Method for predicting sensitivity of cancer cells to helicase inhibitors

Abstract

As a method for predicting the sensitivity of cancer cells to a helicase inhibitor, provided are: a method comprising a step for predicting that cancer cells have sensitivity to a helicase inhibitor when at least one mutation selected from a first group consisting of TTK mutation and RAD50 mutation is detected in the cancer cells; and a method comprising a step for predicting that cancer cells have sensitivity to a helicase inhibitor when at least one mutation selected from a second group consisting of the RAD50 mutation, MRE11 mutation, MBN mutation, DNA2 mutation, and RBBP8 mutation is detected in the cancer cells.

Description

[Technical field]

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

[0002] In recent years, rapid advances in genome sequencing technology have made it possible to decipher genomic information, including specific gene mutations in cancer cells. In the development of anticancer drugs, inhibitors that specifically inhibit the function of cancer cells with gain-of-function gene mutations, such as EGFR gene mutations, BRAF gene mutations, and ALK fusion genes, have been developed (Non-Patent Documents 1 to 3). Therapeutic methods specific to cancer cells with these gene mutations, which target the same cancer cells, are highly selective for cancer and highly effective.

[0003] Furthermore, for example, it has been reported that the survival of cancer cells exhibiting MSI-H (microsatellite instability-high) depends on WRN (Werner syndrome protein) (Non-Patent Documents 4 to 7), and it is believed that treatment that inhibits WRN can specifically target such cancer cells exhibiting MSI-H.

[0004] On the other hand, gene mutations found in human cancer cells include not only the gain-of-function mutations mentioned above, but also loss-of-function mutations. It is difficult to develop drugs specific to loss-of-function mutations, and a different therapeutic strategy is required than the treatment that targets cancer cells with gain-of-function mutations.

[0005] One of the few successful examples of specifically targeting cancer cells with loss-of-function mutations is the use of PARP inhibitors against BRCA1 / 2-deficient tumors (Non-Patent Document 8). However, no other therapeutic strategies have been developed to specifically target cancer cells with loss-of-function mutations. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Makoto Maemondo et al., NEJM 2010 Jun 24;362(25), p.2380-2388 [Non-Patent Document 2] Paul B. Chapman et al., NEJM 2011 Jun 30;364(26), p.2507-2516 [Non-Patent Document 3] D. Ross Camidge et al., J Thorac Oncol.2019 Jul;14(7), p.1233-1243 [Non-Patent Document 4] Lorn Kategaya et al., iScience 13, March 29, 2019, p.488-497 [Non-Patent Document 5] Simone Lieb et al.,eLife 2019,8:e43333,DOI:https: / / doi.org / 10.7554 / eLife.43333 [Non-Patent Document 6] Edmond M. Chan et al., Nature. 2019 April, 568(7753), p.551-556 [Non-Patent Document 7] Fiona M Behan et al., Nature.2019 April, 568(7753), p.511-516 [Non-Patent Document 8] K. Moore et al., NEJM 2018 Dec 27;379(26), p.2495-2505 Summary of the Invention [Problem to be solved by the invention]

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

[0008] The present inventors have conducted extensive research to solve the above problems, and first, they comprehensively suppressed the expression of WRN by siRNA in about 70 cancer cell lines, and analyzed the mutations common to the cell lines in which the suppression of proliferation was confirmed. As a result, they found that, in cancer cells having at least one mutation (preferably a loss-of-function mutation) selected from the first group consisting of TTK mutation and RAD50 mutation, the proliferation of the cancer cells is significantly suppressed when the expression of helicase such as WRN is suppressed or the function is inhibited, whereas such proliferation suppression does not occur in cells having neither TTK mutation nor RAD50 mutation.

[0009] Furthermore, the present inventors comprehensively analyzed about 200 cancer cell lines including the above about 70 cancer cell lines by adding the above cancer cell lines to the above cancer cell lines. As a result, they found that the proliferation of cancer cells in which at least one mutation (preferably a loss-of-function mutation) selected from the second group consisting of RAD50 mutation, MRE11 mutation, NBN mutation, DNA2 mutation, and RBBP8 mutation has occurred in addition to the mutation selected from the above first group is suppressed by suppressing the expression or function of helicase.

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

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

[0012] Furthermore, the present inventors have also found that screening of drugs useful for treating cancer in which the above mutation has occurred can be performed using as an indicator whether or not the drugs inhibit helicase, thereby completing the present invention.

[0013] Therefore, the present invention also relates to a therapeutic method that specifically targets cancer cells having at least one mutation selected from the first group consisting of TTK mutations and RAD50 mutations, and / or at least one mutation selected from the second group consisting of RAD50 mutations, MRE11 mutations, NBN mutations, DNA2 mutations, and RBBP8 mutations, and a companion diagnostic for the therapeutic method, and more specifically, provides the following inventions. [1] A method for predicting sensitivity of a cancer cell to a helicase inhibitor, 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; A method comprising: [2] A method for predicting sensitivity of a cancer cell to a helicase inhibitor, (a) detecting the presence or absence of at least one mutation selected from Group 1 consisting of TTK mutations and RAD50 mutations in cancer cells; (b) predicting that the cancer cell in which the mutation is detected is sensitive to a helicase inhibitor; A method comprising: [3] A method for predicting sensitivity of a cancer patient to treatment with a helicase inhibitor, predicting that a cancer patient in which at least one mutation selected from a first group consisting of a TTK mutation and a RAD50 mutation has been detected in a cancer cell contained in a sample derived from the cancer patient is susceptible to treatment with a helicase inhibitor; A method comprising: [4] A method for predicting sensitivity of a cancer patient to treatment 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; (b) predicting that a cancer patient in whom the mutation has been detected in the cancer cells will be susceptible 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 a cancer patient in which at least one mutation selected from a first group consisting of a TTK mutation and a RAD50 mutation has been detected in cancer cells contained in a sample derived from the cancer patient as a target for cancer treatment using a helicase inhibitor; 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 a 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 a cancer patient in whom the mutation has been detected in the cancer cells as a target for cancer treatment with a helicase inhibitor; A method comprising: [7] A method of treating cancer, administering a helicase inhibitor to a cancer patient in which at least one mutation selected from Group 1 consisting of a TTK mutation and a RAD50 mutation has been detected in cancer cells contained in a sample derived from the cancer patient; A method comprising: [8] A method of treating cancer, (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; (b) administering a helicase inhibitor to a cancer patient in which 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 of [1] to [9], wherein the cancer cells are cancer cells in which an MSH3 mutation is further detected.

[11] A method for screening a compound to be used in the treatment of a cancer comprising 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, A step of selecting a compound based on whether or not it inhibits helicase. A method comprising:

[12] A cancer therapeutic agent comprising a compound that inhibits helicase as an active ingredient, the cancer therapeutic agent including cancer cells in which at least one mutation selected from the group consisting of a TTK mutation and a RAD50 mutation 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 sensitivity of a cancer cell to a helicase inhibitor, predicting that a cancer cell in which at least one mutation selected from a second group consisting of a RAD50 mutation, a MRE11 mutation, a NBN mutation, a DNA2 mutation, and a RBBP8 mutation has been detected is sensitive to a helicase inhibitor; A method comprising:

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

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

[18] A method for predicting sensitivity of a cancer patient to treatment with a helicase inhibitor, (a) detecting the presence or absence of at least one mutation selected from a second group consisting of a RAD50 mutation, a MRE11 mutation, a NBN mutation, a DNA2 mutation, and a RBBP8 mutation in cancer cells contained in a sample derived from a cancer patient; (b) predicting that a cancer patient in whom the mutation has been detected in the cancer cells will be susceptible to treatment with a helicase inhibitor; A method comprising:

[19] A method for selecting a cancer patient to be treated with a helicase inhibitor, selecting a cancer patient 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 has been detected in cancer cells contained in a sample derived from the cancer patient, as a target for cancer treatment using a helicase inhibitor; A method comprising:

[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 a first group consisting of a second group consisting of a RAD50 mutation, a MRE11 mutation, a NBN mutation, a DNA2 mutation, and a RBBP8 mutation in cancer cells contained in a sample derived from a cancer patient; (b) selecting a cancer patient in whom the mutation has been detected in the cancer cells as a target for cancer treatment with a helicase inhibitor; A method comprising: [twenty one] A method of treating cancer, administering a helicase inhibitor to a cancer patient 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 has been detected in cancer cells contained in a sample derived from the cancer patient; A method comprising: [twenty two] A method of treating cancer, (a) detecting the presence or absence of at least one mutation selected from a second group consisting of a RAD50 mutation, a MRE11 mutation, a NBN mutation, a DNA2 mutation, and a RBBP8 mutation in cancer cells contained in a sample derived from a cancer patient; (b) administering a helicase inhibitor to a cancer patient in which the mutation has been detected in the cancer cells; A method comprising: [twenty three] The method according to any one of claims

[15] to

[22] , wherein the helicase inhibitor is a WRN inhibitor. [twenty four] 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 a third group consisting of an EXO1 mutation, an RPA1 mutation, an RPA2 mutation, and an RPA3 mutation is further detected. [twenty five] A method for screening a compound to be used in the treatment of a cancer comprising a cancer cell in which at least one mutation selected from a second group consisting of a RAD50 mutation, a MRE11 mutation, a NBN mutation, a DNA2 mutation, and a RBBP8 mutation is detected, A step of selecting a compound based on whether or not it inhibits helicase. A method comprising:

[26] A cancer therapeutic agent comprising a compound that inhibits helicase as an active ingredient, the cancer therapeutic agent being a cancer therapeutic agent containing cancer cells in which at least one mutation selected from Group 2 consisting of RAD50 mutations, MRE11 mutations, NBN mutations, DNA2 mutations, and RBBP8 mutations has been 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. Effect of the Invention

[0014] According to the present invention, it is possible to efficiently predict the sensitivity to cancer treatment with a helicase inhibitor by using at least one mutation selected from the first group consisting of TTK mutations and RAD50 mutations, and / or at least one mutation selected from the second group consisting of RAD50 mutations, MRE11 mutations, NBN mutations, DNA2 mutations, and RBBP8 mutations as an index. Furthermore, according to the present invention, the presence or absence of the mutations in a sample derived from a cancer patient can be detected, and after selecting a patient in whom the mutation has been detected, the patient can be treated for cancer with a helicase inhibitor. This makes it possible to greatly improve the outcome of cancer treatment. Furthermore, by using oligonucleotide probes or primers 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 (the gene encoding NBS1), DNA2, and RBBP8 (the gene encoding CtIP), as well as antibodies for 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] 1 is a graph showing cell survival rate as a result of suppression of WRN expression in cancer cell lines in Test Example 1. [Diagram 2] 1 is a graph showing cell survival rate as a result of suppression of WRN expression in cancer cell lines in Test Example 2. [Diagram 3] 1 is a graph showing cell survival rate as a result of suppression of WRN expression in cancer cell lines in Test Example 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The present invention will be described in detail below based on preferred embodiments thereof.

[0017] <Method for predicting sensitivity of cancer cells to helicase inhibitors, method for predicting sensitivity of cancer patients to treatment with helicase inhibitors, and method for selecting cancer patients for cancer treatment with helicase inhibitors> In the present invention, it has been found that in cancer cells in which at least one mutation selected from Group 1 consisting of TTK mutations and RAD50 mutations (sometimes referred to herein as "TTK mutations and / or RAD50 mutations" or "Group 1 mutations"), preferably loss-of-function mutations, is present, the proliferation of the cancer cells can be suppressed by inhibiting helicase. Furthermore, it has been found that in cancer cells in which at least one mutation selected from Group 2 consisting of RAD50 mutations, MRE11 mutations, NBN mutations, DNA2 mutations, and RBBP8 mutations (sometimes referred to herein as "Group 2 mutations"), preferably loss-of-function mutations, is present, the proliferation of the cancer cells can be suppressed by inhibiting helicase.

[0018] Based on these findings, it is possible to predict the sensitivity of cancer cells to helicase inhibitors using group 1 mutations and / or group 2 mutations as indicators. (a) detecting the presence or absence of at least one mutation selected from Group 1 consisting of TTK mutations and RAD50 mutations in cancer cells; (b) predicting that the cancer cell in which the mutation is detected is sensitive to a helicase inhibitor; A method for predicting the sensitivity of a cancer cell to a helicase inhibitor, comprising: (a) detecting the presence or absence of at least one mutation selected from a second group consisting of a RAD50 mutation, a MRE11 mutation, a NBN mutation, a DNA2 mutation, and a RBBP8 mutation in a cancer cell; (b) predicting that the cancer cell in which the mutation is detected is sensitive to a helicase inhibitor; The present invention provides a method for predicting the sensitivity of a cancer cell to a helicase inhibitor, comprising the steps of:

[0019] Furthermore, based on the above findings, it is possible to predict sensitivity to cancer treatment with a helicase inhibitor using the first group mutations and / or the second group mutations as an indicator. (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; (b) predicting that a cancer patient in whom the mutation has been detected in the cancer cells will be susceptible to treatment with a helicase inhibitor; 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 a second group consisting of a RAD50 mutation, a MRE11 mutation, a NBN mutation, a DNA2 mutation, and a RBBP8 mutation in cancer cells contained in a sample derived from a cancer patient; (b) predicting that a cancer patient in whom the mutation has been detected in the cancer cells will be susceptible to treatment with a helicase inhibitor; The present invention provides a method for predicting the sensitivity of a cancer patient to treatment with a helicase inhibitor, comprising the steps of:

[0020] Furthermore, since patients in whom group 1 mutations and / or group 2 mutations have been detected in this manner can be said to be suitable for cancer treatment with a helicase inhibitor, it is possible to use group 1 mutations and / or group 2 mutations as an indicator to select patients for whom cancer treatment with a helicase inhibitor is effective and those for whom it is not effective, and to provide efficient treatment. (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; (b) selecting a cancer patient in whom the mutation has been detected in the cancer cells as a target for cancer treatment with a helicase inhibitor; A method for selecting a cancer patient for cancer treatment with a helicase inhibitor, comprising: (a) detecting the presence or absence of at least one mutation selected from a first group consisting of a second group consisting of a RAD50 mutation, a MRE11 mutation, a NBN mutation, a DNA2 mutation, and a RBBP8 mutation in cancer cells contained in a sample derived from a cancer patient; (b) selecting a cancer patient in whom the mutation has been detected in the cancer cells as a target for cancer treatment with a helicase inhibitor; The present invention provides a method for selecting a cancer patient to be treated with a helicase inhibitor, comprising the steps of:

[0021] (Samples, etc.) In the present invention, malignant neoplasms such as cancer (epithelial tumor), leukemia, malignant lymphoma, myeloma, sarcoma, and carcinosarcoma are collectively referred to as "cancer", and cells constituting the cancer are referred to as "cancer cells". Examples of cancers containing cancer cells in which the presence or absence of group 1 mutations and / or group 2 mutations can be detected include, but are not limited to, colon cancer, gastric cancer, cervical cancer, uterine cancer, prostate cancer, esophageal cancer, breast cancer, lung cancer, bladder cancer, head and neck cancer, kidney cancer, ovarian cancer, lymphoma, adenoid cystic carcinoma, and pancreatic cancer. In the present invention, the cancer cells may be cancer cells which further have an MSH3 mutation as described below, or cancer cells which further have at least one mutation selected from the third group consisting of an EXO1 mutation, an RPA1 mutation, an RPA2 mutation, and an RPA3 mutation. When detecting a mutation in the first group, it is preferable that the cancer cells further have an MSH3 mutation, and when detecting a mutation in the second group, it is preferable that the cancer cells further have a mutation in the third group.

[0022] In the present invention, the term "cancer patient" refers not only to a person suffering from the cancer, but also to a person suspected of suffering from the cancer. In the method of the present invention, there is no particular limitation on the cancer patient in which the first group mutation and / or the second group mutation are detected, and all cancer patients can be the target.

[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 group 1 mutations and / or group 2 mutations, but is preferably a sample such as a cancer biopsy specimen, blood, urine, body cavity fluid, or tumor cell-derived circulating tumor DNA (ctDNA). In addition, it may be a protein extract or a nucleic acid extract (e.g., an mRNA extract, a cDNA preparation or a cRNA preparation prepared from an mRNA extract) obtained from the above 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) In the present invention, a "helicase inhibitor" refers to a composition containing at least one compound that inhibits helicase, and may consist of only the compound or a combination thereof, or may further contain the following additive components. In the present invention, a "compound that inhibits helicase" includes a compound that inhibits at least either the activity of helicase or the expression of helicase.

[0025] In the present invention, the "helicase" targeted by the helicase inhibitor is not particularly limited, and examples thereof include RecQ helicases (RecQL1, BLM, WRN, RecQL4 / RTS, RecQL5), preferably RecQ helicase, and more preferably WRN (Werner syndrome protein). A typical base sequence of genomic DNA encoding native human WRN is shown in SEQ ID NO: 1, and a typical amino acid sequence of native human WRN is shown in SEQ ID NO: 2. Note that even in a WRN gene (a gene encoding WRN) that has not undergone a mutation involving substitution, deletion, insertion, addition, or the like of the amino acid sequence, individual differences in the sequence may occur due to polymorphism, etc.

[0026] The inhibition of the activity of the helicase by a compound can be confirmed, for example, by adding a test compound to a system containing a fluorescent molecule, double-stranded DNA in which each strand is labeled with a quencher molecule, and the helicase, in which the fluorescent molecule and the quencher molecule separate and emit fluorescence when the DNA is unwound into single-stranded DNA by the helicase activity, and detecting the inhibition of the generation of the fluorescence (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] Furthermore, inhibition of the expression of the helicase by a compound can be confirmed, for example, by detecting a decrease in the expression of a helicase (preferably WRN) in cells treated with the test compound. A method for detecting a decrease in the expression of a helicase usually includes a method in which the expression level of the helicase is detected at the transcription or translation level, and the expression level is confirmed to be lower than a control (for example, the expression level in cells not treated with the test compound).

[0028] In the method of detecting the expression level of the helicase at the transcription level, first, RNA or cDNA is prepared from cells treated with a test compound. The method of extracting RNA from the cells is not particularly limited, and a known method can be appropriately selected and used, for example, an extraction method using phenol and a chaotropic salt (more specifically, an extraction method using a commercially available kit such as Trizol (Invitrogen) or Isogen (Wako Pure Chemical Industries)) or a method using other commercially available kits (RNAPrep Total RNA Extraction Kit (Beckman Coulter), RNeasy Mini (QIAGEN), RNA Extraction Kit (Pharmacia Biotech), etc.). Furthermore, the reverse transcriptase used to prepare cDNA from the extracted RNA is not particularly limited, and examples thereof 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, the oligonucleotide primer or oligonucleotide probe is used in an amplification reaction or hybridization reaction, respectively, to detect the amplified product or hybrid product. Examples of such a method include RT-PCR, Northern blot, dot blot, DNA array, in situ hybridization, RNase protection assay, and mRNA-seq. Those skilled in the art can routinely design oligonucleotide primers and oligonucleotide probes suitable for each method based on the base sequence of the cDNA encoding the helicase.

[0030] In the method for detecting the expression level of the helicase at the translational level, first, a protein sample is prepared from cells treated with a test compound. Then, an antigen-antibody reaction is carried out using an antibody specific to the helicase to detect the helicase. In such a protein detection method using an antibody, for example, an antibody specific to the helicase is added to the protein sample to carry out an antigen-antibody reaction and detect the binding of the antibody to the helicase. When the antibody specific to the helicase is labeled, the helicase can be detected directly, but when it is not labeled, a labeled molecule that recognizes the antibody (e.g., a secondary antibody or protein A) is further allowed to act, and the helicase can be indirectly detected using the label of the molecule. Examples of such methods that can be used include immunohistochemistry (immunostaining), Western blotting, ELISA, flow cytometry, imaging cytometry, radioimmunoassay, immunoprecipitation, and analysis using an antibody array.

[0031] The type and origin of the antibody to be used are not particularly limited, but a monoclonal antibody is preferable. As long as it is possible to detect the helicase with sufficient specificity, an oligoclonal antibody (a mixture of several to several tens of antibodies) or a polyclonal antibody can also be used. In addition, functional fragments of antibodies and multimers thereof (e.g., dimers, trimers, tetramers, polymers), such as Fab, Fab', F(ab')2, Fv, scFv, sc(Fv)2, dsFv, and diabodies, can also be used. The anti-helicase antibody may be a commercially available product.

[0032] The helicase can also be detected by mass spectrometry (MS). In particular, analysis using a mass spectrometer coupled to liquid chromatography (LC / MS) is advantageous because of its high sensitivity. 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 value. As the label, an isotope labeling reagent known in the art can be used, and a suitable labeling reagent is commercially available. Fractionation can also be performed 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 may be a known compound or a compound identified by the screening described below, but is preferably at least one type selected from the group consisting of compounds, polypeptides, and polynucleotides.

[0034] The compounds include low molecular weight compounds (molecular weight less than 900) and medium molecular weight compounds (molecular weight 900 to 2000). The polypeptides include full-length polypeptides encoded by genes, as well as fragments thereof, synthetic polypeptides, cyclic polypeptides, glycopeptides, and non-natural polypeptides. The polypeptides also include antibodies and antigen peptides, and the antibodies may be polyclonal or monoclonal antibodies. The antibodies include complete antibodies, as well as antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, scFv, sc(Fv)2, dsFv, diabody, etc.) and multimers thereof, and low molecular weight antibodies obtained by binding the variable regions of antibodies. The polynucleotides include DNA, RNA, and siRNA, and include full-length polynucleotides as well as fragments thereof and synthetic polynucleotides.

[0035] The "helicase inhibitor" of the present invention can be made into various dosage forms such as tablets, pills, powders, granules, capsules, and liquids, depending on its properties. In addition, depending on the dosage form, it may further contain pharmacologically acceptable additives such as sterile water, physiological saline, vegetable oil, solvent, base, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, fragrance, excipient, vehicle, preservative, binder, diluent, isotonicity agent, soothing agent, bulking agent, disintegrant, buffer, coating agent, lubricant, colorant, sweetener, thickening agent, flavoring agent, and solubilizing agent, and can be produced using these by known pharmaceutical methods.

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

[0037] (Group 1 mutations, Group 2 mutations) [TTK] In the present invention, "TTK" is a gene encoding a serine-threonine kinase (sometimes referred to as "TTK protein" in the present specification) involved in the control of centrosome duplication and mitotic checkpoint response. A typical base sequence of natural human TTK genomic DNA is shown in SEQ ID NO: 3, and a typical amino acid sequence of natural human TTK protein is shown in SEQ ID NO: 4. Even in TTK that does not have a mutation involving substitution, deletion, insertion, addition, etc. of the amino acid sequence, individual differences in the sequence may occur due to polymorphism, etc.

[0038] In the present invention, the "mutation of TTK" includes substitution, deletion, insertion, and addition of amino acids in the amino acid sequence of TTK protein. Such a mutation of TTK is not particularly limited as long as the original activity of TTK protein is changed, but it is preferably a mutation that causes a decrease in TTK protein activity (including complete loss (inactivation) of TTK protein activity), i.e., a loss-of-function mutation. The decrease in TTK protein activity can be caused by, for example, a missense mutation in TTK, a nonsense mutation across the entire region, or a change in gene structure such as a total or partial deletion of TTK, or a change in gene expression level, but is not limited thereto.

[0039] Whether or not the original gene structure or gene expression level of TTK has changed can be confirmed and determined, for example, by the following methods: obtaining the base sequence of TTK by sequencing genomic DNA; fluorescent detection using an oligonucleotide probe that specifically binds to the nucleotide sequence of TTK; detection by PCR using oligonucleotide primers that specifically bind to the nucleotides of TTK, and the like, and determining whether or not there has been an alteration (preferably whether or not a loss-of-function mutation has occurred, or in the case of mRNA expression level, whether or not there has been a decrease) in comparison with a control (e.g., a healthy subject or non-cancerous tissue of the same patient).

[0040] Whether or not the activity (functional activity) inherent to 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 TTK protein; and a method of determining whether intracellular TTK protein purified by immunoprecipitation or the like phosphorylates a substrate peptide by western blotting or the like, based on whether or not the activity has changed (preferably whether or not it has decreased) in comparison with a control (e.g., a healthy subject or non-cancerous tissue of the same patient), i.e., in the case of the expression level of a protein detected by immunostaining or western blotting, whether or not it has decreased compared to the control, in the case of the molecular weight of a protein detected by western blotting, whether or not it has changed compared to the control, and in the case of phosphorylation activity, whether or not the activity has decreased compared to the control).

[0041] Examples of specific TTK mutations that cause such changes in TTK protein activity include 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), and p.N606Kfs*3 (COSMIC Legacy Mutation ID: COSM150902). 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), etc.

[0042] [RAD50] In the present invention, "RAD50" is a gene that encodes a protein (sometimes referred to as "RAD50 protein" in this specification) that forms a complex (MRN complex) with the MRE11 protein and NBS1 protein described below and is involved in the DNA repair mechanism (particularly, the double-stranded DNA homologous end repair mechanism). A typical base sequence of natural RAD50 genomic DNA derived from humans is shown in SEQ ID NO: 5, and a typical amino acid sequence of natural RAD50 protein derived from humans is shown in SEQ ID NO: 6. Note that even in RAD50 that has not undergone a mutation involving substitution, deletion, insertion, addition, or the like of the amino acid sequence, individual differences in the sequence may occur due to polymorphism, etc.

[0043] In the present invention, the "RAD50 mutation" includes substitution, deletion, insertion, and addition of amino acids in the amino acid sequence of the RAD50 protein. Such a RAD50 mutation is not particularly limited as long as the original activity of the RAD50 protein is changed, but is preferably a mutation that causes a decrease in RAD50 protein activity (including complete loss (inactivation) of RAD50 protein activity), i.e., a loss-of-function mutation. The decrease in RAD50 protein activity can be caused by, for example, a missense mutation in RAD50, a nonsense mutation across the entire region, or a change in gene structure such as a total or partial deletion of RAD50, or a change in gene expression level, but is not limited thereto.

[0044] Whether or not the original gene structure or gene expression level of RAD50 has changed can be confirmed and determined, for example, by the following methods: obtaining the base sequence of RAD50 by sequencing genomic DNA; fluorescent detection using an oligonucleotide probe that specifically binds to the nucleotide sequence of RAD50; and detection by PCR using oligonucleotide primers that specifically bind to the nucleotides of RAD50, and by checking whether or not there has been an change (preferably whether or not a loss-of-function mutation has occurred, or in the case of mRNA expression level, whether or not there has been a decrease) in comparison with a control (e.g., a healthy subject or non-cancerous tissue of the same patient).

[0045] Whether or not the original activity (functional activity) of the RAD50 protein has changed can be determined by, for example, the following methods: detection by immunostaining or Western blotting using an antibody that specifically binds to the RAD50 protein; a method for determining whether the intracellular RAD50 protein purified by immunoprecipitation or the like is bound to the MRE11 protein or NBS1 protein, which are constituent proteins 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. This can be confirmed and judged by comparing whether there is a change (preferably whether there is a decrease, i.e., in the case of the expression level of RAD50 protein detected by immunostaining or Western blotting, whether there is a decrease compared to the control; in the case of the molecular weight of the protein detected by Western blotting, whether there is a change compared to the control; whether the amount of MRE11 protein or NBS1 protein, which is a component protein of the MRN complex bound to the RAD50 protein detected by Western blotting, is decreased compared to the control; and 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 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:COSM9494174), and p. 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), and p.Y1182Lfs*2 (COSMIC Legacy Mutation ID: COSM1633926).

[0047] [MRE11, NBN] In the present invention, "MRE11 (also known as "MRE11A")" is a gene encoding a protein (sometimes referred to herein as "MRE11 protein") that forms a complex (MRN complex) together with the above-mentioned RAD50 protein. The MRE11 protein is a nuclease that can degrade DNA as an exonuclease at the end of DNA and as an endonuclease at internal sites. In addition, in the present invention, "NBN" is a gene encoding NBS1 (sometimes referred to herein as "NBS1 protein"), a protein that forms a complex (MRN complex) together with the above-mentioned RAD50 protein. These proteins are related to each other and are involved in the DNA repair mechanism (particularly the double-stranded DNA homologous end repair mechanism) (e.g., 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.). Even if any one of these functions is lost, the function of the MRN complex itself is reduced and the above-mentioned DNA repair mechanism does not function normally. Therefore, among the second group of mutations, it is preferable to detect at least one of the mutations of RAD50, MRE11, and NBN that encode the proteins that form such MRN complexes, and it is more preferable to detect at least one of the mutations of RAD50 and NBN.

[0048] A typical base sequence of human-derived natural MRE11 genomic DNA is shown in SEQ ID NO: 9, and a typical amino acid sequence of human-derived natural MRE11 protein is shown in SEQ ID NO: 10. A typical base sequence of human-derived natural NBN genomic DNA (genomic DNA encoding NBS1) is shown in SEQ ID NO: 11, and a typical amino acid sequence of human-derived natural NBS1 protein is shown in SEQ ID NO: 12. Even in MRE11 and NBN that have not undergone mutations involving substitution, deletion, insertion, addition, and the like of the amino acid sequence, individual differences in the sequences may occur due to polymorphism, etc.

[0049] In the present invention, the "MRE11 mutation" and the "NBN mutation" include substitution, deletion, insertion, and addition of amino acids in the amino acid sequence of the MRE11 protein and the amino acid sequence of the NBS1 protein, respectively. Such MRE11 mutation and NBN mutation are not particularly limited as long as the original activities of the MRE11 protein and the NBS1 protein are changed, respectively, but are preferably 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), i.e., loss-of-function mutations. The decrease in MRE11 protein activity can be caused by, for example, a missense mutation in MRE11, a nonsense mutation across the entire region, or a change in gene structure such as a total or partial deletion of MRE11, or a change in gene expression level, but is not limited thereto. A decrease in NBS1 protein activity can be caused by, for example, a missense mutation in NBN, a nonsense mutation spanning the entire region, or a change in gene structure such as a total or partial deletion of NBN, or a change in gene expression level, but is not limited to these.

[0050] Whether or not the gene structure and gene expression level inherent to MRE11, the gene structure and gene expression level inherent to NBN have changed, and whether or not the activity (functional activity) inherent to the MRE11 protein and the activity (functional activity) inherent to the NBS1 protein have changed, can be confirmed and determined by, for example, the same method as the method for confirming and determining whether or not the gene structure and gene expression level inherent to RAD50 have changed, and the method for confirming and determining whether or not the activity (functional activity) inherent to the RAD50 protein has changed. Regarding the MRE11 protein, it can also be confirmed and determined by a method for measuring whether the intracellular MRE11 protein purified by immunoprecipitation or the like cleaves substrate DNA, for example, by comparing it with a control (e.g., a healthy person or non-cancer tissue of the same patient) to determine whether or not it has changed (preferably whether or not it has decreased, i.e., whether or not the activity has decreased compared to 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), and p.T481Hfs*43 (COSMIC Legacy Mutation ID: COSM2061331). 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), etc.

[0052] Examples of specific NBN mutations that cause changes in NBS1 protein activity include 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), and p.V153Kfs*17 (COSMIC Legacy Mutation ID: COSM28402). 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. .

[0053] [DNA2] In the present invention, "DNA2" is a gene that encodes a protein (sometimes referred to as "DNA2 protein" in the present specification) that is involved in the DNA repair mechanism (particularly, the double-stranded DNA homologous end repair mechanism) together with the MRN complex. The DNA2 protein is a nuclease that can degrade DNA as an exonuclease at the end of the DNA and as an endonuclease at an internal site, and is also a helicase that can unravel double-stranded DNA into single strands. A typical base sequence of natural DNA2 genomic DNA derived from humans is shown in SEQ ID NO: 13, and a typical amino acid sequence of natural DNA2 protein derived from humans is shown in SEQ ID NO: 14. Note that even in DNA2 that does not have a mutation involving substitution, deletion, insertion, addition, etc. of the amino acid sequence, individual differences in the sequence may occur due to polymorphism, etc.

[0054] In the present invention, the "DNA2 mutation" includes substitution, deletion, insertion, and addition of amino acids in the amino acid sequence of DNA2 protein. Such DNA2 mutations are not particularly limited as long as they are changes in the original activity of DNA2 protein, but are preferably mutations that cause a decrease in DNA2 protein activity (including complete loss (inactivation) of DNA2 protein activity), i.e., loss-of-function mutations. The decrease in DNA2 protein activity can be caused by, for example, a missense mutation in DNA2, a nonsense mutation across the entire region, or a change in gene structure such as a total or partial deletion of DNA2, or a change in gene expression level, but is not limited thereto.

[0055] Whether or not the inherent gene structure or gene expression level of DNA2 has changed, and whether or not the inherent activity (functional activity) of the DNA2 protein has changed, can be confirmed and determined, for example, by methods similar to those listed as the methods for confirming and determining whether or not the inherent gene structure or gene expression level of RAD50 has changed, and the methods for confirming and determining whether or not the inherent activity (functional activity) of the RAD50 protein has changed.

[0056] Examples of specific DNA2 mutations that cause such changes in DNA2 protein activity include 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), and p.S779Hfs*6 (COSMIC Legacy Mutation ID:COSM5092627). 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), and p.S975Vfs*4 (COSMIC Legacy Mutation ID:COSM1727571).

[0057] [RBBP8] In the present invention, "RBBP8" is a gene encoding CtIP (sometimes referred to as "CtIP protein" in this specification), a protein involved in DNA repair mechanisms (particularly double-stranded DNA homologous end repair mechanisms) together with the MRN complex. The CtIP protein is a nuclease that can degrade DNA as an endonuclease at an internal site of DNA. A typical base sequence of natural RBBP8 genomic DNA (genomic DNA encoding CtIP) derived from humans is shown in SEQ ID NO: 15, and a typical amino acid sequence of natural CtIP protein derived from humans is shown in SEQ ID NO: 16. Note that even in RBBP8 that has not undergone mutations involving substitution, deletion, insertion, addition, etc. of the amino acid sequence, individual differences in the sequence may occur due to polymorphism, etc.

[0058] In the present invention, the "mutation of RBBP8" includes substitution, deletion, insertion, and addition of amino acids in the amino acid sequence of the CtIP protein. Such a mutation of RBBP8 is not particularly limited as long as the original activity of the CtIP protein is changed, but is preferably a mutation that causes a decrease in CtIP protein activity (including complete loss (inactivation) of CtIP protein activity), i.e., a loss-of-function mutation. The decrease in CtIP protein activity can be caused by, for example, a missense mutation in RBBP8, a nonsense mutation across the entire region, or a change in gene structure such as a total or partial deletion of RBBP8, or a change in gene expression level, but is not limited thereto.

[0059] Whether or not the inherent gene structure or gene expression level of RBBP8 has changed, and whether or not the inherent activity (functional activity) of the CtIP protein has changed, can be confirmed and determined, for example, by methods similar to those listed as the methods for confirming and determining whether or not the inherent gene structure or gene expression level of RAD50 has changed, and the methods for confirming and determining whether or not the inherent activity (functional activity) of the RAD50 protein has changed.

[0060] Examples of specific RBBP8 mutations that cause such changes in CtIP protein activity include 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), and p.T375Nfs*2 (COSMIC Legacy Mutation ID:COSM1744942). 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), etc.

[0061] [Mutation detection] The methods for "detection of TTK mutations," "detection of RAD50 mutations," "detection of MRE11 mutations," "detection of NBN mutations," "detection of DNA2 mutations," and "detection of RBBP8 mutations" in the present invention are not particularly limited, and each can independently be, for example, the following methods.

[0062] In the present invention, "detection of a mutation" refers to detecting a mutation in each gene on genomic DNA, and when a mutation on the genomic DNA is reflected in a base change in a transcription product or an amino acid change in a translation product, detection of a mutation in the gene also includes detecting the change 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 in cancer cells by directly determining the base sequence of a gene region for detecting each mutation (gene region for detecting a first group of mutations: at least one selected from the TTK gene region and the RAD50 gene region (sometimes referred to as the "first group of gene regions" herein); gene region for detecting a second group of mutations: 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 (sometimes referred to as the "second group of gene regions" herein)). In the present invention, the terms "TTK gene region", "RAD50 gene region", "MRE11 gene region", "NBN gene region", "DNA2 gene region", and "RBBP8 gene region" respectively refer to 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. These regions independently include the expression control regions of each gene (eg, promoter regions, enhancer regions) and the 3'-terminal untranslated regions 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 limitations on the method for extracting genomic DNA or RNA from a biological sample, and any known method can be appropriately selected and used. For example, methods for extracting genomic DNA include the SDS phenol method (a method in which proteins in tissues stored in a solution containing urea or in ethanol are denatured with a protease (proteinase K), a surfactant (SDS), and phenol, and DNA is precipitated and extracted from the tissue with ethanol), and DNA extraction methods using 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), and BuccalQuick (registered trademark, manufactured by TrimGen).

[0066] Furthermore, methods for extracting RNA from a biological sample and preparing cDNA from the extracted RNA include the same methods as those mentioned for detecting the expression level of helicase at the transcription level.

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

[0068] By comparing the determined DNA or cDNA base sequence with a control (for example, when the biological sample is a sample derived from a cancer patient, the base sequence of DNA or cDNA derived from non-cancerous tissue of the same patient), the presence or absence of a mutation in the first group of gene regions or the second group of gene regions in the cancer cells of the biological sample can be determined.

[0069] Methods for detecting mutations in the first group of gene regions or the second group of gene regions can be performed by various methods capable of detecting mutations, in addition to methods for directly determining the base sequence of DNA or cDNA.

[0070] For example, the detection of mutations in the present invention can also be performed by the following method. First, a DNA or cDNA sample is prepared from a biological sample. Then, an oligonucleotide probe is prepared that has a base sequence complementary to the base sequence containing the mutation site of the first group of gene regions or the second group of gene regions and is labeled with a reporter fluorescent dye and a quencher fluorescent dye. Then, the oligonucleotide probe is hybridized to the DNA or cDNA sample, and the base sequence containing the mutation site of the first group of gene regions or the second group of gene regions is amplified using the DNA or cDNA sample hybridized with the oligonucleotide probe as a template. Then, the fluorescence emitted by the reporter fluorescent dye is detected due to the decomposition of the oligonucleotide probe accompanying the amplification, and the detected fluorescence is compared with a control. Examples of such methods include the double dye probe method, so-called TaqMan (registered trademark) probe method.

[0071] In yet another method, a DNA or cDNA sample is prepared from a biological sample. Then, in a reaction system containing an intercalator that emits fluorescence when inserted between DNA double strands, a base sequence containing a mutation site in the first group of gene regions or the second group of gene regions is amplified using the DNA or cDNA sample as a template. The temperature of the reaction system is then changed to detect changes in the intensity of the fluorescence emitted by the intercalator, and the detected changes in the intensity of the fluorescence accompanying the change in temperature are compared with a control. An example of such a method is the high resolution melting (HRM) method.

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

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

[0074] In yet another method, a DNA or cDNA sample is first prepared from a biological sample. Then, DNA containing all or part of the first group of gene regions or the second group of gene regions is amplified. The amplified DNA is then separated on a gel containing increasing concentrations of DNA denaturing agent. The mobility of the separated DNA on the gel is then compared with a control. Such a method includes, for example, denaturant gradient gel electrophoresis (DGGE) method.

[0075] Yet another method is a method using DNA containing a mutation site in the first group of gene regions or the second group of gene regions prepared from a biological sample and a substrate on which an oligonucleotide probe that hybridizes to the DNA is immobilized, such as a DNA array method.

[0076] In yet another method, a DNA or cDNA sample is first prepared from a biological sample. In addition, an "oligonucleotide primer having a base sequence complementary to the base sequence on the 3' side of one base of all or part of the bases of the first group of gene regions or the second group of gene regions" is prepared. Then, a fluorescently labeled ddNTP primer extension reaction is carried out using the primer with the DNA as a template. The primer extension reaction product is then subjected to a DNA sequencer, and the base sequence is determined based on the length and fluorescence of the extension reaction product. The genotype is then determined from the results of the DNA sequencer. The determined genotype is then compared with a control. An example of such a method is the Sanger method.

[0077] In yet another method, first, a DNA or cDNA sample is prepared from a biological sample. Then, 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 a base sequence complementary to the base sequence on the 5' side"-"one base on the 3' side of all or part of the first group of gene regions or the second group of gene regions and a base sequence that does not hybridize to the base sequence on the 3' side"-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 a base sequence on the 3' side" is prepared. Then, the above two types of oligonucleotide probes are hybridized to the prepared DNA or cDNA sample. Then, the hybridized DNA is cleaved with a single-stranded DNA cleavage enzyme to release the flap. There is no particular limitation on the single-stranded DNA cleavage enzyme, and for example, cleavase can be used. In this method, an oligonucleotide probe having a sequence complementary to the flap and labeled with a reporter fluorescent and a quencher fluorescent is then hybridized to the flap. The intensity of the generated fluorescence is then measured. The measured fluorescence intensity is then 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. Then, DNA containing all or a 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 one strand of the dissociated single-stranded DNA is separated. Then, an extension reaction is carried out one base at a time from the vicinity of all or a part of the bases of the first group of gene regions or the second group of gene regions, and the pyrophosphate generated during the reaction is enzymatically made to emit light, and the intensity of the light emission is measured. Then, the measured fluorescence intensity is compared with that of a control. An example of such a method is the pyrosequencing method.

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

[0080] In yet another method, first, a DNA or cDNA sample is prepared from a biological sample. Then, DNA containing all or a part of the first group of gene regions or the second group of gene regions is amplified. Then, "an oligonucleotide primer having a base sequence complementary to the base sequence on the 3' side of one base of all or a part of the first group of gene regions or the second group of gene regions" is prepared. Then, in the presence of a fluorescently labeled nucleotide, a single-base extension reaction is performed using the prepared primer with the amplified DNA as a template. Then, the base type used in the single-base extension reaction is determined. Then, the determined base type is compared with a control. An example of such a method is the SNuPE method.

[0081] In addition, the sample prepared from the biological sample may be a protein, so long as the mutation involves an amino acid change (e.g., substitution, deletion, insertion, addition) in each protein (when detecting a first group mutation: at least one selected from TTK protein and RAD50 protein (sometimes referred to as "first group protein" herein); when detecting a second group mutation: at least one selected from RAD50 protein, MRE11 protein, NBS1 protein, DNA2 protein, and CtIP protein (sometimes referred to as "second group protein" herein)). In such a case, the mutation can be detected by 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 mutation.

[0082] For example, in a method for detecting a protein using an antibody, first, a protein sample is prepared from a biological sample. Then, an antigen-antibody reaction is carried out using an antibody specific to the first group protein or the second group protein to detect the first group protein or the second group protein. As a method for detecting a protein using such an antibody, a method similar to the method for detecting a protein using an antibody in a method for detecting the expression level of helicase at the translation level can be appropriately adopted by adjusting it to suit the first group protein or the second group protein. In this method, immunohistochemistry has the advantage that additional information such as the morphology and distribution state of cancer cells in tissues can be obtained at the same time.

[0083] There is no particular limitation on the type or origin of the antibody used, but it is preferably a monoclonal antibody. As long as it is capable of detecting the first group protein or the second group protein with sufficient specificity, an oligoclonal antibody (a mixture of several to several tens of antibodies) or a polyclonal antibody can also be used. In addition, functional fragments of antibodies and their multimers (e.g., dimers, trimers, tetramers, polymers), such as Fab, Fab', F(ab')2, Fv, scFv, sc(Fv)2, dsFv, and diabody, can also be used. 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 may each be a commercially available product.

[0084] Detection of the first group protein or the second group protein can also be performed using mass spectrometry (MS). In particular, analysis using a mass spectrometer coupled with liquid chromatography (LC / MS) is advantageous because of its sensitivity. As a detection method using mass spectrometry, a method similar to the detection method using mass spectrometry in detecting the expression level of helicase at the translation level can be appropriately adopted by adjusting it to suit the first group protein or the second group protein.

[0085] The detection of the first group protein or the second group protein can also be performed by measuring the activity of each protein. These activities can be measured by appropriately using known methods or methods similar thereto. For example, the phosphorylation activity of the TTK protein can be measured by detecting phosphate groups with a fluorescent substance or the like. The ATPase activity of the RAD50 protein can be measured by utilizing a luminescent ADP assay. For example, the ATPase activity can be measured using ADP-Glo ​​(Promega). Furthermore, the nuclease activity of the MRE11 protein can be measured by the HeLa S3 assay method, the activity gel method, the plasmid assay method, or the like.

[0086] [Prediction of susceptibility and selection of cancer patients] When the first group of mutations and / or the second group of mutations are detected in this manner from a biological sample, if the biological sample is determined to be a cancer cell, the cell can be predicted to be sensitive to a helicase inhibitor, and if the biological sample is a cancer cell contained in a sample derived from a cancer patient, the cancer patient in which the mutations were detected in the cancer cell can be predicted to be sensitive to treatment with a helicase inhibitor, and further, the cancer patient can be selected as a target for cancer treatment with a helicase inhibitor.

[0087] Here, "sensitivity to a helicase inhibitor" and "sensitivity to treatment with a helicase inhibitor" are indicators showing whether or not a helicase inhibitor can exert a therapeutic effect on cancer cells. The sensitivity includes the promotion of death of the cancer cells and the inhibition of 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 an evaluation of whether or not sensitivity can be expected, and an evaluation of the level of sensitivity if there is sensitivity (e.g., an evaluation of whether high sensitivity can be expected, whether moderate sensitivity can be expected, etc.). Therefore, patients who are candidates for cancer treatment may be selected at a level where, for example, moderate sensitivity can be expected, depending on the type and level of the first group mutation and / or the second group mutation.

[0088] On the other hand, if the first group mutation and / or the second group mutation are not detected in the sample from the cancer patient, the patient can be excluded from the cancer treatment with the helicase inhibitor, thereby improving the success rate of the treatment.

[0089] (MSH3) Furthermore, in the present invention, it has been found that when the cancer cells targeted by the helicase inhibitor have a group 1 mutation and / or a group 2 mutation, more preferably a group 1 mutation (TTK mutation and / or RAD50 mutation) and further have an MSH3 mutation, the helicase inhibitor can suppress the proliferation of the cancer cells to the same extent or to a greater extent. Therefore, in the method for predicting cancer cell sensitivity, the method for predicting cancer patient sensitivity, and the method for selecting cancer patients, the presence or absence of detection of an MSH3 mutation can be added as an index, independently of each other, and the present invention provides: A method for predicting sensitivity of a cancer cell to a helicase inhibitor, (a) detecting the presence or absence of at least one mutation selected from Group 1 consisting of TTK mutations and RAD50 mutations in cancer cells; (c) detecting the presence or absence of an MSH3 mutation in the cancer cells; (d) predicting that cancer cells in which a TTK mutation and / or a RAD50 mutation as well as an MSH3 mutation are detected are sensitive to a helicase inhibitor; A method (a method for predicting cancer cell susceptibility); 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 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 an MSH3 mutation in cancer cells contained in a sample derived from a cancer patient; (d) predicting that a cancer patient in which a TTK mutation and / or a RAD50 mutation as well as an MSH3 mutation have been detected in the cancer cells is susceptible to treatment with a helicase inhibitor; A method (a method for predicting cancer patient susceptibility); 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 an MSH3 mutation in cancer cells contained in a sample derived from a cancer patient; (d) selecting cancer patients in whom a TTK mutation and / or a RAD50 mutation as well as an MSH3 mutation have been detected in the cancer cells as subjects for cancer treatment with a helicase inhibitor; A method for selecting cancer patients, comprising: Also provided.

[0090] "MSH3" in the present invention is a gene encoding a mismatch repair protein (sometimes referred to as "MSH3 protein" in the present specification). A typical base sequence of natural human MSH3 genomic DNA is shown in SEQ ID NO: 7, and a typical amino acid sequence of natural human MSH3 protein is shown in SEQ ID NO: 8. Even in MSH3 that does not have a mutation involving substitution, deletion, insertion, addition, or the like of the amino acid sequence, individual differences in the sequence may occur due to polymorphism, etc.

[0091] In the present invention, "MSH3 mutation" includes substitution, deletion, insertion, and addition of amino acids in the amino acid sequence of MSH3 protein. Such MSH3 mutation is not particularly limited as long as it changes the inherent activity of MSH3 protein, but is preferably a mutation that causes a decrease in MSH3 protein activity (including complete loss (inactivation) of MSH3 protein activity), i.e., a loss-of-function mutation. The decrease in MSH3 protein activity can be caused by, for example, a missense mutation in MSH3, a nonsense mutation across the entire region, or a change in gene structure such as a total or partial deletion of MSH3, or a change in gene expression level, but is not limited thereto.

[0092] Whether or not the original gene structure or gene expression level of MSH3 has changed can be confirmed and determined, for example, by the following methods: obtaining the base sequence of MSH3 by sequencing genomic DNA; fluorescent detection using an oligonucleotide probe that specifically binds to the nucleotide sequence of MSH3; and detection by PCR using oligonucleotide primers that specifically bind to the nucleotides of MSH3, and by checking whether or not there has been an change (preferably whether or not a loss-of-function mutation has occurred, or in the case of mRNA expression level, whether or not there has been a decrease) in comparison with a control (e.g., a healthy subject or non-cancerous tissue of the same patient).

[0093] Whether or not the inherent activity (functional activity) of the MSH3 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 MSH3 protein; a method by western blotting or the like to determine whether intracellular MSH3 protein purified by immunoprecipitation or the like is bound to MSH2, a component protein of the MutSβ complex; and a method by ATPase activity measurement to determine whether intracellular MSH3 protein purified by immunoprecipitation or the like has ATPase activity, based on whether or not the activity has changed (preferably whether or not it has decreased, i.e., in the case of the expression level of MSH3 protein detected by immunostaining or western blotting, whether or not it has decreased compared to the control; in the case of the molecular weight of the protein detected by western blotting, whether or not it has changed compared to the control; whether or not the amount of MSH2, a component protein of the MutSβ complex bound to the MSH3 protein detected by western blotting, is decreased compared to the control; and in the case of ATPase activity, whether or not the activity is decreased compared to the control).

[0094] Examples of specific MSH3 mutations that cause such changes in MSH3 protein activity include 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:COSM1438888), and p.L503Wfs*5 (COSMIC Legacy Mutation ID:COSM1438888). 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.Examples include 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), and p.E1092Rfs*24 (COSMIC Legacy Mutation ID: COSM8851685).

[0095] As a method for detecting a mutation in MSH3, a method similar to the method given as the method for "detection of a mutation in TTK" in the above [Detection of Mutations] can be appropriately adopted by adjusting it according to the mutation in MSH3.

[0096] (Group 3 mutations) Furthermore, in the present invention, it has been found that when the cancer cells targeted by the helicase inhibitor have a group 1 mutation and / or a group 2 mutation, more preferably a group 2 mutation, and further have at least one mutation selected from a third group consisting of EXO1 mutations, RPA1 mutations, RPA2 mutations, and RPA3 mutations (sometimes referred to as "group 3 mutations" in this specification), the helicase inhibitor can suppress the proliferation of the cancer cells to the same extent or to a greater extent. Therefore, in the method for predicting cancer cell sensitivity, the method for predicting cancer patient sensitivity, and the method for selecting cancer patients, the presence or absence of detection of a group 3 mutation can each independently be added as an indicator, and the present invention provides: A method for predicting sensitivity of a cancer cell to a helicase inhibitor, (a) detecting the presence or absence of at least one mutation selected from a second group consisting of a RAD50 mutation, a MRE11 mutation, a NBN mutation, a DNA2 mutation, and a RBBP8 mutation in a cancer cell; (c) detecting the presence or absence of a third group consisting of EXO1 mutations, RPA1 mutations, RPA2 mutations, and RPA3 mutations in the cancer cells; (d) predicting that a cancer cell in which a third group of mutations is detected in addition to a second group of mutations is sensitive to a helicase inhibitor; A method (a method for predicting cancer cell susceptibility); 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 a second group consisting of a RAD50 mutation, a MRE11 mutation, a NBN mutation, a DNA2 mutation, and a RBBP8 mutation in cancer cells contained in a sample derived from a cancer patient; (c) detecting the presence or absence of a third group of mutations consisting of EXO1 mutations, RPA1 mutations, RPA2 mutations, and RPA3 mutations in cancer cells contained in a sample derived from a cancer patient; (d) predicting that a cancer patient in which a third group of mutations is detected in addition to the second group of mutations in the cancer cells is susceptible to treatment with a helicase inhibitor; A method (a method for predicting cancer patient susceptibility); 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 second group consisting of a RAD50 mutation, a MRE11 mutation, a NBN mutation, a DNA2 mutation, and a RBBP8 mutation in cancer cells contained in a sample derived from a cancer patient; (c) detecting the presence or absence of a third group of mutations consisting of EXO1 mutations, RPA1 mutations, RPA2 mutations, and RPA3 mutations in cancer cells contained in a sample derived from a cancer patient; (d) selecting cancer patients in whom not only the second group mutation but also the third group mutation has been detected in the cancer cells as subjects for cancer treatment with a helicase inhibitor; A method for selecting cancer patients, comprising: Also provided.

[0097] [EXO1] In the present invention, "EXO1" is a gene that encodes a protein (sometimes referred to as "EXO1 protein" in the present specification) that is a 3'→5' exonuclease that catalyzes the hydrolysis of phosphorylated ester bonds in DNA to release 5'-mononucleotides from the 3'-OH end of single-stranded DNA and has RNase activity. It has been reported that the exonuclease activity of the EXO1 protein is involved in the DNA repair mechanism (particularly the double-stranded DNA homologous end repair mechanism) in cooperation with the endonuclease activity of the MRE11 protein. A typical base sequence of the natural EXO1 genomic DNA derived from humans is shown in SEQ ID NO: 17, and a typical amino acid sequence of the natural EXO1 protein derived from humans is shown in SEQ ID NO: 18. Note that even in EXO1 that does not have a mutation involving substitution, deletion, insertion, addition, etc. of the amino acid sequence, individual differences in the sequence may occur due to polymorphism, etc.

[0098] In the present invention, "EXO1 mutation" includes substitution, deletion, insertion, and addition of amino acids in the amino acid sequence of EXO1 protein. Such EXO1 mutation is not particularly limited as long as the original activity of EXO1 protein is changed, but is preferably a mutation that causes a decrease in EXO1 protein activity (including complete loss (inactivation) of EXO1 protein activity), i.e., a loss-of-function mutation. The decrease in EXO1 protein activity can be caused by, for example, a missense mutation in EXO1, a nonsense mutation across the entire region, or a change in gene structure such as a total or partial deletion of EXO1, or a change in gene expression level, but is not limited thereto.

[0099] Whether or not the original gene structure or gene expression level of EXO1 has changed can be confirmed and determined, for example, by the following methods: obtaining the base sequence of EXO1 by sequencing genomic DNA; fluorescent detection using an oligonucleotide probe that specifically binds to the nucleotide sequence of EXO1; detection by PCR using oligonucleotide primers that specifically bind to the nucleotides of EXO1, and by checking whether or not there has been an change (preferably whether or not a loss-of-function mutation has occurred, or in the case of mRNA expression level, whether or not there has been a decrease) in comparison with a control (e.g., a healthy subject or non-cancerous tissue of the same patient).

[0100] Whether or not the original activity (functional activity) of the EXO1 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 EXO1 protein; and a method of measuring and determining whether intracellular EXO1 protein purified by immunoprecipitation or the like cleaves the 3' end of a substrate single-stranded DNA, based on whether or not the activity has changed (preferably whether or not it has decreased) in comparison with a control (e.g., a healthy subject or non-cancerous tissue of the same patient), i.e., in the case of the expression level of the protein detected by immunostaining or western blotting, whether or not it has decreased compared to the control, in the case of the molecular weight of the protein detected by western blotting, whether or not it has changed compared to the control, and in the case of exonuclease activity, whether or not the activity has decreased compared to the control).

[0101] Examples of specific EXO1 mutations that cause such changes in EXO1 protein activity include 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:COSM1340675), and p.C508Lfs*8 (COSMIC Legacy Mutation ID:COSM1340675). ID:COSM5207739), p.R723Ffs*20 (COSMIC Legacy Mutation ID:COSM9226806), and p.D731Tfs*4 (COSMIC Legacy Mutation ID:COSM6657053).

[0102] As a method for detecting an EXO1 mutation, a method similar to the method given as the method for "detection of a TTK mutation" in the above [Detection of Mutations] can be appropriately adopted by adjusting it according to the EXO1 mutation.

[0103] [RPA1, RPA2, RPA3] In the present invention, "RPA1", "RPA2", and "RPA3" are genes that encode proteins (sometimes referred to as "RPA1 protein", "RPA2 protein", and "RPA1 protein", respectively, and sometimes collectively referred to as "RPA1 to 3 proteins") that have been reported to interact with each other to form the replication protein RPA (replication protein), which then interacts with the MRN complex (e.g., Greg Oakley et al., Biochemistry., 2009 August 11, 48(31), p.7473-7481; Ting Liu et al., Acta Biochim Biophys Sin, 2016, 48(7), p.665-670, etc.). Loss of function of any one of these proteins reduces the function of the complex itself and the DNA repair mechanism does not function normally. Therefore, among the mutations in group 3, it is preferable to detect at least one mutation among RPA1, RPA2, and RPA3 (hereinafter collectively referred to as "RPA1-3"), which encode the proteins that form such a complex.

[0104] Typical base sequences of natural human RPA1 to 3 genomic DNA are shown in SEQ ID NOs: 19, 21, and 23, respectively, and typical amino acid sequences of natural human RPA1 to 3 proteins are shown in SEQ ID NOs: 20, 22, and 24, respectively. Even in RPA1, RPA2, and RPA3 that have not undergone a mutation involving substitution, deletion, insertion, addition, or the like of the amino acid sequence, individual differences in the sequence may occur due to polymorphism, etc.

[0105] In the present invention, "RPA1 mutation", "RPA2 mutation" and "RPA3 mutation" (hereinafter referred to as "RPA1-3 mutation") include substitution, deletion, insertion and addition of amino acids in the amino acid sequences of RPA1-3 proteins, respectively. Such RPA1-3 mutations are not particularly limited as long as they change the original activity of RPA1-3 proteins, respectively, but are preferably mutations that cause a decrease in RPA1-3 protein activity (including complete loss (inactivation) of RPA1-3 protein activity), i.e., loss-of-function mutations. The decrease in RPA1-3 protein activity can be caused by, for example, missense mutations in RPA1-3, nonsense mutations across the entire region, or changes in gene structure such as full or partial deletion of RPA1-3, or changes in gene expression level, respectively, but are not limited thereto.

[0106] Whether or not the original gene structure or gene expression level of RPA1 to 3 has changed can be confirmed and determined, for example, by the following methods: obtaining the base sequence of RPA1 to 3 by sequencing genomic DNA; fluorescent detection using an oligonucleotide probe that specifically binds to the nucleotide sequence of RPA1 to 3; detection by PCR using oligonucleotide primers that specifically bind to the nucleotides of RPA1 to 3, and the like, and by checking whether or not there has been an change (preferably whether or not a loss-of-function mutation has occurred, or in the case of mRNA expression level, whether or not there has been a decrease) in comparison with a control (e.g., a healthy subject or non-cancerous tissue of the same patient).

[0107] Whether or not the original activity (functional activity) of the RPA1-3 proteins 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 RPA1-3 proteins; and a method of determining whether intracellular RPA1-3 proteins purified by immunoprecipitation or the like are bound to other RPA1-3 proteins that are constituent proteins of the complex by western blotting or the like, based on whether or not the activity has changed (preferably whether or not it has decreased) in comparison with a control (e.g., a healthy subject or non-cancerous tissue of the same patient), i.e., in the case of the expression level of RPA1-3 proteins detected by immunostaining or western blotting, whether or not it has decreased compared to the control, in the case of the molecular weight of the protein detected by western blotting, whether or not it has changed compared to the control, and whether or not the amount of bound RPA1-3 proteins that are constituent proteins of the complex detected by western blotting has decreased compared to the control).

[0108] Specific examples of RPA1 mutations that cause such changes in RPA1 to RPA3 protein activity include 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), and p.S609Rfs*46 (COSMIC Legacy Mutation ID: COSM112025). Examples of RPA2 mutations include 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. Examples of RPA3 mutations include 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 to 3, methods similar to those listed as methods for "detection of mutations in TTK" in the above section [Detection of mutations] can be appropriately adopted by adjusting them to suit each mutation in RPA1 to 3.

[0110] (Helicase) Furthermore, when the helicase that is the target of the helicase inhibitor according to the present invention is not normally expressed and / or does not function normally, cancer treatment using the helicase inhibitor may not be effectively carried out. Therefore, in the method for predicting cancer cell susceptibility, the method for predicting cancer patient susceptibility, and the method for selecting cancer patients, detection of a mutation in a gene encoding the helicase and reduced expression of the helicase can also be added as an indicator.

[0111] As a method for detecting a mutation in a gene encoding a helicase, the same method as that mentioned in the above [Detection of Mutations] as the method for "Detection of Mutations in TTK" can be appropriately adopted by adjusting it according to the mutation in a gene encoding a helicase (e.g., WRN gene). In addition, the method for detecting a decrease in expression of a helicase is as described above.

[0112] <How to treat cancer> The present invention relates to (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; (b) administering a helicase inhibitor to a cancer patient in which the mutation has been detected in the cancer cells; A method for treating cancer comprising: (a) detecting the presence or absence of at least one mutation selected from a second group consisting of a RAD50 mutation, a MRE11 mutation, a NBN mutation, a DNA2 mutation, and a RBBP8 mutation in cancer cells contained in a sample derived from a cancer patient; (b) administering a helicase inhibitor to a cancer patient in which the mutation has been detected in the cancer cells; Also provided is a method for treating cancer, comprising the steps of: (collectively, in this specification, sometimes referred to as a "cancer treatment method");

[0113] In the cancer treatment method of the present invention, the detection of group 1 mutations, the detection of group 2 mutations, and the helicase inhibitor are each as described above.

[0114] The helicase inhibitor may be administered to a cancer patient orally or parenterally (for example, intravenously, intraarterially, or locally).

[0115] The dosage of the helicase inhibitor for a cancer patient may be an effective amount for inhibiting helicase and treating cancer, and is selected appropriately depending on the properties of the compound that inhibits helicase, the age, weight, symptoms, health condition, and progression of the cancer of the cancer patient, and cannot be generalized. For example, when administered to a human, the amount of the compound that inhibits helicase is 0.001 to 100,000 mg, preferably 0.01 to 5,000 mg per day. The frequency of administration of the helicase inhibitor to a cancer patient is also not generalized, but it is preferably, for example, administered once a day or in 2 to 4 divided doses, and repeated at appropriate intervals. The dosage and frequency of administration can be appropriately increased or decreased as necessary at the discretion of a physician.

[0116] This further inhibits helicase in cancer cells having group 1 mutations and / or group 2 mutations in a cancer patient, thereby promoting the death of cancer cells and / or suppressing their proliferation, thereby enabling the treatment of cancer.

[0117] Examples of cancers to be treated include, but are not limited to, colorectal cancer, gastric cancer, cervical cancer, uterine 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] <Reagents for detecting the presence or absence of mutations> The present invention also relates to a reagent for detecting the presence or absence of the first group mutations and / or the second group mutations in the above method, (i) an oligonucleotide primer that specifically binds to one gene selected from a first group consisting of TTK and RAD50; (ii) an oligonucleotide probe that specifically binds to one gene selected from a first group consisting of TTK and RAD50; and (iii) an antibody that specifically binds to one protein selected from Group 1 consisting of TTK protein and RAD50 protein; A reagent containing at least one of the molecules listed above as an active ingredient; and (i) an oligonucleotide primer that specifically binds to one gene selected from a second group consisting of RAD50, MRE11, NBN, DNA2, and RBBP8; (ii) an oligonucleotide probe that specifically binds to one gene selected from a second group consisting of RAD50, MRE11, NBN, DNA2, and RBBP8; and (iii) an antibody that specifically binds to one protein selected from a second group consisting of RAD50 protein, MRE11 protein, NBS1 protein, DNA2 protein, and CtIP protein; The present invention provides a reagent containing at least one of the above molecules as an active ingredient.

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

[0120] The above oligonucleotide probes may be designed based on the base sequence information of the genomic DNA or cDNA of each gene (e.g., SEQ ID NOs: 3, 5, 9, 11, 13, 15) so as to be probes suitable for the above-mentioned methods and regions to be hybridized, and so as to minimize hybridization to genes other than the target gene. Such oligonucleotide probe design can be performed by those skilled in the art using standard methods. The length of the oligonucleotide probe is usually 15 to 200 bases, preferably 15 to 100 bases, and more preferably 15 to 50 bases, but may be longer or shorter depending on the method and purpose.

[0121] It is preferable to use the oligonucleotide probe after appropriate labeling. Examples of the labeling method include a method of labeling the 5' end of the oligonucleotide by phosphorylating it with 32P using T4 polynucleotide kinase, and a method of incorporating substrate bases labeled with isotopes such as 32P, fluorescent dyes, biotin, or the like using DNA polymerase such as Klenow enzyme and a random hexamer oligonucleotide as a primer (random prime method, etc.).

[0122] The above-mentioned oligonucleotide primers and oligonucleotide probes can be prepared, for example, by a commercially available oligonucleotide synthesizer. The oligonucleotide probes can also be prepared as double-stranded DNA fragments obtained by restriction enzyme treatment or the like. The oligonucleotide primers and oligonucleotide probes of the present invention do not have to be composed only of natural nucleotides (deoxyribonucleotides (DNA) and ribonucleotides (RNA)), and may be composed partially or entirely of non-natural nucleotides. Examples of non-natural 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] If the antibody that specifically binds to the above-mentioned group 1 protein or group 2 protein is a polyclonal antibody, it can be obtained by immunizing an animal with an antigen (each protein (e.g., TTK protein, etc.), a partial peptide thereof, or a cell expressing these, etc.) and purifying the antiserum by conventional means (e.g., salting out, centrifugation, dialysis, column chromatography, etc.). In addition, a monoclonal antibody can be produced by the hybridoma method or recombinant DNA method.

[0124] A representative example of the hybridoma method is 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 animals (e.g., mice, rats, hamsters, rabbits, monkeys, goats) immunized with an antigen (each protein (e.g., TTK protein, etc.), a partial peptide thereof, or cells expressing these, etc.). It is also possible to use antibody-producing cells obtained by allowing an antigen to act in a medium on the above-mentioned cells or lymphocytes, etc., previously isolated from an unimmunized animal. 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 are preferably of the same animal species origin. Hybridomas can be produced, for example, by cell fusion between spleen cells obtained from a mouse immunized with an antigen and mouse myeloma cells, and then screening can be performed to obtain hybridomas that produce monoclonal antibodies specific to Group 1 proteins or Group 2 proteins. Monoclonal antibodies against Group 1 proteins or Group 2 proteins can be obtained by culturing the hybridomas or from the ascites of a mammal to which the hybridomas have been administered.

[0125] The recombinant DNA method is a method in which DNA encoding the above-mentioned antibody is cloned from hybridomas, B cells, etc., incorporated into an appropriate vector, and then introduced into a host cell (e.g., a mammalian cell line, E. coli, yeast cells, insect cells, plant cells, etc.) to produce the antibody of the present invention as a recombinant antibody (e.g., PJ 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 expressing DNA encoding an antibody, DNA encoding a heavy chain or a light chain may be separately incorporated into an expression vector to transform a host cell, or DNA encoding a heavy chain and a light chain may be incorporated into a single expression vector to transform a host cell (WO94 / 11523, etc.). The antibody can be obtained in a substantially pure and homogeneous form by culturing the above-mentioned host cells and isolating and purifying it from within the host cells or from the culture medium. Methods used for normal polypeptide purification can be used for isolating and purifying the antibody. If a transgenic animal (such as a cow, goat, sheep or pig) incorporating an antibody gene is produced using transgenic animal production technology, it is also possible to obtain a large amount of monoclonal antibodies derived from the antibody gene from the milk of the transgenic animal.

[0126] Based on the antibodies thus obtained or their genes, functional fragments of antibodies, such as Fab, Fab', F(ab')2, Fv, scFv, sc(Fv)2, dsFv, and diabodies, and multimers thereof (e.g., dimers, trimers, tetramers, polymers) can be prepared.

[0127] When directly detecting the amount of antibody bound to the first group protein or the second group protein, 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, or an avidin-biotin system, etc., before use. On the other hand, when performing an indirect detection method in which the amount of antibody bound to the first group protein or the second group protein is detected using a secondary antibody, etc., it is not necessary to label the obtained anti-protein antibody (e.g., anti-TTK protein antibody, etc.) (primary antibody), and a labeled molecule that recognizes the antibody (e.g., secondary antibody or protein A) may be used for detection.

[0128] In addition to the above-mentioned molecules as active ingredients, the reagent of the present invention may contain, as necessary, other ingredients acceptable for a reagent, such as sterilized water, physiological saline, a buffer, a preservative, etc. Furthermore, the reagent may further contain at least any one of the oligonucleotide primers, oligonucleotide probes, and antibodies for detecting MSH3 mutations or group 3 mutations, or may further be combined with a reagent containing such molecules.

[0129] <Screening method for compounds used in cancer treatment / cancer treatment agent> The present invention relates to A step of selecting a compound based on whether or not it inhibits helicase. A method for screening a compound to be used in the treatment of cancer, including cancer cells in which at least one mutation selected from Group 1 consisting of TTK mutations and RAD50 mutations is detected; and A step of selecting a compound based on whether or not it inhibits helicase. The present invention also provides a method for screening for a compound to be used in the treatment of a cancer including cancer cells 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 (hereinafter, these are collectively referred to as "compound screening method" in some cases), the method comprising the steps of:

[0130] In addition, the compounds that inhibit helicase screened by the above compound screening method are used to A cancer therapeutic agent comprising a compound that inhibits helicase as an active ingredient, the cancer therapeutic agent being a cancer therapeutic agent containing cancer cells in which at least one type of mutation selected from Group 1 consisting of TTK mutations and RAD50 mutations is detected; and It is also possible to provide a cancer therapeutic agent (hereinafter, collectively referred to in some cases as "cancer therapeutic agent") which contains a compound that inhibits helicase as an active ingredient and is a therapeutic agent for cancer including cancer cells in which at least one mutation selected from Group 2 consisting of RAD50 mutations, MRE11 mutations, NBN mutations, DNA2 mutations, and RBBP8 mutations has been detected.

[0131] The test compound applied to the compound screening method of the present invention is not particularly limited, and examples thereof include at least one selected from the group consisting of the compounds, polypeptides, and polynucleotides cited as examples of compounds that inhibit helicases (however, it is not necessary to know whether or not they inhibit helicases). More specific examples of the test compound include synthetic low molecular weight compound libraries, expression products of gene libraries, peptide libraries, siRNAs, antibodies, bacteria-released substances, extracts and culture supernatants of cells (microorganisms, plant cells, animal cells), purified or partially purified polypeptides, extracts derived from marine organisms, plants, or animals, and random phage peptide display libraries. The test compound may also 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, a test compound may be allowed to act on the above-mentioned confirmation system for inhibition of helicase activity or expression, and the helicase activity or expression thereafter may be detected. If the detection result shows that the helicase activity or expression is reduced compared to that in a control (e.g., when the test compound is not added), it can be evaluated that the helicase is inhibited.

[0133] In the screening, the "helicase" to be evaluated for the presence or absence of inhibition by a compound is not particularly limited, and examples thereof include RecQ helicases (RecQL1, BLM, WRN, RecQL4 / RTS, RecQL5), preferably RecQ helicase, and more preferably WRN (Werner syndrome protein).

[0134] The compound identified by the compound screening method of the present invention can be used as a pharmaceutical agent for treating cancer by appropriately mixing it with pharmacologically acceptable additive components such as those listed for the above-mentioned helicase inhibitors and formulating it using a known pharmaceutical method. EXAMPLES

[0135] The present invention will be described in more detail below 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 line First, WRN expression was suppressed using siRNA in approximately 70 cancer cell lines, and cell lines in which growth inhibition was confirmed and cell lines in which growth was not suppressed were selected. In the following tests, some of the selected cell lines were used as test subject cancer cell lines, including HCT 116, KM12, SW48, CW-2, HT-29, NCI-H716 (colon cancer), RL95-2, C-33A, COLO-684 (uterine cancer), SNU-1, and GSU (gastric cancer).

[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 mutations in at least TTK. CW-2 is a cancer cell line with mutations in at least RAD50. COLO-684, GSU, HT-29, and NCI-H716 are cancer cell lines with no mutations in either TTK or RAD50, and no reported mutations in MSH3. These cancer cell lines and their tissues of origin, as well as information on TTK, RAD50, and MSH3 mutations 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 a mutation disclosed in the Cancer Cell Line Encyclopedia (CCLE), a cancer cell line database created by the Broad Institute; "*2" indicates a mutation disclosed in Mol Cell. 2018 Jul 19; 71(2), p. 319-331.e3; "*3" indicates an mutation disclosed in Eur J Cancer. 2000 May; 36(7), p. 925-931; and "NI" indicates that information regarding the mutation is not publicly known.

[0140] (2) Short interfering RNA (si) ON-TARGETplus individual siRNA (Dharmacon) was used to suppress the expression of WRN. Lipofectamine RNAiMAX (Invitrogen) was used for transfection. 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 in 1× siRNA buffer prepared by diluting 5× siRNA buffer (Dharmacon: B-002000-UB-100) 5-fold with nuclease-free water (Ambion: AM9932).

[0141] (3) Inhibition of WRN expression The cytotoxic activity due to the suppression of WRN expression was evaluated in each cancer cell line. Each cell line was cultured in a cell culture flask (Corning: 430641U) using the culture medium listed in Table 2 below, and the cell surface was washed with PBS (Nacalai tesque: 14249-24), and then trypsin (Nacalai tesque: 35554-64) was added. After incubation at 37°C for 5 minutes, the cells were suspended in each culture medium. The number of cells was measured using an automatic cell counter (Chemometec: NC-200) and Via1-Cassette (Chemometec: 941-0011), and the cell solution was adjusted with each culture medium to achieve the cell concentration listed in Table 3 below. Each cell line solution was seeded in a 96-well plate (Greiner bio-one) at 100 μL / well, and the cell numbers were seeded to the seeding numbers listed in Table 3.

[0142] Next, Lipofectamine RNAiMAX (Thermo Fischer Scientific: 13778150) was diluted 50-fold using Opti-MeM (Thermo Fischer Scientific: 31985-062), and then mixed with an equal amount of 120 nM of the test substance (WRN siRNA1) or non-targeting siRNA (non-target siRNA1). The mixture was added to the plate seeded with the above cells at 20 μL / well, and the plate was shaken to transfect each well with siRNA at a final concentration of 10 nM.

[0143] After 7 days of culture in a 37°C incubator, 50 μL / well of CellTiter-Glo2.0 Cell Viability Assay (Promega: G9243) was added and incubated at room temperature for 5 minutes, after which luminescence was measured using EnVision (PerkinElmer) to measure the amount of intracellular ATP, a marker of cell survival. The cell survival rate due to WRN expression inhibition was calculated by taking the amount of intracellular ATP in each cancer cell line after 7 days of culture after transfection with the negative control as 100%. The cell survival rate (% survival) due to WRN expression inhibition for each cancer cell line is shown in Figure 1.

[0144] [Table 2]

[0145] [Table 3]

[0146] 2.Results As shown in Figure 1, in the WRN expression inhibition test, the cell viability was 70% or less (particularly 40% or less in Figure 1) in all seven cancer cell lines with at least one of TTK and RAD50 mutations, and a significant decrease in cell viability was observed. Similarly, in the other cancer cell lines tested, a significant decrease in cell viability was observed in the cancer cell lines with at least one of TTK and RAD50 mutations. These results indicate that the survival of cancer cell lines with at least one mutation selected from the group consisting of TTK and RAD50 mutations (group 1) is strongly dependent on the function of helicase, and it has been revealed that the proliferation of these cancer cells is significantly inhibited by inhibiting the expression of helicase in these cancer cells.

[0147] (Test Example 2) 1. Experimental Materials and Methods (1) Cell line Furthermore, WRN expression was suppressed by siRNA for approximately 200 cancer cell lines, including the approximately 70 cancer cell lines mentioned above, and cell lines in which growth was suppressed and cell lines in which growth was not suppressed were selected. In the following tests, examples of the selected cell lines used as the cancer cell lines to be tested are shown below: HCT 116, KM12, LS411N, SNU-407, SNU-C5, RKO, CW-2, CCK-81, HT-29 (colon cancer), RL95-2, AN3 CA, C-33A, SIHA, JHUEM-3 (uterine cancer), IM95, MKN1 (gastric cancer), TOV-21G, and PA-1 (ovarian cancer).

[0148] HCT 116 is a cancer cell line having a mutation (truncating mutation) in at least DNA2 in addition to the above TTK, RAD50, and MSH3. KM12 is a cancer cell line having a mutation (truncating mutation) in at least DNA2, RBBP8, and EXO1 in addition to the above TTK and RAD50. LS411N is a cancer cell line having a mutation (truncating mutation) in at least MRE11, DNA2, RBBP8, and EXO1. SNU-407 is a cancer cell line having a mutation (truncating mutation) 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 a mutation (truncating mutation) 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 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 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 a mutation (truncating mutation) in at least DNA2 and RPA1 in addition to the above 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 that do not have mutations in any of TTK, RAD50, MRE11, NBN, DNA2, RBBP8, EXO1, RPA1, RPA2, and RPA3. These cancer cell lines and their tissues of origin, as well as information on the presence or absence of truncating mutations in RAD50, MRE11, NBN, DNA2, RBBP8, EXO1, RPA1, and RPA3 in each cancer cell line, are shown in Table 4 below.

[0149] Each mutation listed in Table 4 is a mutation disclosed in the cancer cell line database "Cancer Cell Line Encyclopedia (CCLE)" created by the Broad Institute. In addition, in Table 4, "NI" indicates that at least information on truncating mutations that cause loss-of-function mutations is not publicly known.

[0150] [Table 4]

[0151] (2) Short interfering RNA (si) ON-TARGETplus individual siRNA (Dharmacon) was used to suppress the expression of WRN. Lipofectamine RNAiMAX (Invitrogen) was used for transfection. 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) or non-targeted siRNA (non-target siRNA2, Dharmacon product code: D-001810-03, SEQ ID NO: 27) was used as the negative control. Each siRNA was dissolved in 1× siRNA buffer prepared by diluting 5× siRNA buffer (Dharmacon: B-002000-UB-100) 5-fold with nuclease-free water (Ambion: AM9932).

[0152] (3) Inhibition of WRN expression The cytotoxic activity due to the suppression of WRN expression was evaluated in each cancer cell line. Each cell line was cultured in a cell culture flask (Corning: 430641U) using the culture medium shown in Table 5 below, and the cell surface was washed with PBS (Nacalai tesque: 14249-24), and then trypsin (Nacalai tesque: 35554-64) was added. After incubation at 37°C for 5 minutes, the cells were suspended in each culture medium. The number of cells was measured using an automatic cell counter (Chemometec: NC-200) and Via1-Cassette (Chemometec: 941-0011), and the cells were seeded in a 96-well plate (Greiner bio-one) at 100 μL / well so that the number of cells was 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 (Thermo Fischer Scientific: 13778150) was diluted 50-fold using Opti-MeM (Thermo Fischer Scientific: 31985-062), and then mixed with an equal amount of 120 nM of the test substance (WRN siRNA1) or non-targeting siRNA (non-target siRNA1). The mixture was added to the plate seeded with the above cells at 20 μL / well, and the plate was shaken, and siRNA at a final concentration of 10 nM was transfected. For MKN1 and PA-1, Lipofectamine RNAiMAX (Thermo Fischer Scientific: 13778150) was diluted 50-fold using Opti-MeM (Thermo Fischer Scientific: 31985-062) and mixed with an equal amount of 12 nM of the test substance (WRN siRNA1) or non-targeting siRNA (non-target siRNA2). The mixture was added to the plate seeded with the above cells at 20 μL / well, and the plate was shaken to transfect each siRNA at a final concentration of 1 nM.

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

[0155] [Table 5]

[0156] [Table 6]

[0157] 2.Results As shown in Figure 2, in the WRN expression suppression test, the cell viability was reduced to 70% or less in all 13 cancer cell lines with at least one of RAD50 mutation, MRE11 mutation, NBN mutation, DNA2 mutation, and RBBP8 mutation, particularly at least one of RAD50 mutation, NBN mutation, DNA2 mutation, and RBBP8 mutation. Similarly, in the other cancer cell lines tested, a significant reduction in cell viability was observed in cancer cell lines with at least one of RAD50 mutation, MRE11 mutation, NBN mutation, DNA2 mutation, and RBBP8 mutation. These results indicate that the survival of cancer cell lines harboring at least one mutation selected from the group consisting of RAD50 mutations, MRE11 mutations, NBN mutations, DNA2 mutations, and RBBP8 mutations (group 2) is strongly dependent on the function of helicase, and that inhibition of helicase expression in these cancer cells significantly suppresses the proliferation of the cancer cells.

[0158] (Test Example 3) 1. Experimental Materials and Methods (1) Cell line 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 RNA (si) ON-TARGETplus individual siRNA (Dharmacon) was used to suppress the expression of WRN. Lipofectamine RNAiMAX (Invitrogen) was used for transfection. WRN siRNA (WRN siRNA2, Dharmacon product code: J-010378-07, SEQ ID NO: 28) was used as siRNA. siRNA was dissolved in 1× siRNA buffer prepared by diluting 5× siRNA buffer (Dharmacon: B-002000-UB-100) 5-fold with nuclease-free water (Ambion: AM9932).

[0160] (3) Transfection ViaFect Transfection Reagent (Promega) was used for overexpression of WRN. A plasmid in which an siRNA-resistant wild-type WRN gene (SEQ ID NO: 29) was cloned into the pCMV-3Tag-1a vector was used as a control substance, a plasmid in which a typical base sequence (SEQ ID NO: 1) of genomic DNA encoding the above-mentioned human-derived natural 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 an siRNA-resistant K577MWRN gene (K577MWRN, SEQ ID NO: 30) lacking helicase activity and having a K577M mutation was cloned into the pCMV-3Tag-1a vector was used as test substance 2. (4) WRN Rescue Test In the HCT 116 cancer cell line, the cytotoxic activity was evaluated when the siRNA-resistant wild-type WRN gene, the siRNA-non-resistant wild-type WRN gene, or the siRNA-resistant K577MWRN gene was overexpressed while suppressing WRN expression. First, the HCT 116 cell line was cultured in a cell culture flask (Corning: 430641U) using the culture medium listed in Table 2 above, and the cell surface was washed with PBS (Nacalai tesque: 14249-24), after which trypsin (Nacalai tesque: 35554-64) was added, and the cells were incubated at 37°C for 5 minutes, and then suspended in the culture medium. The number of cells was measured using an automatic cell counter (Chemometec: NC-200) and a Via1-Cassette (Chemometec: 941-0011), and the cells were then adjusted to 7,500 cells / 76 μL in each culture medium and seeded at 76 μL / well.

[0161] Next, using Opti-MeM (Thermo Fischer Scientific: 31985-062), siRNA (WRN siRNA2) was mixed to a final concentration of 5 nM, and Lipofectamine RNAiMAX (Thermo Fischer Scientific: 13778150) was diluted 100-fold. After incubating the mixture at room temperature for 20 minutes, 19 μL / well of the mixture was added to the plate seeded with the above cells, and the plate was shaken to transfect the siRNA to 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), test substance 1 (plasmid of siRNA-non-resistant wild-type WRN gene), or test substance 2 (plasmid of siRNA-resistant K577MWRN gene) was mixed with 0.3 μL of ViaFect Transfection Reagent in 10 μL of Opti-MeM. The mixture was incubated at room temperature for 20 minutes, and then 10 μL / well of the mixture was added to the wells transfected with the above siRNA. The plate was shaken to overexpress the siRNA-resistant wild-type WRN gene, the siRNA-non-resistant wild-type WRN gene, or the siRNA-resistant K577MWRN gene.

[0163] After 6 days of culture in a 37°C incubator, 50 μL / well of CellTiter-Glo2.0 Cell Viability Assay (Promega: G9243) was added and incubated at room temperature for 5 minutes. Luminescence was measured using EnVision (PerkinElmer), and the amount of intracellular ATP, 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 inhibition was calculated by taking the amount of intracellular ATP in each cancer cell line after 6 days of culture after transfection with the control substance as 100%. The cell survival rate (% survival) when each cancer cell line was transfected with the control substance, test substance 1, or test substance 2 is shown in Figure 3. T-tests were performed between the control substance and test substance 1 or 2, and between test substance 1 and test substance 2. A significant difference was determined when p<0.01 was obtained from the T-test.

[0164] 2.Results As shown in Figure 3, a significant difference was observed between the control substance (strain transfected with a plasmid of an siRNA-resistant wild-type WRN gene) and test substance 1 (strain transfected with a plasmid of an siRNA-non-resistant wild-type WRN gene) or test substance 2 (strain transfected with a plasmid of an siRNA-resistant K577MWRN gene), but no significant difference was observed between test substance 1 and test substance 2. These results and the results of Test Examples 1 and 2 above indicate that the survival of at least cancer cells in which the above mutations have occurred is indeed strongly dependent on the function of WRN helicase, and that inhibition of helicase expression in such cancer cells significantly inhibits the proliferation of the cancer cells. [Industrial Applicability]

[0165] As described above, according to the present invention, it is possible to efficiently predict the sensitivity to cancer treatment with a helicase inhibitor by using at least one mutation selected from the first group consisting of TTK mutations and RAD50 mutations, and / or at least one mutation selected from the second group consisting of RAD50 mutations, MRE11 mutations, NBN mutations, DNA2 mutations, and RBBP8 mutations as an index. Furthermore, according to the present invention, the presence or absence of the mutations in a sample derived from a cancer patient can be detected, and after selecting a patient in whom the mutation has been detected, the patient can be treated for cancer with a helicase inhibitor. This makes it possible to greatly improve the outcome of cancer treatment. Furthermore, by using oligonucleotide probes or primers 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 antibodies for 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 sensitivity of a cancer cell to a helicase inhibitor, predicting that a cancer cell in which at least one mutation selected from a first group consisting of a loss-of-function mutation in TTK and a loss-of-function mutation in RAD50 has been detected is sensitive to a helicase inhibitor; Including, The method, wherein said helicase inhibitor is a WRN inhibitor.

2. A method for predicting sensitivity of a cancer cell to a helicase inhibitor, (a) detecting the presence or absence of at least one mutation selected from a first group consisting of loss-of-function mutations of TTK and loss-of-function mutations of RAD50 in cancer cells; (b) predicting that the cancer cell in which the mutation is detected is sensitive to a helicase inhibitor; Including, The method, wherein said helicase inhibitor is a WRN inhibitor.

3. A method for predicting sensitivity of a cancer patient to treatment with a helicase inhibitor, predicting that a cancer patient in which at least one mutation selected from a first group consisting of a loss-of-function mutation in TTK and a loss-of-function mutation in RAD50 has been detected in a cancer cell contained in a sample derived from the cancer patient is susceptible to treatment with a helicase inhibitor; Including, The method, wherein said helicase inhibitor is a WRN inhibitor.

4. A method for predicting sensitivity of a cancer patient to treatment with a helicase inhibitor, (a) detecting the presence or absence of at least one mutation selected from a first group consisting of loss-of-function mutations in TTK and loss-of-function mutations in RAD50 in cancer cells contained in a sample derived from a cancer patient; (b) predicting that a cancer patient in whom the mutation has been detected in the cancer cells will be susceptible to treatment with a helicase inhibitor; Including, The method, wherein said helicase inhibitor is a WRN inhibitor.

5. A method for selecting a cancer patient to be treated with a helicase inhibitor, selecting a cancer patient in which at least one mutation selected from a first group consisting of a loss-of-function mutation in TTK and a loss-of-function mutation in RAD50 has been detected in cancer cells contained in a sample derived from the cancer patient as a target for cancer treatment using a helicase inhibitor; Including, The method, wherein said helicase inhibitor is a WRN inhibitor.

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 a first group consisting of loss-of-function mutations in TTK and loss-of-function mutations in RAD50 in cancer cells contained in a sample derived from a cancer patient; (b) selecting a cancer patient in whom the mutation has been detected in the cancer cells as a target for cancer treatment with a helicase inhibitor; Including, The method, wherein said helicase inhibitor is a WRN inhibitor.

7. The method according to any one of claims 1 to 6, wherein the cancer cells are cancer cells in which a loss-of-function mutation in MSH3 is further detected.

8. A method for screening a compound to be used in the treatment of a cancer comprising a cancer cell in which at least one mutation selected from a first group consisting of a loss-of-function mutation of TTK and a loss-of-function mutation of RAD50 is detected, A step of selecting a compound based on whether or not it inhibits helicase. Including, The method, wherein the helicase is WRN.

9. Contains a compound that inhibits helicase as an active ingredient, A cancer therapeutic agent for detecting the presence or absence of at least one mutation selected from a first group consisting of loss-of-function mutations of TTK and loss-of-function mutations of RAD50 in cancer cells contained in a sample derived from a cancer patient, and for administering to a cancer patient in which said mutation has been detected in the cancer cells, A cancer therapeutic agent, wherein the helicase is WRN.

10. A method for predicting sensitivity of a cancer cell to a helicase inhibitor, predicting that a cancer cell in which at least one mutation selected from a second group consisting of a loss-of-function mutation in RAD50, a loss-of-function mutation in MRE11, a loss-of-function mutation in NBN, a loss-of-function mutation in DNA2, and a loss-of-function mutation in RBBP8 has been detected is sensitive to a helicase inhibitor; Including, The method, wherein said helicase inhibitor is a WRN inhibitor.

11. A method for predicting sensitivity of a cancer cell to a helicase inhibitor, (a) detecting the presence or absence of at least one mutation selected from a second group consisting of loss-of-function mutations of RAD50, loss-of-function mutations of MRE11, loss-of-function mutations of NBN, loss-of-function mutations of DNA2, and loss-of-function mutations of RBBP8 in cancer cells; (b) predicting that the cancer cell in which the mutation is detected is sensitive to a helicase inhibitor; Including, The method, wherein said helicase inhibitor is a WRN inhibitor.

12. A method for predicting sensitivity of a cancer patient to treatment with a helicase inhibitor, predicting that a cancer patient in which at least one mutation selected from a second group consisting of a loss-of-function mutation in RAD50, a loss-of-function mutation in MRE11, a loss-of-function mutation in NBN, a loss-of-function mutation in DNA2, and a loss-of-function mutation in RBBP8 has been detected in cancer cells contained in a sample derived from the cancer patient, is susceptible to treatment with a helicase inhibitor; Including, The method, wherein said helicase inhibitor is a WRN inhibitor.

13. A method for predicting sensitivity of a cancer patient to treatment with a helicase inhibitor, (a) detecting the presence or absence of at least one mutation selected from a second group consisting of loss-of-function mutations of RAD50, loss-of-function mutations of MRE11, loss-of-function mutations of NBN, loss-of-function mutations of DNA2, and loss-of-function mutations of RBBP8 in cancer cells contained in a sample derived from a cancer patient; (b) predicting that a cancer patient in whom the mutation has been detected in the cancer cells will be susceptible to treatment with a helicase inhibitor; Including, The method, wherein said helicase inhibitor is a WRN inhibitor.

14. A method for selecting a cancer patient to be treated with a helicase inhibitor, selecting a cancer patient in which at least one mutation selected from a second group consisting of a loss-of-function mutation in RAD50, a loss-of-function mutation in MRE11, a loss-of-function mutation in NBN, a loss-of-function mutation in DNA2, and a loss-of-function mutation in RBBP8 has been detected in cancer cells contained in a sample derived from the cancer patient, as a target for cancer treatment using a helicase inhibitor; Including, The method, wherein said helicase inhibitor is a WRN inhibitor.

15. 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 second group consisting of loss-of-function mutations of RAD50, loss-of-function mutations of MRE11, loss-of-function mutations of NBN, loss-of-function mutations of DNA2, and loss-of-function mutations of RBBP8 in cancer cells contained in a sample derived from a cancer patient; (b) selecting a cancer patient in whom the mutation has been detected in the cancer cells as a target for cancer treatment with a helicase inhibitor; Including, The method, wherein said helicase inhibitor is a WRN inhibitor.

16. The method according to any one of claims 10 to 15, wherein the cancer cells are cancer cells in which at least one mutation selected from a third group consisting of loss-of-function mutations in EXO1, loss-of-function mutations in RPA1, loss-of-function mutations in RPA2, and loss-of-function mutations in RPA3 is further detected.

17. A method for screening a compound to be used in the treatment of a cancer comprising cancer cells in which at least one mutation selected from a second group consisting of a loss-of-function mutation in RAD50, a loss-of-function mutation in MRE11, a loss-of-function mutation in NBN, a loss-of-function mutation in DNA2, and a loss-of-function mutation in RBBP8 is detected, A step of selecting a compound based on whether or not it inhibits helicase. Including, The method, wherein the helicase is WRN.

18. Contains a compound that inhibits helicase as an active ingredient, a cancer therapeutic agent for detecting the presence or absence of at least one mutation selected from a second group consisting of a loss-of-function mutation of RAD50, a loss-of-function mutation of MRE11, a loss-of-function mutation of NBN, a loss-of-function mutation of DNA2, and a loss-of-function mutation of RBBP8 in cancer cells contained in a sample derived from a cancer patient, and for administration to a cancer patient in which said mutation has been detected in the cancer cells; A cancer therapeutic agent, wherein the helicase is WRN.

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