Method for predicting sensitivity of cancer cell to GPX4 inhibitor

Abstract

To develop a therapeutic strategy capable of specifically targeting cancer cells in which depression of the function of the SWI / SNF complex occurs, and more specifically, to develop a therapeutic strategy that specifically targets cancer cells in which depression of the function of an SWI / SNF complex factor is detected.SOLUTION: A therapeutic agent for cancer comprising, as an active ingredient, a compound that inhibits GPX4 and containing a cancer cell in which depression of the function of an SWI / SNF complex factor is detected, and a method for predicting the sensitivity of a cancer cell to a GPX4 inhibitor, the method comprising a step for predicting a cancer cell in which depression of the function of an SWI / SNF complex factor is detected as having sensitivity to the GPX4 inhibitor.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for predicting the sensitivity of cancer cells to a GPX4 inhibitor, and also to a method for predicting the sensitivity of a cancer patient to treatment with a GPX4 inhibitor, a method for selecting a cancer patient for cancer treatment with a GPX4 inhibitor, a method for suppressing the proliferation of cancer cells, a method for treating cancer, a method for screening a compound to be used in cancer treatment, 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, efforts are being made to develop inhibitors that specifically inhibit the function of cancer cells with gain-of-function gene mutations, such as EGFR gene mutations, BRAF gene mutations, ALK fusion genes, and ROS1 fusion genes (Non-Patent Documents 1 to 3, etc.). Treatment methods that target cancer cells with these gene mutations and are specific to these cancer cells are expected to be highly selective and effective treatment methods for cancer.

[0003] On the other hand, genetic mutations found in human cancer cells include not only the gain-of-function mutations mentioned above, but also loss-of-function mutations. Loss-of-function mutations are difficult to develop drugs specific to these mutations, and require a different treatment strategy than treatments that target cancer cells with gain-of-function mutations.

[0004] The SWI / SNF complex is a chromatin remodeling factor composed of 12 to 15 subunits (complex factors). In recent years, functional inhibition of SWI / SNF complex factors, such as loss-of-function gene mutations or expression suppression, has been reported in many human cancers, drawing attention to its involvement in tumor initiation and progression. These mutations are thought to disrupt key SWI / SNF complex functions, such as dissociation of DNA stored in nucleosomes and recruitment of transcriptional regulators, including transcription factors and histone deacetylases, to DNA. Therefore, it is speculated that such functional inhibition may be closely related to cancer initiation and progression, and research is currently being conducted.

[0005] For example, Non-Patent Document 4 describes that in non-small cell lung cancer, deficiency of SMARCA4, a SWI / SNF complex factor, is synthetically lethal to CDK4 / 6 inhibitors; Non-Patent Document 5 describes that lung cancer lacking SMARCA4 has increased sensitivity to OXPHOS (mitochondrial oxidative phosphorylation) inhibitors; Non-Patent Document 6 describes that mutations in the SMARCA4 gene in non-small cell lung cancer have increased sensitivity to Aurora kinase inhibitors; and Non-Patent Document 7 describes that mutations in the ARID1A gene, another SWI / SNF complex factor, are synthetically lethal to GSH synthesis inhibitors.

[0006] However, there are still few therapeutic strategies that specifically target cancer cells in which the function of the SWI / SNF complex is suppressed. [Prior art documents] [Non-patent literature]

[0007] [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] Yibo Xue et al., Nat. Commun. 10:557, 2019 [Non-patent document 5] Yonathan Lissanu Deribe et al., Nat.Med.Vol 24, July 2018, p.1047-1057 [Non-patent document 6] Vural Tagal et al., Nat.Commun.8:14098,2017 [Non-Patent Document 7] Hideaki Ogiwara et al., 2019, Cancer Cell 35, p.177-190 Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention has been made in light of the above-mentioned circumstances, and aims to develop a therapeutic strategy that can specifically target cancer cells in which the function of the SWI / SNF complex is suppressed, more specifically, to develop a therapeutic strategy that specifically targets cancer cells in which the function of SWI / SNF complex factors is detected to be suppressed. [Means for solving the problem]

[0009] As a result of extensive research conducted by the inventors to solve the above-mentioned problems, they found that when the expression or function of GPX4 is inhibited in cancer cells in which functional inhibition of SWI / SNF complex factors is detected, the proliferation of the cancer cells is significantly inhibited and / or cell death is induced, whereas such proliferation inhibition and / or cell death does not occur in cells in which functional inhibition of SWI / SNF complex factors is not observed.

[0010] More specifically, the present inventors conducted comprehensive knockdown experiments on human cancer cell lines to investigate which protein-inhibiting drugs would have antitumor activity against human cancer cells in which the function of SWI / SNF complex factors was inhibited. As a result, they found that suppression of GPX4 expression in cancer cell lines that have deletion mutations in the SMARCA4 gene, a core component of the SWI / SNF complex, and / or in which SMARCA4 protein expression is undetectable, induced cell death in the cancer cells and significantly inhibited cell proliferation.

[0011] Furthermore, the inventors have found that inhibiting GPX4 activity using compounds (e.g., ML210, RSL3) that have been reported to inhibit the enzymatic activity of GPX4 also induces cell death in cancer cells in which SMARCA4 function has been suppressed, and significantly suppresses cell proliferation, similar to the case of suppressing GPX4 expression.

[0012] Furthermore, eight types of molecules belonging to the GPX family have been reported in humans: GPX1, GPX2, GPX3, GPX4, GPX5, GPX6, GPX7, and GPX8. The present inventors have found that inhibition of GPX4 in particular exhibits specific antitumor activity against cancer cells in which SMARCA4 function is suppressed.

[0013] On the other hand, because SMARCA4 is a component of the SWI / SNF complex, we suspected that GPX4 might also be effective against cells in which the function of other SWI / SNF complex proteins is suppressed. We therefore evaluated the antitumor activity of GPX4 by inhibiting the expression and / or activity of GPX4 in cancer cell lines in which the function of other SWI / SNF complex proteins (e.g., SMARCA2, ARID1A, ARID1B, ARID2, and BCL11B) is suppressed in addition to SMARCA4. We found that GPX4 inhibition significantly suppressed cell proliferation and cell death in cancer cell lines in which the function of other SWI / SNF complex proteins was suppressed, as well as in cancer cell lines in which the function of SMARCA4 was suppressed.

[0014] Furthermore, since GPX4 is an enzyme that consumes intracellular glutathione and hydrolyzes it, it is possible that other factors (proteins) involved in glutathione synthesis may also exhibit antitumor activity against cancer cells in which the function of SWI / SNF complex factors is suppressed. However, inhibition of other glutathione synthesis-related factors (e.g., gamma-glutamylcysteine ​​synthetase (GCLC), glutathione synthetase (GSS), glutamate-cysteine ​​ligase modifier subunit (GCLM), microsomal glutathione S-transferase 1 (MGST1), microsomal glutathione S-transferase 3 (MGST3), glutathione-disulfide reductase (GSR), glucose-6-phosphate dehydrogenase (G6PD)) did not result in sufficient antitumor activity compared to inhibition of GPX4. Therefore, it has become clear that inhibition of GPX4 in particular is important for achieving antitumor activity targeting cancer cells in which the function of SWI / SNF complex factors is suppressed.

[0015] Furthermore, the inventors demonstrated that in tumor-bearing mice transplanted with cancer cells in which the function of SWI / SNF complex factors was suppressed, the increase in tumor volume was significantly suppressed by administration of a compound (GPX4 inhibitor) that has been reported to inhibit the enzymatic activity of GPX4, demonstrating that GPX4 inhibition indeed exhibits antitumor effects in vivo.

[0016] Therefore, the present inventors have found that inhibiting GPX4 (by suppressing its expression or activity) is a promising approach for targeting cancer cells in which the function of SWI / SNF complex factors is suppressed. Furthermore, this therapeutic strategy allows cancer patients to be selected based on the suppression of SWI / SNF complex factors and then administered a GPX4 inhibitor, making it possible to provide efficient treatment based on companion diagnostics.

[0017] Furthermore, the inventors have discovered that screening for drugs useful for treating cancers with suppressed function of SWI / SNF complex factors can be carried out using whether or not they inhibit GPX4 as an indicator, thereby completing the present invention.

[0018] Therefore, the present invention relates to a therapeutic method that specifically targets cancer cells in which the function of SWI / SNF complex factors is suppressed, and a companion diagnostic for the therapeutic method, and more specifically, provides the following inventions.

[0019] [1] A cancer therapeutic agent containing a compound that inhibits GPX4 as an active ingredient, the agent being a therapeutic agent for cancer including cancer cells in which functional inhibition of SWI / SNF complex factors is detected.

[0020] [2] A cancer therapeutic agent containing a compound that inhibits GPX4 as an active ingredient, which is a therapeutic agent for treating cancer patients in which functional inhibition of SWI / SNF complex factors has been detected in cancer cells contained in samples derived from cancer patients.

[0021] [3] A cancer therapeutic agent containing a compound that inhibits GPX4 as an active ingredient, which is to be administered to a cancer patient in whom functional inhibition of SWI / SNF complex factors has been detected in cancer cells contained in a sample derived from the cancer patient.

[0022] [4] A method for predicting the sensitivity of cancer cells to a GPX4 inhibitor, predicting that cancer cells in which functional inhibition of SWI / SNF complex factors has been detected are sensitive to a GPX4 inhibitor; A method comprising:

[0023] [5] A method for predicting the sensitivity of cancer cells to a GPX4 inhibitor, (a) detecting whether or not the function of a SWI / SNF complex factor is inhibited in cancer cells; (b) predicting that cancer cells in which functional inhibition of the SWI / SNF complex factor is detected are sensitive to a GPX4 inhibitor; A method comprising:

[0024] [6] A method for predicting the sensitivity of a cancer patient to treatment with a GPX4 inhibitor, predicting that a cancer patient in which functional inhibition of a SWI / SNF complex factor has been detected in cancer cells contained in a sample derived from the cancer patient will be sensitive to treatment with a GPX4 inhibitor; A method comprising:

[0025] [7] A method for predicting the sensitivity of a cancer patient to treatment with a GPX4 inhibitor, (a) detecting whether or not the function of a SWI / SNF complex factor is inhibited in cancer cells contained in a sample derived from a cancer patient; (b) predicting that a cancer patient in whom functional inhibition of the SWI / SNF complex factor is detected in the cancer cells will be sensitive to treatment with a GPX4 inhibitor; A method comprising:

[0026] [8] A method for selecting cancer patients who are candidates for cancer treatment with a GPX4 inhibitor, selecting cancer patients in whom functional inhibition of SWI / SNF complex factors has been detected in cancer cells contained in a sample derived from the cancer patients as targets for cancer treatment with a GPX4 inhibitor; A method comprising:

[0027] [9] A method for selecting cancer patients who are candidates for cancer treatment with a GPX4 inhibitor, (a) detecting whether or not the function of a SWI / SNF complex factor is inhibited in cancer cells contained in a sample derived from a cancer patient; (b) selecting cancer patients in whom functional inhibition of the SWI / SNF complex factor is detected in the cancer cells as targets for cancer treatment with a GPX4 inhibitor; A method comprising:

[0028]

[10] A method for suppressing the proliferation of cancer cells in which functional inhibition of SWI / SNF complex factors is detected, contacting a GPX4 inhibitor with cancer cells in which functional inhibition of the SWI / SNF complex factor is detected; A method comprising:

[0029]

[11] A method of treating cancer, administering a GPX4 inhibitor to a cancer patient in which functional inhibition of a SWI / SNF complex factor has been detected in cancer cells contained in a sample derived from the cancer patient; A method comprising:

[0030]

[12] A method of treating cancer, (a) detecting whether or not the function of a SWI / SNF complex factor is inhibited in cancer cells contained in a sample derived from a cancer patient; (b) administering a GPX4 inhibitor to a cancer patient in which functional inhibition of the SWI / SNF complex factor has been detected in the cancer cells; A method comprising:

[0031]

[13] A method for screening compounds to be used in the treatment of cancer, including cancer cells in which functional inhibition of SWI / SNF complex factors is detected, a step of selecting a compound based on whether or not it inhibits GPX4; A method comprising:

[0032]

[14] The cancer therapeutic agent according to any one of [1] to [3], wherein the SWI / SNF complex factor is a BAF complex factor.

[0033]

[15] The method according to any one of [4] to

[13] , wherein the SWI / SNF complex factor is a BAF complex factor.

[0034]

[16] The cancer therapeutic agent according to any one of [1] to [3], wherein the SWI / SNF complex factor is at least one selected from the group consisting of SMARCA2, SMARCA4, ARID1A, ARID1B, ARID2, and BCL11B.

[0035]

[17] The method according to any one of [4] to

[13] , wherein the SWI / SNF complex factor is at least one selected from the group consisting of SMARCA2, SMARCA4, ARID1A, ARID1B, ARID2, and BCL11B.

[0036]

[18] The cancer therapeutic agent according to any one of [1] to [3], wherein the functional inhibition of the SWI / SNF complex factor is a decrease in the activity of a SWI / SNF complex of which the SWI / SNF complex factor is a component factor and / or a decrease in the expression of the SWI / SNF complex factor.

[0037]

[19] The method according to any one of [4] to

[13] , wherein the functional inhibition of the SWI / SNF complex factor is a decrease in the activity of a SWI / SNF complex of which the SWI / SNF complex factor is a component factor and / or a decrease in the expression of the SWI / SNF complex factor.

[0038]

[20] The cancer therapeutic agent according to any one of [1] to [3], wherein the function of the SWI / SNF complex factor is inhibited by a loss-of-function mutation in the gene of the SWI / SNF complex factor.

[0039] [twenty one] The method according to any one of [4] to

[13] , wherein the functional inhibition of the SWI / SNF complex factor is a loss-of-function mutation in the gene of the SWI / SNF complex factor.

[0040] [twenty two] A cancer therapeutic agent containing a compound that inhibits GPX4 as an active ingredient, which is a therapeutic agent for cancer including cancer cells in which loss-of-function mutations in genes of SWI / SNF complex factors have been detected.

[0041] [twenty three] A cancer therapeutic agent containing a compound that inhibits GPX4 as an active ingredient, for treating cancer patients in whom loss-of-function mutations in the genes of SWI / SNF complex factors have been detected in cancer cells contained in samples derived from cancer patients.

[0042] [twenty four] A cancer therapeutic agent containing a compound that inhibits GPX4 as an active ingredient, which is to be administered to cancer patients in whom loss-of-function mutations in the genes of SWI / SNF complex factors have been detected in cancer cells contained in samples derived from the cancer patients.

[0043] [twenty five] A method for predicting the sensitivity of cancer cells to a GPX4 inhibitor, a step of predicting that cancer cells in which loss-of-function mutations in SWI / SNF complex factor genes have been detected are sensitive to a GPX4 inhibitor; A method comprising:

[0044]

[26] A method for predicting the sensitivity of cancer cells to a GPX4 inhibitor, (a) detecting the presence or absence of a loss-of-function mutation in a gene encoding a SWI / SNF complex factor in a cancer cell; (b) predicting that cancer cells in which loss-of-function mutations in the genes of the SWI / SNF complex factor have been detected are sensitive to a GPX4 inhibitor; A method comprising:

[0045]

[27] A method for predicting the sensitivity of a cancer patient to treatment with a GPX4 inhibitor, predicting that a cancer patient in which a loss-of-function mutation in a gene of a SWI / SNF complex factor has been detected in cancer cells contained in a sample derived from the cancer patient will be sensitive to treatment with a GPX4 inhibitor; A method comprising:

[0046]

[28] A method for predicting the sensitivity of a cancer patient to treatment with a GPX4 inhibitor, (a) detecting the presence or absence of loss-of-function mutations in genes of SWI / SNF complex factors in cancer cells contained in a sample derived from a cancer patient; (b) predicting that a cancer patient in whom a loss-of-function mutation in the gene of the SWI / SNF complex factor has been detected in the cancer cells will be sensitive to treatment with a GPX4 inhibitor; A method comprising:

[0047]

[29] A method for selecting cancer patients who are candidates for cancer treatment with a GPX4 inhibitor, a step of selecting cancer patients in whom loss-of-function mutations in genes encoding SWI / SNF complex factors have been detected in cancer cells contained in a sample derived from the cancer patients as targets for cancer treatment with a GPX4 inhibitor; A method comprising:

[0048]

[30] A method for selecting cancer patients who are candidates for cancer treatment with a GPX4 inhibitor, (a) detecting the presence or absence of loss-of-function mutations in genes of SWI / SNF complex factors in cancer cells contained in a sample derived from a cancer patient; (b) selecting cancer patients in whom loss-of-function mutations in the genes of the SWI / SNF complex factor are detected in the cancer cells as targets for cancer treatment with a GPX4 inhibitor; A method comprising:

[0049]

[31] This method inhibits the proliferation of cancer cells in which loss-of-function mutations in the genes of SWI / SNF complex factors are detected. contacting a GPX4 inhibitor with cancer cells in which loss-of-function mutations in the genes of the SWI / SNF complex factors are detected; A method comprising:

[0050]

[32] A method of treating cancer, administering a GPX4 inhibitor to a cancer patient in which a loss-of-function mutation in a gene of a SWI / SNF complex factor has been detected in cancer cells contained in a sample derived from the cancer patient; A method comprising:

[0051]

[33] A method of treating cancer, (a) detecting the presence or absence of loss-of-function mutations in genes of SWI / SNF complex factors in cancer cells contained in a sample derived from a cancer patient; (b) administering a GPX4 inhibitor to a cancer patient in which a loss-of-function mutation in the gene of the SWI / SNF complex factor has been detected in the cancer cells; A method comprising:

[0052]

[34] A method for screening compounds to be used in the treatment of cancer, including cancer cells in which loss-of-function mutations in genes of SWI / SNF complex factors are detected, a step of selecting a compound based on whether or not it inhibits GPX4; A method comprising:

[0053]

[35] The cancer therapeutic agent according to any one of

[22] to

[24] , wherein the SWI / SNF complex factor is a BAF complex factor.

[0054]

[36] The cancer therapeutic agent according to any one of

[25] to

[34] , wherein the SWI / SNF complex factor is a BAF complex factor.

[0055]

[37] The cancer therapeutic agent according to any one of

[22] to

[24] , wherein the SWI / SNF complex factor is at least one selected from the group consisting of SMARCA2, SMARCA4, ARID1A, ARID1B, ARID2, and BCL11B.

[0056]

[38] The method according to any one of

[25] to

[34] , wherein the SWI / SNF complex factor is at least one selected from the group consisting of SMARCA2, SMARCA4, ARID1A, ARID1B, ARID2, and BCL11B. [Effects of the Invention]

[0057] According to the present invention, it is possible to efficiently predict sensitivity to cancer treatment with a GPX4 inhibitor by using functional inhibition of a SWI / SNF complex factor (e.g., genetic mutation or reduced protein expression of a SWI / SNF complex factor, such as SMARCA4 or ARID1A) as an indicator. Furthermore, according to the present invention, the presence or absence of functional inhibition of a SWI / SNF complex factor in a sample from a cancer patient can be detected, and patients in whom such mutations are detected can be selected and then treated with a GPX4 inhibitor. This makes it possible to significantly improve the outcome of cancer treatment. Furthermore, by using probes or primers for genes of SWI / SNF complex factors and antibodies against SWI / SNF complex factors, it is possible to efficiently perform companion diagnostics by detecting the presence or absence of functional inhibition of such SWI / SNF complex factors. [Brief explanation of the drawings]

[0058] [Figure 1] This is a graph showing the cell growth inhibition rate (CGI (%)) when the expression of GPX4 (#1, #2) was suppressed in each of the cell lines NCI-H1792, MOR, NCI-H2110, NCI-H522, NCI-H23, and SNU-1327. [Figure 2] 1 is a graph showing the cell growth inhibition rate (CGI (%)) when the expression of GPX4 (#2) was suppressed in each cell line shown in Table 4. [Figure 3] It is a graph showing the cell growth inhibition rate (CGI (%)) when the expression of GPX4 (#3) is suppressed in each cell line shown in Table 4. [Figure 4] It is a graph showing the IC50 (μM) in each cell line shown in Table 4 when ML210 is used as a GPX4 inhibitor. [Figure 5] It is a graph showing the IC50 (μM) in each cell line shown in Table 4 when RSL3 is used as a GPX4 inhibitor. [Figure 6] It is a graph showing the cell growth inhibition rate (CGI (%)) when the expression of each GPX family protein is suppressed in each of the cell lines NCI-H2110, NCI-H522, and NCI-H23. [Figure 7] It is a graph showing the cell growth inhibition rate (CGI (%)) when the expression of each glutathione synthesis-related protein is suppressed in each of the cell lines NCI-H2110, NCI-H522, and NCI-H23. [Figure 8] It is a graph showing the IC50 (μM) in each of the cell lines MOR, NCI-H358, NCI-H23, and NCI-H522 when ML210, RSL3, or BSO is used. [Figure 9] It is a graph showing the relationship between the volume of tumors in each group (ML210 or Vehicle) of SK-HEP-1 tumor-bearing mice and the time after SK-HEP-1 transplantation.

Mode for Carrying Out the Invention

[0059] Hereinafter, the present invention will be described in detail according to its preferred embodiments.

[0060] <Method for predicting the sensitivity of cancer cells to a GPX4 inhibitor, method for predicting the sensitivity of a cancer patient to treatment with a GPX4 inhibitor, method for selecting a cancer patient to be treated with a GPX4 inhibitor> In the present invention, it has been found that inhibiting GPX4 in cancer cells in which the function of SWI / SNF complex factors is suppressed can suppress the proliferation of the cancer cells. Based on this finding, the sensitivity of cancer cells to GPX4 inhibitors can be predicted using the function suppression of SWI / SNF complex factors as an indicator. Therefore, the present invention provides: (a) detecting whether or not the function of a SWI / SNF complex factor is inhibited in cancer cells; (b) predicting that cancer cells in which functional inhibition of the SWI / SNF complex factor is detected are sensitive to a GPX4 inhibitor; The present invention provides a method for predicting the sensitivity of cancer cells to a GPX4 inhibitor (hereinafter sometimes referred to as a "method for predicting cancer cell sensitivity"), which comprises:

[0061] Furthermore, based on the above findings, it is possible to predict sensitivity to cancer treatment with a GPX4 inhibitor using functional inhibition of SWI / SNF complex factors as an indicator. (a) detecting whether or not the function of a SWI / SNF complex factor is inhibited in cancer cells contained in a sample derived from a cancer patient; (b) predicting that a cancer patient in whom functional inhibition of the SWI / SNF complex factor is detected in the cancer cells will be sensitive to treatment with a GPX4 inhibitor; The present invention provides a method for predicting the sensitivity of a cancer patient to treatment with a GPX4 inhibitor (hereinafter sometimes referred to as a "method for predicting sensitivity in a cancer patient"), which comprises:

[0062] Furthermore, patients in whom functional inhibition of SWI / SNF complex factors is detected in this manner can be said to be suitable for cancer treatment with GPX4 inhibitors, and therefore, using functional inhibition of SWI / SNF complex factors as an indicator, patients for whom cancer treatment with GPX4 inhibitors is effective and those for whom it is not effective can be selected, and efficient treatment can be carried out. (a) detecting whether or not the function of a SWI / SNF complex factor is inhibited in cancer cells contained in a sample derived from a cancer patient; (b) selecting cancer patients in whom functional inhibition of the SWI / SNF complex factor is detected in the cancer cells as targets for cancer treatment with a GPX4 inhibitor; The present invention provides a method for selecting cancer patients to be treated with a GPX4 inhibitor (hereinafter sometimes referred to as a "cancer patient selection method"), which comprises:

[0063] (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" or "tumor," and cells constituting the cancer are referred to as "cancer cells." Cancers containing cancer cells for which the presence or absence of functional inhibition of SWI / SNF complex factors can be detected include, but are not limited to, lung cancer, ovarian cancer, uterine cancer, liver cancer, gastric cancer, esophageal cancer, colon cancer, pancreatic cancer, prostate cancer, bladder cancer, and kidney cancer.

[0064] In the present invention, the term "cancer patient" refers not only to a person currently suffering from the cancer, but also to a person suspected of suffering from the cancer. In the method of the present invention, there are no particular limitations on the cancer patients who are the targets for detecting the functional inhibition of SWI / SNF complex factors, and all cancer patients can be the targets.

[0065] The "sample derived from a cancer patient" used in the present invention is not particularly limited as long as it is a biological sample that can detect the presence or absence of functional inhibition of a SWI / SNF complex factor, but is preferably a cancer biopsy specimen, blood, urine, body cavity fluid, tumor cell-derived circulating tumor DNA (ctDNA), or other specimen. Protein extracts or nucleic acid extracts (e.g., mRNA extracts, cDNA preparations or cRNA preparations prepared from mRNA extracts) obtained from the specimens may also be used. As used herein, the term "biological sample" includes samples derived from cancer patients and samples derived from cancer cell cultures.

[0066] (GPX4 inhibitor) In the present invention, a "GPX4 inhibitor" refers to a composition containing at least one compound that inhibits GPX4 (glutathione peroxidase 4), and may consist solely of the compound or a combination thereof, or may further contain the following additional components. In the present invention, a "GPX4 inhibitor" includes compounds that inhibit at least either the activity of GPX4 or the expression of GPX4.

[0067] In the present invention, "GPX4" (also referred to herein as "GPX4 protein"), the target of GPX4 inhibitors, is one of eight known isozymes of glutathione peroxidase (GPX1 to GPX8) that possess peroxidase activity by oxidizing and cleaving the peroxide structure of glutathione to two hydroxyl groups. While other GPXs can reduce hydrogen peroxide or fatty acid peroxides as substrates, GPX4 is an enzyme that also functions to directly reduce phospholipid peroxides. The nucleotide sequence of a typical human cDNA encoding GPX4 is shown in SEQ ID NO: 1 (NCBI Reference No. NM_002085.5), and the typical amino acid sequence of a human GPX4 protein is shown in SEQ ID NO: 2 (NCBI Reference No. NP_002076.2). Note that even DNA encoding GPX4 without mutations such as substitutions, deletions, insertions, or additions in the amino acid sequence may vary between individuals due to polymorphisms.

[0068] Whether a compound inhibits GPX4 activity can be confirmed, for example, by adding phosphatidylcholine hydroperoxide, a GPX4 substrate, to a cell lysate treated with the test compound and measuring its reduction as an indicator (see, for example, Viswanathan et al., Nature. 2017 July 27;547(7664):453-457, "Dependency of a therapy-resistant state of cancer cells on a lipid peroxidase pathway"; Yang et al., Cell. 2014 January 16 156(0):317-331, "Regulation of Ferroptotic Cancer Cell Death by GPX4"). When GPX4 activity is inhibited by the test compound, reduction of the substrate does not occur or occurs less easily (the amount of reduction decreases).

[0069] Inhibition of GPX4 activity by a compound can also be confirmed by treating a purified GPX4 preparation with a test compound, adding GPX4 substrates GSH and peroxide, glutathione reductase, and NADPH, and measuring activity by an enzyme recycling method (Shinome et al., Antioxid. Redox Signal. 22(4), 281-293, Expression of Inactive Glutathione Peroxidase 4 Leads to Embryonic Lethality, and Inactivation of the Alox15 Gene Does Not Rescue Such Knock-In Mice, etc.). When GPX4 activity is inhibited by the test compound, the consumption of GSH or NADPH decreases.

[0070] Furthermore, whether a compound inhibits GPX4 expression can be confirmed, for example, by detecting a decrease in GPX4 expression in cells treated with the test compound. A typical method for detecting a decrease in GPX4 expression is to detect the expression level of GPX4 at the transcriptional or translational level and confirm that the expression level is lower than that of a control (e.g., the expression level in cells not treated with the test compound).

[0071] In a method for detecting the expression level of GPX4 at the transcription level, RNA or cDNA is first prepared from cells treated with a test compound. The method for extracting RNA from the cells is not particularly limited, and any known method can be appropriately selected and used. Examples include extraction methods using phenol and chaotropic salts (more specifically, extraction methods using commercially available kits such as Trizol (Invitrogen) and Isogen (Wako Pure Chemical Industries, Ltd.)) and methods 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 include reverse transcriptases derived from retroviruses such as Rous associated virus (RAV) and Avian myeloblastosis virus (AMV), and reverse transcriptases derived from murine retroviruses such as Moloney murine leukemia virus (MMLV).

[0072] The oligonucleotide primers or oligonucleotide probes are then used in an amplification reaction or hybridization reaction, respectively, to detect the amplified or hybridized products. Examples of such methods include RT-PCR, Northern blotting, dot blotting, DNA array analysis, in situ hybridization, RNase protection assay, and mRNA-seq. Those skilled in the art can routinely design oligonucleotide primers or oligonucleotide probes suitable for each method based on the nucleotide sequence of the GPX4 cDNA.

[0073] In a method for detecting GPX4 expression at the translational level, a protein sample is first prepared from cells treated with a test compound. Next, an antigen-antibody reaction is carried out using an antibody specific to GPX4 protein to detect GPX4 protein. In such an antibody-based protein detection method, for example, an antibody specific to GPX4 protein is added to the protein sample to carry out an antigen-antibody reaction, and binding of the antibody to GPX4 protein is detected. When the antibody specific to GPX4 protein is labeled, GPX4 protein can be detected directly. However, when the antibody is unlabeled, GPX4 protein can be detected indirectly by further reacting it with a labeled molecule that recognizes the antibody (e.g., a secondary antibody or protein A) and utilizing the label of the molecule. Examples of such methods include immunohistochemistry (immunostaining), Western blotting, ELISA, flow cytometry, imaging cytometry, radioimmunoassay, immunoprecipitation, and analysis using an antibody array.

[0074] There are no particular limitations on the type or origin of the antibody used, but a monoclonal antibody is preferred. Oligoclonal antibodies (a mixture of several to several dozen types of antibodies) or polyclonal antibodies can also be used, as long as they are capable of detecting GPX4 protein with sufficient specificity. Functional fragments of antibodies, such as Fab, Fab', F(ab')2, Fv, scFv, sc(Fv)2, dsFv, and diabodies, or multimers thereof (e.g., dimers, trimers, tetramers, polymers), can also be used. Such anti-GPX4 protein antibodies may be commercially available.

[0075] GPX4 protein can also be detected using mass spectrometry (MS). Analysis using a mass spectrometer coupled to liquid chromatography (LC / MS) is particularly advantageous due to its 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 GPX4 protein from the mass spectrometry values. Isotopic labeling reagents known in the art can be used for labeling, and suitable labeling reagents are commercially available. Fractionation can also be performed by methods known in the art, for example, using commercially available ion exchange columns.

[0076] The "compound that inhibits GPX4" 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 selected from the group consisting of organic or inorganic compound molecules, polypeptides, and polynucleotides.

[0077] Examples of the compound molecules include low-molecular-weight compounds (molecular weight less than 800) and medium-molecular-weight compounds (molecular weight 800 to 2,000). The polypeptides include full-length polypeptides encoded by genes, as well as fragments thereof, synthetic polypeptides, cyclic polypeptides, and glycopeptides. The polypeptides also include antibodies and antigen peptides, and the antibodies may be polyclonal or monoclonal. The antibodies include complete antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, scFv, sc(Fv)2, dsFv, diabodies, etc.), multimers thereof, and minibodies formed by binding antibody variable regions. The polynucleotides include DNA, RNA, and siRNA, and include full-length polynucleotides, fragments thereof, and synthetic polynucleotides.

[0078] The "GPX4 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. Furthermore, 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, isotonic agent, soothing agent, bulking agent, disintegrant, buffer, coating agent, lubricant, colorant, sweetener, thickener, flavoring agent, and solubilizing agent, and can be manufactured using these by known pharmaceutical methods.

[0079] Furthermore, in the GPX4 inhibitor of the present invention, the content of the compound that inhibits GPX4 (if there are two or more types of compounds, the total content of these compounds) can be adjusted appropriately depending on the dosage form and purpose of use.

[0080] (Suppression of SWI / SNF complex factors) [SWI / SNF complex factor] The "SWI / SNF complex" in this invention is one of the chromatin modeling factors, and is a complex consisting of multiple subunits that mainly functions to transform chromatin structure by moving or removing nucleosomes using the energy of ATP hydrolysis.

[0081] In the present invention, "SWI / SNF complex factors (sometimes referred to herein as "SWI / SNF complex factor proteins")" refer to subunits constituting the SWI / SNF complex, such as SMARCA2 (sometimes referred to herein as "SMARCA2 protein"; the same applies to the following factors), SMARCA4, ARID1A, ARID1B, ARID2, ACTL6A, ACTL6B, DPF1, DPF2, DPF3, SMARCB1, SMARCC1, SMARCC2, SMARCD1, SMARCD2, SMARCD3, SMARCE1, SS18, SS18L1, BCL7A, BCL7B, BCL7C, BCL11A, BCL11B, BRD7, BRD9, PBRM1, and PHF10. The SWI / SNF complex factors whose functional inhibition is to be detected may be one of these alone or a combination of two or more of them.

[0082] Among the SWI / SNF complex factors, SMARCA2, SMARCA4, SMARCC1, SMARCC2, SMARCB1, ARID1A, ARID1B, SMARCD1, SMARCD2, SMARCD3, BCL11B, SMARCE1, PHF10, DPF1, DPF3, ACTL6A, and ACTL6B are BAF complex factors that constitute the BAF complex, and SMARCA4, SMARCC1, SMARCC2, SMARCB1, ARID2, PBRM1, BRD7, BCL11B, SMARCE1, PHF10, DPF1, DPF3, ACTL6A, and ACTL6B are PBAF complex factors that constitute the PBAF complex. Of these, the SWI / SNF complex factor of the present invention is preferably at least one selected from the group consisting of SMARCA2, SMARCA4, ARID1A, ARID1B, ARID2, and BCL11B, more preferably at least one selected from the group consisting of BAF complex factors, and even more preferably at least one selected from the group consisting of SMARCA2, SMARCA4, ARID1A, ARID1B, and BCL11B.

[0083] For the above-mentioned SWI / SNF complex factors, the SEQ ID NOs of the nucleotide sequences of typical human-derived DNA (cDNA) encoding each SWI / SNF complex factor and the SEQ ID NOs of typical human-derived amino acid sequences of each SWI / SNF complex factor protein are shown in Tables 1 to 3 below, respectively, but are not limited thereto. Tables 1 to 3 also show the name and reference ID of each SWI / SNF complex factor in the NCBI RefSeq database (National Center for Biotechnology Information Reference Sequence Database). Note that even DNA encoding a SWI / SNF complex factor that does not contain mutations such as substitutions, deletions, insertions, or additions in the amino acid sequence may vary between individuals due to polymorphisms, etc.

[0084] [Table 1]

[0085] [Table 2]

[0086] [Table 3]

[0087] In the present invention, "inhibition of the function of a SWI / SNF complex factor" includes a reduction in activity, including inactivation, of the SWI / SNF complex containing the factor (having the factor as a constitutive factor), and a reduction in expression of the SWI / SNF complex factor.

[0088] [Detection of decreased activity of SWI / SNF complex] "Decreased activity of the SWI / SNF complex" means that the activity inherent to the SWI / SNF complex is reduced, and typically means that the activity level is lower than that of a control (e.g., the activity level in a healthy individual or non-cancerous tissue of the same patient). "Decreased activity of the SWI / SNF complex" also includes both partial or complete inactivation and reduced activity of the SWI / SNF complex.

[0089] Detection of direct reduction in activity of SWI / SNF complex The method for "detecting a direct decrease in the activity of the SWI / SNF complex" in the present invention is not particularly limited, and can be confirmed, for example, by purifying the SWI / SNF complex from a biological sample by a technique such as immunoprecipitation and measuring its chromatin remodeling activity (Michael L. Phelan et al., Molecular Cell, Vol. 3, pp. 247-253, February 1999, "Reconstitution of a Core Chromatin Remodeling Complex from SWI / SNF Subunits," etc.). If the activity is decreased compared to a control, it can be determined that a decrease in the activity of the SWI / SNF complex has been detected.

[0090] Alternatively, a direct decrease in the activity of the SWI / SNF complex can be detected by, for example, measuring its ATPase activity. For example, the SWI / SNF complex is purified from a biological sample by immunoprecipitation or other techniques, and its ATP consumption is detected using a commercially available product, such as ADP-glo (Promega). If the detection results show a decrease in ATP consumption compared to a control, it can be determined that a decrease in the activity of the SWI / SNF complex has been detected.

[0091] <Detection of mutations in SWI / SNF complex factor genes> Decreased activity of the SWI / SNF complex is typically caused by loss-of-function mutations in the genes of SWI / SNF complex factors (DNA encoding the SWI / SNF complex factors) that make up the complex. Therefore, detection of mutations in the genes of SWI / SNF complex factors can be used to detect decreased activity of the SWI / SNF complex.

[0092] Loss-of-function mutations can be caused by, for example, missense mutations, global nonsense mutations, frameshift mutations, or total or partial deletion of the gene in the SWI / SNF complex factor gene, but are not limited to these, as long as they cause a decrease in the activity of the SWI / SNF complex. Examples of mutations in SWI / SNF complex factor genes in major cancer cell lines are shown in Table 4 below. Table 4 also shows examples of the presence or absence of expression of SWI / SNF complex factors in major cancer cell lines.

[0093] [Table 4]

[0094] In Table 4, "Reference" indicates databases and literature in which the gene mutation or protein expression is reported. "CCLE" indicates the database created by the Broad Institute: "Cancer Cell Line Encyclopedia (https: / / portals.broadinstitute.org / ccle)." "Hoffman et al. 2014" indicates the literature: "Hoffman et al., PNAS February 25, 2014 111(8), pp. 3128-3133." "Matsubara et al. 2012" indicates the literature: "Matsubara et al., Cancer Sci, February 2013, vol. 104, no. 2, pp. 266-273." "Ogiwara et al. 2019" indicates the literature: "Ogiwara et al., Volume 35, Issue 2, 11 February 2019, pp. 177-190.e8." "patent" indicates that this is information on mutations or expression that have not been reported in papers, and that the inventors have confirmed that the gene has a truncated deletion mutation.

[0095] Other examples of specific mutations in genes of SWI / SNF complex factors that cause decreased activity of the SWI / SNF complex include, but are not limited to, ARID1A:p.Y551Lfs*72 (Insertion-Frameshift) (COSMIC Legacy Mutation ID: COSM51423).

[0096] The method for "detecting a mutation in a gene encoding an SWI / SNF complex factor" in the present invention is not particularly limited, and examples thereof include the following methods.

[0097] In the present invention, "detecting a mutation" generally means detecting a mutation in genomic DNA, but when the mutation in the genomic DNA is reflected in a base change in a transcription product or an amino acid change in a translation product, it also includes detecting the change in these transcription products or translation products (i.e., indirect detection).

[0098] A preferred embodiment of the method of the present invention is a method for detecting mutations by directly determining the nucleotide sequence of a gene region encoding a SWI / SNF complex factor in a cancer cell. In the present invention, "a gene region encoding a SWI / SNF complex factor" refers to a certain region on genomic DNA that contains the gene encoding the SWI / SNF complex factor. This region independently includes, in addition to the translated region, untranslated regions such as expression control regions of each gene (e.g., promoter regions and enhancer regions) and the 3'-terminal untranslated region of each gene.

[0099] In this method, a DNA sample is first prepared from a biological sample, including genomic DNA samples and cDNA samples prepared by reverse transcription from RNA.

[0100] 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 tissue stored in a solution containing urea or ethanol is denatured with a protease (proteinase K), a surfactant (SDS), and phenol to denature the proteins in the tissue, and then 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 MoBiTec), prepGEM (registered trademark, manufactured by ZyGEM), and BuccalQuick (registered trademark, manufactured by TrimGen).

[0101] Furthermore, methods for extracting RNA from biological samples and preparing cDNA from the extracted RNA include the same methods as those mentioned for detecting the expression level of GPX4 at the transcription level.

[0102] In this embodiment, DNA containing the gene region of the SWI / SNF complex factor is then isolated, and the nucleotide sequence of the isolated DNA is determined. The DNA can be isolated, for example, by PCR using genomic DNA or RNA as a template, and a pair of oligonucleotide primers designed to flank all or part of the gene region of the SWI / SNF complex factor. The nucleotide sequence of the isolated DNA can be determined by methods known to those skilled in the art, such as the Maxam-Gilbert method or the Sanger method. For the isolated or extracted DNA, a next-generation sequencer or the like can also be used, which is capable of rapid and comprehensive analysis of the nucleotide sequence of genes.

[0103] By comparing the determined DNA or cDNA base sequence with a control (for example, if the biological sample is derived from a cancer patient, with the DNA or cDNA base sequence derived from non-cancerous tissue of the same patient or with a publicly known database), it is possible to determine whether or not there is a mutation in the gene region of the SWI / SNF complex factor in the cancer cells of the biological sample.

[0104] Mutations in the gene region of an SWI / SNF complex factor can be detected by various methods that allow detection of mutations, in addition to methods that directly determine the base sequence of DNA or cDNA.

[0105] For example, mutation detection in the present invention can also be performed by the following method. First, a DNA or cDNA sample is prepared from a biological sample. Next, an oligonucleotide probe is prepared, which has a base sequence complementary to a base sequence containing the mutation site in the gene region of the SWI / SNF complex factor and is labeled with a reporter fluorescent dye and a quencher fluorescent dye. The oligonucleotide probe is then hybridized to the DNA or cDNA sample. The DNA or cDNA sample hybridized with the oligonucleotide probe is then used as a template to amplify the base sequence containing the mutation site in the gene region of the SWI / SNF complex factor. Fluorescence emitted by the reporter fluorescent dye due to degradation of the oligonucleotide probe during amplification is then detected, and the detected fluorescence is then compared with that of a control. Examples of such methods include the double-dye probe method, also known as the TaqMan® probe method.

[0106] In yet another method, a DNA or cDNA sample is prepared from a biological sample. Next, a base sequence containing a mutation site in the gene region of a SWI / SNF complex factor is amplified using the DNA or cDNA sample as a template in a reaction system containing an intercalator that emits fluorescence when inserted into the DNA double strand. The temperature of the reaction system is then changed, and changes in the intensity of the fluorescence emitted by the intercalator are detected. The detected changes in the intensity of the fluorescence associated with the temperature change are compared with a control. Examples of such methods include high-resolution melting (HRM) analysis.

[0107] In yet another method, a DNA or cDNA sample is first prepared from a biological sample. DNA containing all or part of the gene region of a SWI / SNF complex factor is then amplified. The amplified DNA is then cleaved with a restriction enzyme. The DNA fragments are then separated according to size. The sizes of the detected DNA fragments are then compared with a control. Examples of such methods include methods using restriction fragment length polymorphism (RFLP) and PCR-RFLP.

[0108] In yet another method, a DNA or cDNA sample is first prepared from a biological sample. DNA containing all or part of the gene region of a SWI / SNF complex factor is then 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. Examples of such methods include PCR-SSCP (single-strand conformation polymorphism).

[0109] In yet another method, a DNA or cDNA sample is first prepared from a biological sample. DNA containing all or part of the gene region of a SWI / SNF complex factor is then 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 that of a control. Such a method includes, for example, denaturant gradient gel electrophoresis (DGGE).

[0110] Yet another method is to use DNA containing a mutation site in the gene region of a SWI / SNF complex factor, 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.

[0111] In yet another method, a DNA or cDNA sample is first prepared from a biological sample. An oligonucleotide primer is also prepared, which has a base sequence complementary to the base 3' of all or part of the gene region of the SWI / SNF complex factor and the base sequence 3' of that base. Next, a dNTP primer extension reaction is carried out using the DNA as a template and the primer. The primer extension reaction product is then subjected to a mass spectrometer to measure the mass. The genotype is then determined based on the mass measurement results. The determined genotype is then compared with a control. Examples of such methods include MALDI-TOF / MS.

[0112] In yet another method, a DNA or cDNA sample is first prepared from a biological sample. An oligonucleotide probe is then prepared, consisting of 5'-"all or a portion of the gene region of the SWI / SNF complex factor and a base sequence complementary to the base sequence on the 5' side"-"all or a portion of the gene region of the SWI / SNF complex factor, one base 3' to the nucleotide sequence and a base sequence that does not hybridize to the nucleotide sequence on the 3' side"-3' (flap). Also, an "oligonucleotide probe having a base sequence complementary to all or a portion of the gene region of the SWI / SNF complex factor and the base sequence on the 3' side" is prepared. The two oligonucleotide probes are then hybridized to the prepared DNA or cDNA sample. The hybridized DNA is then cleaved with a single-stranded DNA cleaving enzyme to release the flap. There are no particular limitations on the single-stranded DNA cleaving enzyme, and examples include cleavase. 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 emitted fluorescence is then measured. The measured fluorescence intensity is then compared with a control. Examples of such methods include the Invader method.

[0113] In yet another method, a DNA or cDNA sample is first prepared from a biological sample. DNA containing all or part of the gene region of the SWI / SNF complex factor is then amplified. The amplified DNA is then dissociated into single strands, and one strand of the dissociated single-stranded DNA is isolated. An extension reaction is then carried out one base at a time, starting from the vicinity of all or part of the base in the gene region of the SWI / SNF complex factor. The pyrophosphate produced during this process is enzymatically induced to emit light, and the intensity of the luminescence is measured. The measured fluorescence intensity is then compared with that of a control. Examples of such methods include pyrosequencing.

[0114] In yet another method, a DNA or cDNA sample is first prepared from a biological sample. Then, DNA containing all or part of the gene region of the SWI / SNF complex factor is amplified. Next, an "oligonucleotide primer having a base one base 3' of all or part of the gene region of the SWI / SNF complex factor and a base sequence complementary to the base sequence 3' of that base" is prepared. Next, a single-base extension reaction is carried out using the prepared primer and the amplified DNA as a template in the presence of fluorescently labeled nucleotides. The degree of fluorescence polarization is then measured. The measured degree of fluorescence polarization is then compared with a control. Examples of such methods include the AcycloPrime method.

[0115] In yet another method, a DNA or cDNA sample is first prepared from a biological sample. Then, DNA containing all or part of the gene region of a SWI / SNF complex factor is amplified. Next, an "oligonucleotide primer having a base one base 3' of all or part of the gene region of a SWI / SNF complex factor and a base sequence complementary to the base sequence 3' of that base" is prepared. Next, a single-base extension reaction is carried out using the prepared primer and the amplified DNA as a template in the presence of a fluorescently labeled nucleotide. The base type used in the single-base extension reaction is then determined. The determined base type is then compared with a control. An example of such a method is the SNuPE method.

[0116] If the mutation involves an amino acid change (e.g., substitution, deletion, insertion, or addition) in the SWI / SNF complex factor protein, the sample prepared from the biological sample may be a protein. In such cases, 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 occurred due to the mutation, peptide mass fingerprinting (PMF), protein sequencer (Edman degradation), or the like.

[0117] For example, in antibody-based protein detection methods, a protein sample is first prepared from a biological sample. Then, an antibody specific to the SWI / SNF complex protein (anti-SWI / SNF complex protein antibody) is used to detect the SWI / SNF complex protein through an antigen-antibody reaction. Such antibody-based protein detection methods can be appropriately adapted to suit SWI / SNF complex protein, using methods similar to those listed as antibody-based protein detection methods for detecting GPX4 expression levels at the translational level. This method also has the advantage of simultaneously obtaining additional information, such as the morphology and distribution of cancer cells in tissue, through immunohistochemistry.

[0118] There are no particular limitations on the type or origin of the antibody used, but monoclonal antibodies are preferred. Oligoclonal antibodies (a mixture of several to several dozen antibodies) or polyclonal antibodies can also be used, as long as they can detect the SWI / SNF complex protein with sufficient specificity. Functional antibody fragments 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. Commercially available anti-SWI / SNF complex protein antibodies may also be used.

[0119] SWI / SNF complex proteins can also be detected using mass spectrometry (MS). Liquid chromatography-mass spectrometry (LC / MS) is particularly advantageous due to its sensitivity. Mass spectrometry detection methods can be used to detect GPX4 expression at the translational level, using methods similar to those listed above, but adjusted for SWI / SNF complex proteins.

[0120] [Detection of decreased expression of SWI / SNF complex factors] "Decreased expression of a SWI / SNF complex factor" generally means that the expression level is lower than that of a control (e.g., the expression level in a healthy subject or in non-cancerous tissue from the same patient). Methods for detecting "decreased expression of a SWI / SNF complex factor" include detecting the expression level of a SWI / SNF complex factor at the transcriptional or translational level and comparing it with the control.

[0121] In a method for detecting the expression level of a SWI / SNF complex factor at the transcriptional level, RNA or cDNA is first prepared from a biological sample using the method described above. Then, the oligonucleotide primers or oligonucleotide probes are used in an amplification reaction or hybridization reaction, respectively, and the resulting amplification product or hybrid product is detected. Such a method can be similar to the method described for detecting the expression level of GPX4 at the transcriptional level, but adjusted to suit the SWI / SNF complex factor.

[0122] In a method for detecting the expression level of a SWI / SNF complex factor at the translational level, a protein sample is first prepared from a biological sample. Then, an antigen-antibody reaction is carried out using an antibody specific to the SWI / SNF complex factor protein to detect the SWI / SNF complex factor protein. The method for detecting a protein using such an antibody is as described above in the method for detecting a SWI / SNF complex factor protein.

[0123] In the method for detecting the expression level of a SWI / SNF complex factor at the translational level, the SWI / SNF complex factor protein can also be detected using mass spectrometry (MS), as described above in the method for detecting a SWI / SNF complex factor protein.

[0124] It is also known in the art that promoter hypermethylation is one of the factors that contribute to decreased gene expression. Therefore, when detecting whether or not a SWI / SNF complex factor is functionally inhibited, it is possible to detect the methylation of the gene promoter of the SWI / SNF complex factor as an indicator. Promoter methylation can be detected by known methods, such as a direct method of detecting changes in the base sequence after bisulfite treatment, which has the activity of converting methylated cytosine to uracil, by base sequencing, or an indirect method using a restriction endonuclease that can recognize (cleave) the base sequence before bisulfite treatment but cannot recognize (cleave) the base sequence after bisulfite treatment.

[0125] (Prediction of susceptibility and selection of cancer patients) When functional inhibition of a SWI / SNF complex factor is detected in a biological sample in this manner, if the biological sample is a cancer cell, the cancer cell can be predicted to be sensitive to a GPX4 inhibitor.Furthermore, if the biological sample is a cancer cell contained in a sample derived from a cancer patient, the cancer patient in whose cancer cells the above mutation is detected can be predicted to be sensitive to treatment with a GPX4 inhibitor, and further, the cancer patient can be selected as a target for cancer treatment with a GPX4 inhibitor.

[0126] Here, "sensitivity to a GPX4 inhibitor" and "sensitivity to treatment with a GPX4 inhibitor" are indicators of whether a GPX4 inhibitor can exert a therapeutic effect on cancer cells. The sensitivity includes the promotion of cancer cell death and the inhibition of proliferation by the GPX4 inhibitor. The prediction of sensitivity includes not only determining whether or not a patient is sensitive, but also evaluating whether or not sensitivity can be expected, and, if sensitivity is present, evaluating the level of sensitivity (e.g., 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 based on the type and level of functional inhibition of SWI / SNF complex factors, for example, at a level where moderate sensitivity can be expected.

[0127] On the other hand, if no functional inhibition of SWI / SNF complex factors is detected in samples from cancer patients, the patient can be excluded from cancer treatment with GPX4 inhibitors, which can improve the success rate of treatment.

[0128] (GPX4) Furthermore, if GPX4, the target of the GPX4 inhibitor of the present invention, is not normally expressed, cancer treatment using the GPX4 inhibitor may not be effective. 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 GPX4 gene mutations and decreased expression can also be added as an indicator.

[0129] The method for detecting GPX4 gene mutations can be the same as the method for "detecting SWI / SNF complex factor gene mutations" and can be appropriately adapted to the GPX4 gene mutation. The method for detecting decreased GPX4 expression is as described above.

[0130] <Methods for suppressing cancer cell growth and treating cancer> The present invention provides A step of contacting a GPX4 inhibitor with cancer cells in which functional inhibition of the SWI / SNF complex factor is detected. A method for inhibiting the proliferation of cancer cells in which functional inhibition of SWI / SNF complex factors is detected (hereinafter sometimes referred to as a "method for inhibiting cancer cell proliferation"), comprising: (a) detecting whether or not the function of a SWI / SNF complex factor is inhibited in cancer cells contained in a sample derived from a cancer patient; (b) administering a GPX4 inhibitor to a cancer patient in which functional inhibition of the SWI / SNF complex factor has been detected in the cancer cells; Also provided is a method for treating cancer (sometimes referred to herein as a "cancer treatment method"), comprising:

[0131] In the method for inhibiting cancer cell proliferation and the method for treating cancer of the present invention, the method for detecting the inhibition of the function of an SWI / SNF complex factor and the GPX4 inhibitor are as described above.

[0132] The inhibition of cancer cell proliferation includes promoting the death of the cancer cells and inhibiting their proliferation. The method for contacting the cancer cells with the GPX4 inhibitor is not particularly limited, and examples thereof include a method in which the GPX4 inhibitor is added to a culture medium for the cancer cells.

[0133] Furthermore, the amount of GPX4 inhibitor to be contacted with cancer cells need only be an amount effective in inhibiting GPX4 and suppressing the proliferation of cancer cells, and is selected appropriately depending on the properties of the GPX4-inhibiting compound, the type of cancer cells, etc.

[0134] The administration of GPX4 inhibitors to cancer patients may be oral or parenteral (eg, intravenous, arterial, or local) administration.

[0135] The dosage of a GPX4 inhibitor administered to a cancer patient should be an amount effective for inhibiting GPX4 and treating cancer. It is not universally specified; it is chosen based on factors such as the properties of the GPX4-inhibiting compound, the age, weight, symptoms, and health status of the cancer patient, and the progression of the cancer. For example, when administered to a human, the amount of the GPX4-inhibiting compound per day is 0.001 to 100,000 mg, preferably 0.01 to 5,000 mg. The frequency of administration of a GPX4 inhibitor to a cancer patient is also not universally specified; however, it is preferred that the administration be once a day or in 2 to 4 divided doses, repeated at appropriate intervals. The dosage and frequency of administration can be adjusted as needed at the physician's discretion.

[0136] This further inhibits GPX4 in cancer cells in cancer patients that have functional suppression of SWI / SNF complex factors, thereby promoting the death of cancer cells and / or suppressing their proliferation, thereby enabling cancer treatment.

[0137] The cancer to be treated is not particularly limited, and examples thereof include at least one type selected from the group consisting of lung cancer, ovarian cancer, uterine cancer, liver cancer, stomach cancer, esophageal cancer, colon cancer, pancreatic cancer, prostate cancer, bladder cancer, and kidney cancer. Among these, at least one type selected from the group consisting of lung cancer, ovarian cancer, liver cancer, stomach cancer, and pancreatic cancer is preferred.

[0138] <Reagents for detecting the presence or absence of mutations> The present invention also provides a reagent for detecting the presence or absence of functional inhibition of an SWI / SNF complex factor in the above-mentioned method, (i) an oligonucleotide primer that specifically binds to the gene for a SWI / SNF complex factor; (ii) an oligonucleotide probe that specifically binds to the gene for a SWI / SNF complex factor; and (iii) an antibody that specifically binds to a SWI / SNF complex factor protein; The present invention provides a reagent containing any one of the molecules listed above as an active ingredient.

[0139] The oligonucleotide primers can be designed based on the nucleotide sequence information of the genomic DNA or cDNA of the SWI / SNF complex factor (e.g., Tables 1 to 3) so as to be 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 SWI / SNF complex factor gene. Such oligonucleotide primer design can be carried out 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.

[0140] The above-mentioned oligonucleotide probes may be designed based on the base sequence information of the genomic DNA or cDNA of the SWI / SNF complex factor (e.g., Tables 1 to 3) so as to be suitable for the above-mentioned methods and for the region to be hybridized, and so as to minimize hybridization to genes other than the gene for the SWI / SNF complex factor. Such oligonucleotide probe design can be carried out by a person skilled in the art using conventional 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.

[0141] The oligonucleotide probe is preferably appropriately labeled before use. Examples of labeling methods include a method in which the 5' end of the oligonucleotide is phosphorylated with 32P using T4 polynucleotide kinase, and a method in which a substrate base labeled with an isotope such as 32P, a fluorescent dye, biotin, or the like is incorporated using a DNA polymerase such as Klenow enzyme and a random hexamer oligonucleotide as a primer (random prime method, etc.).

[0142] The above-mentioned oligonucleotide primers and oligonucleotide probes can be prepared, for example, using a commercially available oligonucleotide synthesizer. Oligonucleotide probes can also be prepared as double-stranded DNA fragments obtained by restriction enzyme treatment or the like. Furthermore, the oligonucleotide primers and oligonucleotide probes of the present invention do not have to be composed solely 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.

[0143] Polyclonal antibodies that specifically bind to the SWI / SNF complex protein can be obtained by immunizing an animal with an antigen (such as the SWI / SNF complex protein, a partial peptide thereof, or cells expressing either or both) and purifying the resulting antiserum by conventional means (e.g., salting out, centrifugation, dialysis, column chromatography, etc.). Monoclonal antibodies can be produced by hybridoma or recombinant DNA techniques.

[0144] A representative example of the hybridoma method is the method of Kohler and Milstein (Nature 1975;256:495). The antibody-producing cells used in the cell fusion step in this method include spleen cells, lymph node cells, peripheral blood leukocytes, etc. from animals (e.g., mice, rats, hamsters, rabbits, monkeys, and goats) immunized with an antigen (such as an SWI / SNF complex factor protein, a partial peptide thereof, or cells expressing these). Antibody-producing cells obtained by reacting an antigen in a culture medium with the above-mentioned cells or lymphocytes previously isolated from an unimmunized animal can also be used. Various known cell lines can be used as myeloma cells. The antibody-producing cells and myeloma cells may be derived from different animal species, as long as they are fusible; however, they are preferably derived from the same animal species. 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 SWI / SNF complex factor proteins. Monoclonal antibodies against SWI / SNF complex factor proteins can be obtained by culturing hybridomas or from the ascites of a mammal to which the hybridoma has been administered.

[0145] In recombinant DNA techniques, DNA encoding the antibody is cloned from hybridomas, B cells, or the like, inserted into an appropriate vector, and then introduced into host cells (e.g., mammalian cell lines, Escherichia coli, yeast cells, insect cells, plant cells, etc.) to produce the antibody of the present invention as a recombinant antibody (e.g., P. J. Delves, Antibody Production: Essential Techniques, 1997; WILEY, P. Shepherd and C. Dean Monoclonal Antibodies, 2000 OXFORD UNIVERSITY PRESS; Vandamme AM et al., Eur. J. Biochem. 1990;192:767-775). For expression of the antibody-encoding DNA, DNA encoding the heavy chain and DNA encoding the light chain may be inserted separately into expression vectors and used to transform host cells, or DNA encoding the heavy and light chains may be inserted into a single expression vector and used to transform host cells (e.g., WO94 / 11523). Antibodies can be obtained in a substantially pure and homogeneous form by culturing the above-mentioned host cells and isolating and purifying them from within the host cells or from the culture medium. Antibodies can be isolated and purified using methods commonly used for purifying polypeptides. By using transgenic animal production technology to create transgenic animals (such as cows, goats, sheep, or pigs) incorporating antibody genes, it is possible to obtain large quantities of monoclonal antibodies derived from the antibody genes from the milk of the transgenic animals.

[0146] 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, as well as multimers thereof (e.g., dimers, trimers, tetramers, polymers) can be prepared.

[0147] When directly detecting the amount of antibody bound to the SWI / SNF complex protein, the obtained anti-SWI / SNF complex protein antibody is directly labeled with an enzyme, radioisotope, fluorescent dye, avidin-biotin system, etc. On the other hand, when performing an indirect detection method in which the amount of antibody bound to the SWI / SNF complex protein is detected using a secondary antibody, etc., the obtained anti-SWI / SNF complex protein antibody (primary antibody) does not need to be labeled, and detection can be performed using a labeled molecule that recognizes the antibody (e.g., a secondary antibody or protein A).

[0148] The reagent of the present invention may contain, in addition to the above molecule as an active ingredient, other ingredients acceptable as a reagent, such as sterilized water, physiological saline, a buffer, a preservative, etc., as needed.

[0149] <Screening method for compounds used in cancer treatment / cancer therapeutic agents> The present invention provides A step of selecting compounds based on whether or not they inhibit GPX4. The present invention also provides a method for screening for a compound to be used in the treatment of cancer, including cancer cells in which functional inhibition of a SWI / SNF complex factor is detected (hereinafter sometimes referred to as a "method for screening for a compound"), comprising:

[0150] Furthermore, using the GPX4-inhibiting compound screened by the above compound screening method, It is also possible to provide a cancer therapeutic agent that contains a compound that inhibits GPX4 as an active ingredient, and that is a therapeutic agent for cancer including cancer cells in which functional inhibition of SWI / SNF complex factors has been detected.

[0151] The test compound to be used in the compound screening method of the present invention is not particularly limited and may include, for example, at least one selected from the group consisting of the compound molecules, polypeptides, and polynucleotides listed above as compounds that inhibit GPX4. More specific examples of the test compound include synthetic low-molecular-weight compound libraries, expression products of gene libraries, peptide libraries, siRNAs, antibodies, substances released by bacteria, 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 GPX4 inhibitor.

[0152] In a method (screening) for selecting a compound using whether or not it inhibits GPX4 as an index, a test compound is allowed to act on the above-mentioned confirmation system for inhibition of GPX4 activity or expression, and then GPX4 activity or expression is detected. If the result of the detection shows that the activity or expression is reduced compared to the GPX4 activity or expression in a control (e.g., when the test compound is not added), it can be evaluated that GPX4 is inhibited.

[0153] In the screening, the "GPX4" to be evaluated for the presence or absence of inhibition by the compound is as described above.

[0154] The compounds identified by the compound screening method of the present invention can be mixed appropriately with pharmacologically acceptable additives such as those listed for the GPX inhibitors and formulated by known pharmaceutical methods to be used as pharmaceuticals to treat cancer. In particular, the compounds are effective against cancers including cancer cells in which functional inhibition of SWI / SNF complex factors has been detected, and can be used as therapeutic agents for treating and / or administering to cancer patients in which functional inhibition of SWI / SNF complex factors has been detected. [Example]

[0155] The present invention will be described in more detail below based on examples, but the present invention is not limited to the following examples.

[0156] 1. Experimental Materials and Methods (1) Cell line The cancer cell lines used in the present study were NCI-H358, NCI-H2122, NCI-H2110, NCI-H1792, NCI-H1437, MOR, COV362, MKN-45, NCI-H838, B1203L (Int J Cancer. 2006; 118:1992-7), SBC-5, TOV-21G, NCI-H1703, SNU-1327, NCI-H23, OVISE, NCI-H522, and SK-HEP-1, as listed in Table 4 above. The source of each cell line is listed in Table 5 below. As shown in Table 4 above, NCI-H23, NCI-H522, SBC-5, SK-HEP-1, and SNU-1327 are human cancer cell lines with truncated mutations in the SMARCA4 gene, and NCI-H1703 is a human cancer cell line with a mutation at the splicing site of the SMARCA4 gene. Furthermore, NCI-H1703, NCI-H23, NCI-H522, SBC-5, SK-HEP-1, and NCI-H838 are human cancer cell lines in which SMARCA4 protein expression is not detected by immunoblotting analysis. Furthermore, NCI-H1703, NCI-H23, NCI-H522, and SBC-5 are human cancer cell lines in which SMARCA2 protein expression is not detected by immunoblotting analysis. B1203L, OVISE, and TOV-21G are human cancer cell lines harboring truncating mutations in the ARID1A gene, B1203L is a human cancer cell line harboring truncating mutations in the ARID2 gene, OVISE is a human cancer cell line harboring truncating mutations in the ARID1B gene, and NCI-H838 is a human cancer cell line harboring truncating mutations in the BCL11B gene.On the other hand, NCI-H358, NCI-H2122, NCI-H2110, NCI-H1792, NCI-H1437, MOR, COV362, and MKN-45 are human cancer cell lines for which information on loss-of-function mutations in SWI / SNF complex factor genes and protein expression has not been published.

[0157] (2) Short interfering (si) RNA GPX4 expression was suppressed using ON-TARGET plus individual siRNA (Dharmacon). Lipofectamine RNAiMAX (Thermo Fisher Scientific, product code: 13778150) was used for transfection. A non-targeting siRNA (ON-TARGET plus Non-targeting Control, Dharmacon product code: D-001810-01, SEQ ID NO: 60) was used as a negative control, and PLK1 (Dharmacon product code: J-003290-09, SEQ ID NO: 59) was used as a positive control. Each siRNA was diluted with 1x siRNA buffer, which was prepared by diluting 5x siRNA buffer (Dharmacon: B-002000-UB-100) 5-fold with nuclease-free water (Ambion: AM9932).

[0158] (3) GPX4 expression inhibition test The cell growth inhibitory activity of GPX4 expression suppression was evaluated for each cancer cell line listed in Table 4 (1) above. Each cell line was seeded at 100 μL / well in a 96-well plate (Greiner Bio-One) at the seeding number (1) or (2) listed in Table 5 below. For transfection evaluation, GPX4 siRNA (#1, #2, #3), a negative control (ON-TARGET plus Non-targeting Control), and a positive control (PLK1 siRNA) were used, as shown in Table 6 below. Each siRNA was adjusted to a final concentration of 0.1–10 nM, and Lipofectamine RNAiMAX (Thermo Fisher Scientific, product code: 13778150) was used for transfection. After 7 days of culture, intracellular ATP, a marker of cell viability, was measured using the CellTiter-Glo 2.0 Cell Viability Assay (Promega). The cell growth inhibition rate when GPX4 expression was suppressed was calculated by setting the values ​​after 7 days of culture following transfection with the negative control and the values ​​after 7 days of culture following transfection with the positive control as 0% and 100%, respectively.

[0159] The cell growth inhibition rates (CGI (%)) of GPX4 expression suppression by GPX4 siRNA #1 or #2 in the NCI-H1792, MOR, NCI-H2110, NCI-H522, NCI-H23, and SNU-1327 cell lines (lung cancer cell lines) are shown in Figure 1. The cell growth inhibition rates (CGI (%)) of GPX4 expression suppression by GPX4 siRNA #2 in the cell lines (including lung cancer cell lines, ovarian cancer cell lines, liver cancer cell lines, and gastric cancer cell lines) shown in Table 4, which were tested separately from those in Figure 1, are shown in Figure 2, and the cell growth inhibition rates (CGI (%)) of GPX4 expression suppression by GPX4 siRNA #3 are shown in Figure 3.

[0160] (4) GPX4 activity inhibition test 1 The cell growth inhibitory activity of GPX4 inhibitors was evaluated in each cancer cell line. The GPX4 inhibitors included ML210 (CAS number: 1360705-96-9), a small molecule GPX4 inhibitor that has been reported to inhibit the enzymatic activity of GPX4.

[0161] [ka]

[0162] or RSL3 (CAS number: 1219810-16-8):

[0163] [ka]

[0164] was used.

[0165] These compounds were added to each cell line, and 100 μL / well of each was seeded to achieve the seeding number (2) listed in Table 5 below. After 7 days of culture, intracellular ATP, a marker of cell viability, was measured using the CellTiter-Glo 2.0 Cell Viability Assay (Promega). Cell viability was calculated by defining the amount of intracellular ATP in each cancer cell line after 7 days of culture in the presence of only the compound's solvent (dimethyl sulfoxide (DMSO) or water) as 100%. IC50 (50% Inhibition Concentration (μM)) was calculated from the cell survival curve obtained from the concentration of each compound and cell viability. Figure 4 shows the IC50 (μM) for each cell line when ML210 was used as a GPX4 inhibitor, and Figure 5 shows the IC50 (μM) for each cell line when RSL3 was used as a GPX4 inhibitor.

[0166] (5) GPX family protein expression inhibition test The cell growth inhibitory activity of each cancer cell line due to the suppression of expression of GPX family proteins GPX1 to GPX8 was evaluated. Each cell line was seeded at 100 μL / well in a 96-well plate with the seeding number (2) listed in Table 5 below. For transfection evaluation, GPX1 siRNA (#1, #2), GPX2 siRNA (#1, #2), GPX3 siRNA (#1, #2), GPX4 siRNA (#1, #2), GPX5 siRNA (#1, #2), GPX6 siRNA (#1, #2), GPX7 siRNA (#1, #2), and GPX8 siRNA (#1, #2) (all Dharmacon) were used, as shown in Table 6 below. Each siRNA was used at a final concentration of 5 nM or 10 nM, and transfection was performed using Lipofectamine RNAiMAX (Thermo Fisher Scientific, product code: 13778150). After 7 days of culture, intracellular ATP, a marker of cell viability, was measured using the CellTiter-Glo 2.0 Cell Viability Assay (Promega). The percentage of cell growth inhibition following inhibition of each GPX family protein was calculated by setting the values ​​after 7 days of culture in the negative control and the positive control, respectively, as 0% and 100%. Figure 6 shows the percentage of cell growth inhibition (CGI (%)) following inhibition of each GPX family protein in the NCI-H2110, NCI-H522, and NCI-H23 cell lines.

[0167] (6) Test for suppression of expression of glutathione synthesis-related proteins The cell proliferation inhibitory activity of each cancer cell line was evaluated by suppressing the expression of proteins involved in glutathione synthesis (glutathione synthesis-related proteins). Each cell line was seeded at 100 μL / well in a 96-well plate, with the seeding number (2) listed in Table 5 below. For transfection evaluation, GPX4 siRNA (#1, #2), GCLC siRNA (#1, #2), GCLM siRNA (#1, #2), GSS siRNA (#1, #2), MGST1 siRNA (#1, #2), MGST3 siRNA (#1, #2), GSR siRNA (#1, #2), and G6PD siRNA (#1, #2) (all Dharmacon) were used, as listed in Table 6 below. Each siRNA was used at a final concentration of 5 nM, and transfection was performed using Lipofectamine RNAiMAX (Thermo Fisher Scientific, product code: 13778150). After 7 days of culture, intracellular ATP, a marker of cell viability, was measured using the CellTiter-Glo 2.0 Cell Viability Assay (Promega). The cell growth inhibition rate when the expression of each glutathione synthesis-related protein was suppressed was calculated by setting the values ​​after 7 days of culture after transfection with the negative control and the values ​​after 7 days of culture after transfection with the positive control as 0% and 100%, respectively. Figure 7 shows the cell growth inhibition rate (CGI (%)) when the expression of each glutathione synthesis-related protein was suppressed in the NCI-H2110, NCI-H522, and NCI-H23 cell lines.

[0168] [Table 5]

[0169] [Table 6]

[0170] Table 5 shows the seeding number for each cell line in each test, as well as the culture medium and source of each cell line. Table 6 shows the sequence number of each siRNA, as well as the ID (GENE ID), reference ID (NCBI reference ID), and GI number in the NCBI RefSeq database of the gene whose expression is suppressed by each siRNA.

[0171] (7) GPX4 activity inhibition test 2 (comparison test with GCLC inhibitor) The cell growth inhibitory activity of GPX4 inhibitors was evaluated in each cancer cell line. ML210 or RSL3 was used as the GPX4 inhibitor. As a control, buthionine sulphoximine (BSO, CAS number: 83730-53-4), an inhibitor of GCLC (catalytic subunit of glutamate-cysteine ​​ligase (GCL) which is the rate-limiting enzyme in glutathione synthesis), was also used.

[0172] [ka]

[0173] These compounds were added to each cell line, and each cell line was seeded at 100 μL / well to achieve the seeding number (2) listed in Table 5 above. After 7 days of culture, intracellular ATP, a marker of cell viability, was measured using the CellTiter-Glo 2.0 Cell Viability Assay (Promega). Cell viability was calculated by defining the amount of intracellular ATP in each cancer cell line after 7 days of culture in the presence of only the solvent for the compound (dimethyl sulfoxide (DMSO) or water) as 100%. The IC50 (50% Inhibition Concentration (μM)) was calculated from the cell viability curve obtained from the concentration of each compound and the cell viability.

[0174] The IC50 (μM) in each of the MOR, NCI-H358, NCI-H23, and NCI-H522 cell lines when ML210, RSL3, or BSO was used is shown in FIG.

[0175] (8) Efficacy test of ML210 on SK-HEP-1 tumor-bearing mice To investigate the efficacy of GPX4 inhibitors against SWI / SNF complex factor-deficient cancers in vivo, we administered ML210 to mice (SK-HEP-1 tumor-bearing mice) transplanted with SK-HEP-1 (a human cancer cell line with SMARCA4 function suppression). Specifically, SK-HEP-1 cells were cultured in vitro and harvested on the day of transplantation using 2.5 g / L Tripsin / 1 mmol EDTA Solution (Nacalai Tesque, product code: 35554-64). The cells were then suspended at a final concentration of 2.5E7 cells / mL in Matrigel (Corning, product code: 356234) diluted with Hanks' Balanced Salt solution (Sigma-Aldrich, product code: H9269) (50%). The resulting cell suspension was then transferred to BALB / c-nu / nu mice (CAnN.Cg-Foxn1). <nu>0.2 mL of each solution was implanted into the groin of a mouse (CrlCrlj, Charles River).

[0176] Next, for mice (SK-HEP-1 tumor-bearing mice) in which tumor (cancer cell) engraftment was confirmed, as soon as this was confirmed, ML210 was suspended at a predetermined concentration in a mixture of 10% dimethyl sulfoxide (Wako, product code: 043-07216), 10% Cremophor (Sigma-Aldrich, product code: C5135), 15% Polyethylene Glycol 400 (Wako, product code: 161-09065), and 15% hydroxypropyl-β-cyclodextrin (Nihon Shokuhin Kako, product code: 7585-39-9). ML210 was orally administered once daily at a predetermined volume (20 mL / kg) at a dose of 100 mg / kg per day. The day of SK-HEP-1 implantation was considered day 0, and ML210 administration began on day 11. Tumor volumes were measured on days 11, 14, and 18. Tumor volume was calculated by least squares regression using electronic calipers (Mitutoyo, product code: CD-15AX) to measure the minor and major axes of the tumor. Similarly, for SK-HEP-1 tumor-bearing mice administered only the mixture (Vehicle) without ML210, the day of SK-HEP-1 tumor implantation was designated as day 0. Vehicle administration was initiated on day 11, and tumor volume was measured on days 11, 14, and 18. Figure 9 shows the relationship between tumor volume and the time after SK-HEP-1 tumor implantation (days after inoculation) in the group of SK-HEP-1 tumor-bearing mice administered with ML210 (ML210) or the group of SK-HEP-1 tumor-bearing mice administered with the mixture (Vehicle: no ML210 administration).

[0177] 2.Results (1) GPX4 expression inhibition test As shown in Figure 1, GPX4 expression inhibition demonstrated significant cell growth inhibition (>90%) in cancer cell lines harboring a deletion mutation in the gene encoding the SWI / SNF complex factor SMARCA4 and / or in which SMARCA4 protein was not detected by immunoblot analysis. Furthermore, as shown in Figures 2 and 3, GPX4 expression inhibition also demonstrated significant cell growth inhibition (>90%) in cancer cell lines harboring a deletion mutation in the gene encoding at least one of other SWI / SNF complex factors (e.g., SMARCA2, ARID1A, ARID1B, ARID2, and BCL11B) and / or in which the expression of at least one of these factors was not detected by immunoblot analysis. These results indicate that the survival of cancer cell lines with SWI / SNF complex factor function inhibition is strongly dependent on GPX4 function.

[0178] (2) GPX4 activity inhibition test 1 Furthermore, as shown in Figures 4 and 5, in GPX4 activity inhibition test 1, when a GPX4 inhibitor was added to cancer cell lines that had deletion mutations in at least one gene of SWI / SNF complex factors (e.g., SMARCA4, SMARCA2, ARID1A, ARID1B, ARID2, BCL11B) and / or in which the expression of at least one of these factors was not detected by immunoblot analysis, significant cell growth inhibition was observed compared to cell lines in which the function of SWI / SNF complex factors was not suppressed.

[0179] The results of (1) and (2) above showed that inhibiting GPX4 (by suppressing its expression or activity) is effective in suppressing the proliferation of cancer cells in which the function of SWI / SNF complex factors is suppressed (to obtain antitumor activity).

[0180] (3) GPX family protein expression inhibition test As shown in Figure 6, in cancer cell lines with deletion mutations in the gene encoding SMARCA4, a SWI / SNF complex factor, and / or in which SMARCA4 protein was not detected by immunoblotting analysis, significant cell growth inhibition was observed only when the expression of GPX4, among GPX family proteins, was suppressed. These results indicate that inhibition of GPX4 is important for achieving antitumor activity against cancer cells in which the function of SWI / SNF complex factors (e.g., SMARCA4) is suppressed.

[0181] (4) Test for suppression of expression of glutathione synthesis-related proteins As shown in Figure 7, in a test to inhibit the expression of glutathione synthesis-related proteins, significant cell growth inhibition was observed only when the expression of glutathione synthesis-related proteins, particularly GPX4, was inhibited in cancer cell lines that had deletion mutations in the gene for SMARCA4, a SWI / SNF complex factor, and / or in which SMARCA4 protein was not detected by immunoblotting analysis. These results also demonstrate that inhibiting GPX4 is important for achieving antitumor activity against cancer cells in which the function of SWI / SNF complex factors (e.g., SMARCA4) is inhibited.

[0182] (5) GPX4 activity inhibition test 2 (comparison test with GCLC inhibitor) As shown in Figure 8, in GPX4 activity inhibition test 2, when a GPX4 inhibitor was added to cancer cell lines that had a deletion mutation in the gene for SMARCA4, a SWI / SNF complex factor, and / or in which SMARCA4 protein was not detected by immunoblot analysis, significant cell growth inhibition was observed compared to the GCLC inhibitor BSO. This result also demonstrated that inhibiting GPX4 is effective in obtaining antitumor activity against cancer cells in which the function of SWI / SNF complex factors (e.g., SMARCA4) is suppressed.

[0183] (6) Efficacy test of ML210 on SK-HEP-1 tumor-bearing mice As shown in Figure 9, GPX4 inhibitors also exhibited in vivo antitumor activity against cancers (tumors), including those with deletion mutations in the SMARCA4 gene, a SWI / SNF complex factor, and / or cancer cells in which SMARCA4 protein was not detected by immunoblotting (SMARCA4 function-inhibited cancer cells). Specifically, in SK-HEP-1 tumor-bearing mice treated with ML210, tumor volume growth was suppressed compared to the vehicle-treated group, demonstrating significant antitumor activity. [Industrial Applicability]

[0184] As described above, according to the present invention, it is possible to efficiently predict sensitivity to cancer treatment with a GPX4 inhibitor using functional inhibition of a SWI / SNF complex factor as an indicator. Furthermore, according to the present invention, the presence or absence of functional inhibition of a SWI / SNF complex factor in a sample from a cancer patient can be detected, and patients in whom such mutations are detected can be selected and then treated with a GPX4 inhibitor. This makes it possible to significantly improve the outcome of cancer treatment. Furthermore, by using probes or primers for genes encoding SWI / SNF complex factors and antibodies against SWI / SNF complex factors, it is possible to efficiently perform companion diagnostics by detecting the presence or absence of functional inhibition of such SWI / SNF complex factors.< / nu>

Claims

1. A cancer therapeutic agent for treating a cancer patient in which functional inhibition of a SWI / SNF complex factor has been detected in cancer cells contained in a sample derived from the cancer patient, the cancer therapeutic agent containing a compound that inhibits GPX4 as an active ingredient, wherein the compound that inhibits GPX4 is a compound that inhibits at least one of the activity of GPX4 and the expression of GPX4.

2. A cancer therapeutic agent containing a compound that inhibits GPX4 as an active ingredient, for administration to a cancer patient in whom functional inhibition of a SWI / SNF complex factor has been detected in cancer cells contained in a sample derived from the cancer patient, wherein the compound that inhibits GPX4 is a compound that inhibits at least one of the activity of GPX4 and the expression of GPX4.

3. A method for predicting the sensitivity of cancer cells to a GPX4 inhibitor, comprising: predicting that cancer cells in which functional inhibition of SWI / SNF complex factors has been detected are sensitive to a GPX4 inhibitor; wherein the GPX4 inhibitor is a composition containing a compound that inhibits GPX4, and the compound that inhibits GPX4 is a compound that inhibits at least one of the activity of GPX4 and the expression of GPX4.

4. A method for predicting the sensitivity of a cancer patient to treatment with a GPX4 inhibitor, predicting that a cancer patient in which functional inhibition of a SWI / SNF complex factor has been detected in cancer cells contained in a sample derived from the cancer patient will be sensitive to treatment with a GPX4 inhibitor; wherein the GPX4 inhibitor is a composition containing a compound that inhibits GPX4, and the compound that inhibits GPX4 is a compound that inhibits at least one of the activity of GPX4 and the expression of GPX4.

5. A method for selecting cancer patients to be treated with a GPX4 inhibitor, selecting cancer patients in whom functional inhibition of SWI / SNF complex factors has been detected in cancer cells contained in a sample derived from the cancer patients as targets for cancer treatment with a GPX4 inhibitor; wherein the GPX4 inhibitor is a composition containing a compound that inhibits GPX4, and the compound that inhibits GPX4 is a compound that inhibits at least one of the activity of GPX4 and the expression of GPX4.

6. A method for inhibiting the proliferation of cancer cells in which functional inhibition of a SWI / SNF complex factor has been detected, contacting a GPX4 inhibitor with cancer cells in which functional inhibition of the SWI / SNF complex factor has been detected outside the human body; wherein the GPX4 inhibitor is a composition containing a compound that inhibits GPX4, and the compound that inhibits GPX4 is a compound that inhibits at least one of the activity of GPX4 and the expression of GPX4.

7. A method for screening a compound to be used in the treatment of cancer, including cancer cells in which functional inhibition of a SWI / SNF complex factor has been detected, comprising: a step of selecting a compound based on whether or not it inhibits GPX4; A method comprising:

8. The cancer therapeutic agent according to claim 1 or 2, wherein the SWI / SNF complex factor is a BAF complex factor.

9. The method of any one of claims 3 to 7, wherein the SWI / SNF complex factor is a BAF complex factor.

10. The cancer therapeutic agent according to claim 1 or 2, wherein the SWI / SNF complex factor is at least one selected from the group consisting of SMARCA2, SMARCA4, ARID1A, ARID1B, ARID2, and BCL11B.

11. The method according to any one of claims 3 to 7, wherein the SWI / SNF complex factor is at least one selected from the group consisting of SMARCA2, SMARCA4, ARID1A, ARID1B, ARID2, and BCL11B.

12. The cancer therapeutic agent according to claim 1 or 2, wherein the functional inhibition of the SWI / SNF complex factor is a reduction in the activity of the SWI / SNF complex, of which the SWI / SNF complex factor is a constituent factor, and / or a reduction in the expression of the SWI / SNF complex factor.

13. The method according to any one of claims 3 to 7, wherein the functional inhibition of the SWI / SNF complex factor is a reduction in the activity of a SWI / SNF complex of which the SWI / SNF complex factor is a constitutive factor and / or a reduction in the expression of the SWI / SNF complex factor.

14. The cancer therapeutic agent according to claim 1 or 2, wherein the functional inhibition of the SWI / SNF complex factor is a loss-of-function mutation in the gene encoding the SWI / SNF complex factor.

15. The method according to any one of claims 3 to 7, wherein the functional inhibition of the SWI / SNF complex factor is a loss-of-function mutation in the gene of the SWI / SNF complex factor.

Images