell survival, was measured, and the average was calculated (n = 3 (N1, N2, N3)). The cell survival rate due to WRN expression suppression was calculated with the amount of intracellular ATP in each cancer cell line after transfection with the control substance and culturing for 6 days as 100%. The cell survival rates (% survival) when the control substance, test substance 1, or test substance 2 was transfected into each cancer cell line are shown in Figure 3. A t-test was performed between the control substance and test substance 1 or 2, and between test substance 1 and test substance 2, respectively. As a result of the t-test, a significant difference was determined when p < 0.01.

[0164] 2. Results As shown in Fig. 3, a significant difference was observed between the control substance (a strain transfected with a plasmid of siRNA-resistant wild-type WRN gene) and the test substance 1 (a strain transfected with a plasmid of siRNA-non-resistant wild-type WRN gene) or the test substance 2 (a strain transfected with a plasmid of siRNA-resistant K577M WRN gene), and no significant difference was observed between the test substance 1 and the test substance 2. From these results and the results of the above Test Examples 1 to 2, it was shown that the survival of cancer cells in which at least the above mutations occurred was indeed strongly dependent on the helicase function of WRN, and that when the expression of helicase was suppressed in such cancer cells, the proliferation of the cancer cells was significantly suppressed.

Industrial Applicability

[0165] As described above, according to the present invention, at least one mutation selected from the first group consisting of mutations of TTK and mutations of RAD50, and / or at least one mutation selected from the second group consisting of mutations of RAD50, mutations of MRE11, mutations of NBN, mutations of DNA2, and mutations of RBBP8 are used as indicators, and it becomes possible to efficiently predict the sensitivity to cancer treatment with a helicase inhibitor. Further, according to the present invention, the presence or absence of the above mutations in a sample derived from a cancer patient is detected, and after selecting the patient in whom the mutation is detected, the patient can be treated with a helicase inhibitor for cancer. Therefore, it becomes possible to greatly improve the treatment outcome of cancer. Further, by using an oligonucleotide probe or primer for at least one gene selected from the first group consisting of TTK and RAD50, and / or at least one gene selected from the second group consisting of RAD50, MRE11, NBN, DNA2, and RBBP8, and an antibody against at least one protein selected from the first group consisting of TTK protein and RAD50 protein, and / or at least one protein selected from the second group consisting of RAD50 protein, MRE11 protein, NBS1 protein, DNA2 protein, and CtIP protein, it becomes possible to efficiently perform companion diagnosis by detecting the presence or absence of the above mutations.

Claims

1. A method for predicting the sensitivity of cancer cells to a helicase inhibitor, comprising the step of predicting that cancer cells in which at least one mutation selected from the first group consisting of a TTK mutation and a RAD50 mutation is detected are sensitive to a helicase inhibitor. A method comprising the above.

2. A method for predicting the sensitivity of cancer cells to a helicase inhibitor, comprising: (a) detecting the presence or absence of at least one mutation selected from the first group consisting of a TTK mutation and a RAD50 mutation in cancer cells; and (b) predicting that the cancer cells in which the mutation is detected are sensitive to a helicase inhibitor. A method comprising the above.

3. A method for predicting the sensitivity of a cancer patient to treatment with a helicase inhibitor, comprising the step of predicting that a cancer patient in which at least one mutation selected from the first group consisting of a TTK mutation and a RAD50 mutation is detected in cancer cells contained in a sample derived from the cancer patient is sensitive to treatment with a helicase inhibitor. A method comprising the above.

4. A method for predicting the sensitivity of a cancer patient to treatment with a helicase inhibitor, comprising: (a) detecting the presence or absence of at least one mutation selected from the first group consisting of a TTK mutation and a RAD50 mutation in cancer cells contained in a sample derived from the cancer patient; and (b) predicting that the cancer patient in which the mutation is detected in the cancer cells is sensitive to treatment with a helicase inhibitor. A method comprising the above.

5. A method for selecting a cancer patient to be treated with a helicase inhibitor, comprising the step of selecting, as a subject for cancer treatment with a helicase inhibitor, a cancer patient in which at least one mutation selected from the first group consisting of a TTK mutation and a RAD50 mutation is detected in cancer cells contained in a sample derived from the cancer patient. A method comprising the above.

6. A method for selecting a cancer patient to be treated with a helicase inhibitor, comprising: (a) detecting the presence or absence of at least one mutation selected from the first group consisting of a TTK mutation and a RAD50 mutation in cancer cells contained in a sample derived from the cancer patient; and (b) selecting, as a subject for cancer treatment with a helicase inhibitor, the cancer patient in which the mutation is detected in the cancer cells. A method comprising the above.

7. A method for treating cancer, For a cancer patient in whom at least one mutation selected from the first group consisting of a TTK mutation and a RAD50 mutation is detected in cancer cells contained in a sample derived from the cancer patient, administering a helicase inhibitor; A method comprising the above. **Claim 8** A method for treating cancer, (a) detecting the presence or absence of at least one mutation selected from the first group consisting of a TTK mutation and a RAD50 mutation in cancer cells contained in a sample derived from a cancer patient; and (b) administering a helicase inhibitor to a cancer patient in whom the mutation is detected in the cancer cells; A method comprising the above. **Claim 9** The method according to any one of claims 1 to 8, wherein the helicase inhibitor is a WRN inhibitor. **Claim 10** The method according to any one of claims 1 to 9, wherein the cancer cells are cancer cells in which a mutation in MSH3 is further detected. **Claim 11** A method for screening a compound for use in treating cancer, the method comprising cancer cells in which at least one mutation selected from the first group consisting of a TTK mutation and a RAD50 mutation is detected, and selecting a compound using inhibition of helicase as an indicator. A method comprising the above. **Claim 12** A cancer therapeutic agent comprising, as an active ingredient, a compound that inhibits helicase and comprising cancer cells in which at least one mutation selected from the first group consisting of a TTK mutation and a RAD50 mutation is detected. **Claim 13** The method according to claim 11, wherein the helicase is WRN. **Claim 14** The cancer therapeutic agent according to claim 12, wherein the helicase is WRN. **Claim 15** A method for predicting the sensitivity of cancer cells to a helicase inhibitor, the method comprising predicting that cancer cells in which at least one mutation selected from the second group consisting of a RAD50 mutation, an MRE11 mutation, an NBN mutation, a DNA2 mutation, and an RBBP8 mutation is detected are sensitive to a helicase inhibitor. A method comprising the above. **Claim 16** A method for predicting the sensitivity of cancer cells to a helicase inhibitor, the method comprising (a) detecting the presence or absence of at least one mutation selected from the second group consisting of a RAD50 mutation, an MRE11 mutation, an NBN mutation, a DNA2 mutation, and an RBBP8 mutation in cancer cells; and (b) predicting that the cancer cells in which the mutation is detected are sensitive to a helicase inhibitor. A method comprising the above.

17. A method for predicting the sensitivity of a cancer patient to treatment with a helicase inhibitor, comprising the step of predicting that a cancer patient in whom at least one mutation selected from the second group consisting of mutations of RAD50, mutations of MRE11, mutations of NBN, mutations of DNA2, and mutations of RBBP8 is detected in cancer cells contained in a sample derived from the cancer patient is sensitive to treatment with a helicase inhibitor; A method comprising the above.

18. A method for predicting the sensitivity of a cancer patient to treatment with a helicase inhibitor, comprising: (a) a step of detecting the presence or absence of at least one mutation selected from the second group consisting of mutations of RAD50, mutations of MRE11, mutations of NBN, mutations of DNA2, and mutations of RBBP8 in cancer cells contained in a sample derived from the cancer patient; and (b) a step of predicting that a cancer patient in whom the mutation is detected in the cancer cells is sensitive to treatment with a helicase inhibitor. A method comprising the above.

19. A method for selecting a cancer patient to be treated with a helicase inhibitor, comprising the step of selecting, as a subject for cancer treatment with a helicase inhibitor, a cancer patient in whom at least one mutation selected from the second group consisting of mutations of RAD50, mutations of MRE11, mutations of NBN, mutations of DNA2, and mutations of RBBP8 is detected in cancer cells contained in a sample derived from the cancer patient. A method comprising the above.

20. A method for selecting a cancer patient to be treated with a helicase inhibitor, comprising: (a) a step of detecting the presence or absence of at least one mutation selected from the second group consisting of mutations of RAD50, mutations of MRE11, mutations of NBN, mutations of DNA2, and mutations of RBBP8 in cancer cells contained in a sample derived from the cancer patient; and (b) a step of selecting, as a subject for cancer treatment with a helicase inhibitor, a cancer patient in whom the mutation is detected in the cancer cells. A method comprising the above.

21. A method for treating cancer, comprising the step of administering a helicase inhibitor to a cancer patient in whom at least one mutation selected from the second group consisting of mutations of RAD50, mutations of MRE11, mutations of NBN, mutations of DNA2, and mutations of RBBP8 is detected in cancer cells contained in a sample derived from the cancer patient. A method comprising the above.

22. A method for treating cancer, Step of detecting the presence or absence of at least one mutation selected from the second group consisting of mutations of RAD50, mutations of MRE11, mutations of NBN, mutations of DNA2, and mutations of RBBP8 in cancer cells contained in a sample derived from a cancer patient; Step of administering a helicase inhibitor to a cancer patient in whom the mutation has been detected in the cancer cells; A method comprising the steps.

23. The method according to any one of claims 15 to 22, wherein the helicase inhibitor is a WRN inhibitor.

24. The method according to any one of claims 15 to 23, wherein the cancer cells are cancer cells in which at least one mutation selected from the third group consisting of mutations of EXO1, mutations of RPA1, mutations of RPA2, and mutations of RPA3 is further detected.

25. A method for screening a compound for use in the treatment of cancer, the method comprising cancer cells in which at least one mutation selected from the second group consisting of mutations of RAD50, mutations of MRE11, mutations of NBN, mutations of DNA2, and mutations of RBBP8 is detected, Step of selecting a compound using the inhibition or non-inhibition of helicase as an indicator A method comprising the steps.

26. A cancer therapeutic agent containing, as an active ingredient, a compound that inhibits helicase and comprising cancer cells in which at least one mutation selected from the second group consisting of mutations of RAD50, mutations of MRE11, mutations of NBN, mutations of DNA2, and mutations of RBBP8 is detected.

27. The method according to claim 25, wherein the helicase is WRN.

28. The cancer therapeutic agent according to claim 26, wherein the helicase is WRN.

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