Patent Application
20220087987
The present invention relates to a method for treating SWI/SNF complex-deficient cancers comprising a glutathione (GSH) metabolic pathway inhibitor, a pharmaceutical composition comprising a glutathione (GSH) metabolic pathway inhibitor for therapy in SWI/SNF complex-deficient cancers, and a glutathione (GSH) metabolic pathway inhibitor for use in treating SWI/SNF complex-deficient cancers.
The SWI/SNF complex was originally described in yeast as the protein complex critical for cellular responses to mating-type switching (SWI) or sucrose fermentation (SNF). Recently, the SWI/SNF complex has been linked to a number of human diseases, most notably, cancer. Loss-of-function mutations of genes encoding subunits of the SWI/SNF chromatin-remodeling complex are found in approximately 20% of all human cancers. Such mutations are thought to promote tumorigenesis by disturbing transcriptional homeostasis due to impairment of chromatin remodeling for transcription, DNA damage repair, DNA replication and chromatin segregation. The ARID1A gene, which encodes a component of the SWI/SNF chromatin-remodeling complex, is frequently mutated in several intractable cancers; it is mutated in about 46% of ovarian clear cell carcinoma (OCCC), 33% of gastric carcinoma, 27% of cholangiocarcinoma and 15% of pancreatic carcinoma [see, NPL 1-4], all of which lack effective molecular targeting therapies. Furthermore, ARID1A deficiency is associated with poor prognosis of a variety of cancers [see, NPL 5]. Thus, much effort has been devoted to identifying vulnerabilities caused by ARID1A deficiency for developing effective therapeutic modalities against those intractable cancers [see, NPL 6].
Similarly, the other components of the SWI/SNF complex, such as SMARCA2 (BRM), SMARCA4 (BRG1), SMARCB1, PBRM1, are also known to be mutated in the various cancers [see, NPL 7].
Regulation of oxidative stress homeostasis is important for cell survival. Reactive oxygen species (ROS) cause oxidative stress. Cellular ROS levels are determined by the balance between ROS generation and elimination, and are regulated by antioxidant defense mechanisms. High levels of ROS cause cell damage and death. Therefore, targeting antioxidant defense systems may be an anti-cancer therapeutic strategy. Glutathione (GSH) is an abundant antioxidant tripeptide molecule synthesized from cysteine, glutamate, and glycine by the ATP-dependent enzyme glutamate-cysteine ligase synthetase (GCL), which is composed of the glutamate-cysteine ligase catalytic subunit (GCLC) and the glutamate-cysteine ligase modifier subunit (GCLM), and GSH synthetase (GSS) [see, NPL 8]. GCL catalyzes the rate-limiting step of glutamate ligation with cysteine during GSH synthesis. Cysteine is the rate-limiting precursor substrate for GSH synthesis. Intracellular cysteine levels are controlled by SLC7A11, which encodes the cystine/glutamate transporter XCT.
Conventional molecular target drugs can selectively inhibit gene mutations which activate prolifiration of cancer cells, but many patients in which gene mutations cannot be detected are not targets of the molecular target drugs. However, molecular target drugs cannot be applied to loss-of-function mutations of genes. Therefore, a new therapy has been required for loss-of-function mutations of genes.
Some loss-of-function mutations of genes confer druggable vulnerabilities on cancer cells. “Synthetic lethality,” which is defined by an interdependent relationship between two genes, which means that simultaneous loss of two genes, but not loss of either gene alone, leads to cell death. Therefore, cancer cells harboring such a deleterious gene mutation would be vulnerable to inhibition of the synthetic lethal target, as epitomized by the success of PARP1-targeted therapy against hereditary breast and ovarian cancers harboring BRCA1 and BRCA2 mutations [see, NPL 9].
However, no efficient therapeutic strategy in SWI/SNF complex-deficient cancers has been established. The goal of the invention is to develop effective therapeutic strategy for treating SWI/SNF complex-deficient cancers by identifying druggable targets.
It has unexpectedly been discovered that since the expression of SCL7A11 protein which carries cystein, an element constituting Glutathione (GSH), is suppressed in SWI/SNF complex-deficient cancer, the level of GSH is also lowered (referred to as “metabolome aberration due to loss-of-function”). This phenomenon cannot be found in normal cells.
In addition, when GSH itself or GSH synthesis enzymes are inhibited, the level of GSH is easily reduced, and then ROS is in excess, which induces apoptosis of SWI/SNF complex-deficient cancer cells. We have discovered that various cancers having SWI/SNF complex gene mutations can be treated using inhibitors of GSH or GSH synthesis enzymes (“synthetic lethal targets”). This new therapy is referred to as a “synthetic lethality therapy.” Specifically, in the first embodiment, the present invention provides a method for treating SWI/SNF complex-deficient cancer, comprising administering an effective amount of a glutathione (GSH) metabolic pathway inhibitor to a mammal in need thereof.
In the second embodiment, the present invention provides a method for detecting and/or selecting a susceptible patient to a glutathione (GSH) metabolic pathway inhibitor, comprising detecting the presence of SWI/SNF complex protein deficiency in a patient having cancer.
In the third embodiment, the present invention provides a pharmaceutical composition comprising an effective amount of a glutathione (GSH) metabolic pathway inhibitor for treating SWI/SNF complex-deficient cancer.
In the fourth embodiment, the present invention provides a glutathione (GSH) metabolic pathway inhibitor for use in treating SWI/SNF complex-deficient cancer.
In the fifth embodiment, the present invention provides a kit comprising (a) a glutathione (GSH) metabolic pathway inhibitor, and (b) instructions for treating SWI/SNF complex-deficient cancer.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.
The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “an” and “the” include plural references unless the context clearly dictates otherwise.
The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.
The term “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (for example, it includes at least the degree of error associated with the measurement of the particular quantity). The term “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. The term “about” may refer to plus or minus 10% of the indicated number.
The present invention relates to a method for treating SWI/SNF complex-deficient cancer, comprising administering an effective amount of a glutathione (GSH) metabolic pathway inhibitor to a mammal in need thereof.
A “glutathione (GSH) metabolic pathway inhibitor” includes a substance for inhibiting GSH itself, or GSH synthesis enzymes. For example, a glutathione (GSH) metabolic pathway inhibitor includes a glutamate-cysteine ligase synthetase (GCL) inhibitor, a glutamate-cysteine ligase catalytic subunit (GCLC) inhibitor, a glutamate-cysteine ligase modifier (GCLM) inhibitor, a GSH synthetase (GSS) inhibitor, an SLC7A11 inhibitor, or a combination thereof.
Among them, a GCLC inhibitor, which inhibits a glutamate-cysteine ligase catalytic subunit (GCLC), i.e., a rate-limiting enzyme in a GSH biosyntheis pathway, is particularly preferable. The representative example of a GCLC inhibitor is buthionine sulfoximine (BSO) (Cayman Chemical Co.) (CAS Number: 83730-53-4). Among them, a GCL inhibitor is also preferable.
SLC7A11 inhibitor is, for example, sulfasalazine (Pfizer).
A glutathione (GSH) metabolic pathway inhibitor may be also antibody, small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), antisense oligonucleotide (ASO), low-molecular compound, a combination thereof, and the like.
As used herein, a “low-molecular compound” means a low molecular weight organic compound that may serve as an enzyme substrate or regulator of biological processes. In general, a low-molecular compound is less than about 5 kilodaltons (kD) in size. In some embodiments, in accordance with the present invention, a low-molecular compound is not proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, polysaccharides, glycoproteins, proteoglycans, and the like. In some embodiments, a small molecule may be a therapeutic, an adjuvant, or a drug.
A glutathione (GSH) metabolic pathway inhibitor may be a “GSH reduction compound,” which inhibits GSH itself, and as a result, reduces the GSH level in cells. A GSH reduction compound is, for example, APR-017 (also known as Anti-P2Y13 Receptor Antibody or PRIMA-1; Alomone Labs.), or APR-246 (also known as PRIMA-1MET; Aprea Therapeutic) (CAS Number: 5291-32-7).
As used herein, component(s) of “SWI/SNF complex(es)” include(s), for example, ACTB, ACTL6A (BAF53A), ACTL6B (BAF53B), ARID1A (BAF250A), ARID1B (BAF250B), ARID2 (BAF200), BCL11A, BCL11B, BCL7A, BCL7B, BCL7C, BICRA (GLTSCR1), BRD7, BRD9, DPF1 (BAF45B), DPF2 (BAF45D), DPF3 (BAF45C), PBRM1 (BAF180), PHF10 (BAF45A), SMARCA2 (BRM), SMARCA4 (BRG1, BAF190), SMARCB1 (BAF47, hSNF5, INI1), SMARCC1 (BAF155), SMARCC2 (BAF170), SMARCD1 (BAF60A), SMARCD2 (BAF60B), SMARCD3 (BAF60C), SMARCE1 (BAF57), SS18, or a combination thereof. Among them, ARID1A, SMARCA2, SMARCA4, SMARCB1, PBRM1 or a combination thereof is preferable. In particular, ARID1A is more preferable.
As used herein, in some cases, “SWI/SNF complex(es)” mean(s) each component(s) or a combination thereof.
As used herein, “SWI/SNF complex(es) deficiency” means SWI/SNF complex(es) gene(s) deficiency or SWI/SNF complex(es) protein(s) deficiency caused by SWI/SNF complex(es) gene mutation. Herein, “SWI/SNF complex(es) deficiency” may be caused by which each of genes belonging to SWI/SNF complex(es) or a combination of genes belonging to SWI/SNF complex(es) are mutated. “SWI/SNF complex(es)-deficient cancer” means cancer having SWI/SNF complex(es) deficiency.
As used herein, “ARID1A deficiency” means ARID1A gene deficiency or ARID1A protein deficiency caused by ARID1A gene mutation. “ARID1A-deficient cancer” means cancer having ARID1A deficiency. Examples of ARID1A-deficient cancer include ovarian cancer, uterine cancer, gastric cancer, bladder cancer, bile duct cancer, liver cancer, esophageal cancer, lung cancer, colon cancer, pancreatic cancer, breast cancer, neuroblastoma, glioma, skin cancer, B-cell lymphoma, renal cancer, and the like.
As used herein, “SMARCA2 (BRM) deficiency” means SMARCA2 (BRM) gene deficiency or SMARCA2 (BRM) protein deficiency caused by SMARCA2 (BRM) gene mutation. “SMARCA2 (BRM)-deficient cancer” means cancer having SMARCA2 (BRM) deficiency. Examples of SMARCA2 (BRM)-deficient cancer include adenoid cystic carcinoma, bladder cancer, breast cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, glioma, head and neck cancer, lung cancer, medul-loblastoma, melanoma, pancreatic cancer, ovarian cancer, prostate cancer, SMARCA4-deficient thoracic sarcoma, uterine cancer, sarcoma, and the like.
As used herein, “SMARCA4 (BRG1) deficiency” means SMARCA4 (BRG1) gene deficiency or SMARCA4 (BRG1) protein deficiency caused by SMARCA4 (BRG1) gene mutation. “SMARCA4 (BRG1)-deficient cancer” means cancer having SMARCA4 (BRG1) deficiency. Examples of SMARCA4 (BRG1)-deficient cancer include bladder cancer, breast cancer, cervical cancer, colon cancer, B-cell lymphoma, esophageal cancer, gastric cancer, glioma, head and neck cancer, liver cancer, lung cancer, melanoma, pancreatic cancer, prostate cancer, renal cancer, rhabdoid tumor, ovarian cancer, SMARCA4-deficient thoracic sarcoma, uterine cancer and the like.
As used herein, “SMARCB1 deficiency” means SMARCB1 gene deficiency or SMARCB1 protein deficiency caused by SMARCB1 gene mutation. “SMARCB1-deficient cancer” means cancer having SMARCB1 deficiency. Examples of SMARCB1-deficient cancer include epithelioid sarcoma, familial schwannomatosis, gastric cancer, renal cancer, rhabdoid tumor, sinonasal carcinoma, synovial sarcoma, undifferentiated chordomas, uterine cancer and the like.
As used herein, “PBRM1 deficiency” means PBRM1 gene deficiency or PBRM1 protein deficiency caused by PBRM1 gene mutation. “PBRM1-deficient cancer” means cancer having PBRM1 deficiency. Examples of PBRM1-deficient cancer include bladder cancer, bile duct cancer, colon cancer, B-cell lymphoma, esophageal cancer, gastric cancer, head and neck cancer, lung cancer, melanoma, mesothelioma, pancreatic cancer, renal cancer, uterine cancer and the like.
As used herein, ovarian cancer, includes, for example, ovarian clear cell carcinoma or ovarian endometrioid carcinoma.
There are a large number of patients suffering from ovarian clear cell carcinoma in Japan, and ARID1A-deficient ovarian cancer is found at a high rate, i.e., in one of two of ovarian cancer patients.
As used herein, uterine cancer is, for example, uterine corpus endometrial carcinoma. As used herein, the term “treatment” (also “treat” or “treating”) refers to any administration of a glutathione (GSH) metabolic pathway inhibitor that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and/or reduces frequency, incidence or severity of one or more symptoms, features, and/or causes of SWI/SNF complex(es)-deficient cancer. Such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition (e.g., may be prophylactic) and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition. Alternatively or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition (e.g., may be therapeutic).
As used herein, the term “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. An effective amount of the composition may be determined by a person skilled in the art and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the composition to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of an inhibitor are outweighed by the therapeutically beneficial effects.
The effective amount of a glutathione (GSH) metabolic pathway inhibitor may be adjusted appropriately to achieve desired drug levels, local or systemic, depending upon the mode of administration. Specifically, the effective amount is preferably about 0.5 ng to about 2000 mg per day at a time, or about 1 ng to about 1000 mg per day at a time, and it may be administered to patients once to several times per day.
As used herein, the term “mammal” refers to humans or non-human animals. In some embodiments, the non-human animal includes, for example, a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, a pig, and the like.
As used herein, the term “administer” (also, administration, administering) refers to administering the present inhibitor or a composition comprising the same to the subject (mammal). The route by which the disclosed inhibitor or composition is administered and the form thereof will dictate the type of carrier to be used. The inhibitor or composition may be in a variety of forms, suitable, for example, for systemic administration (e.g., oral, rectal, nasal, sublingual, buccal, implants, or parenteral) or topical administration (e.g., dermal, pulmonary, nasal, aural, ocular, liposome delivery systems, or iontophoresis).
The inhibitor or the pharmaceutical compositions according to the present invention can be prepared by processes which are known per se and familiar to the person skilled in the art. As pharmaceutical compositions, the active agents are either employed as such, or preferably in combination with suitable pharmaceutical auxiliaries and/or excipients, e.g. in the form of tablets, coated tablets, capsules, caplets, suppositories, patches, emulsions, suspensions, gels or solutions. A person skilled in the art is familiar with auxiliaries, vehicles, excipients, diluents, carriers or adjuvants which are suitable for the desired pharmaceutical formulations, preparations or compositions on account of his/her expert knowledge. In addition to solvents, gel formers, ointment bases and other excipients, for example antioxidants, dispersants, emulsifiers, preservatives, solubilizers, colorants, complexing agents or permeation promoters, can be used.
The present invention also relates to a method for detecting and/or selecting a susceptible patient to a GSH metabolic pathway inhibitor, comprising detecting the presence of SWI/SNF complex(es) deficiency in a patient having cancer.
As used herein, “susceptible patient to a GSH metabolic pathway inhibitor” means patient having cancers which are sensitive to a GSH metabolic pathway inhibitor.
As used herein, the term “sample” includes a tissue sample (brain, hair, buccal swabs, blood, saliva, semen, muscle, any internal organs, or cancer, precancerous, tumor cells), cell sample, a fluid sample (urine, blood, ascites, pleural fluid, spinal fluid), and the like.
Specifically, in some embodiment, a method for detecting and/or selecting a susceptible patient to a GSH metabolic pathway inhibitor, according to the present invention, comprises (i) providing a sample derived from a patient having cancer, (ii) measuring the level of SWI/SNF complex(es) in the sample, (iii) comparing the measued level with the predetermined value, and (iv) it is determined that if the measued level is below the predetermined value, SWI/SNF complex(es) is deficient and that the patient is likely to be sensitive to a GSH metabolic pathway inhibitor, while if the measued level is above the predetermined value, it is determined that SWI/SNF complex(es) is not deficient and that the patient is unlikely to be sensitive to a GSH metabolic pathway inhibitor.
In some embodiment, a method for detecting and/or selecting a susceptible patient to a GSH metabolic pathway inhibitor, according to the present invention, comprises (i) providing a sample derived from a patient having cancer, (ii) measuring the level of SWI/SNF complex(es) gene expression in the sample, (iii) comparing the measured level with the predetermined value, and (iv) it is determined that if the measured level is below the predetermined value, SWI/SNF complex(es) gene is deficient and that the patient is likely to be sensitive to a GSH metabolic pathway inhibitor, while if the measued level is above the predetermined value, it is determined that SWI/SNF complex(es) gene is not deficient and that the patient is unlikely to be sensitive to a GSH metabolic pathway inhibitor.
The predetermined value may be the level of SWI/SNF complex(es) gene or protein in normal tissue sample from unaffected individuals.
The presence of SWI/SNF complex(es) deficiency in the sample is determined by, for example, Cancer Gene Panel Test (OncoGuide™ NCC Oncopanel System).
The present invention also relates to a kit. The kit may include information, instructions, or both that use of the kit will provide treatment for medical conditions in mammals (particularly humans). The information and instructions may be in the form of words, pictures, or both, and the like. Additionally or alternatively, the kit may include the inhibitor, the composition, or both; and information, instructions, or both, regarding methods of application of the inhibitor or the composition, preferably with the benefit of treating or preventing medical conditions in mammals (e.g., humans).
In this study, selective sensitivity to GSH metabolic pathway inhibitors such as APR-246 according to SWI/SNF complex(es) deficiency, was observed in several cancer cell lines. For example, with regard to ARID1A deficiency, HCT116 colon cancer cells with or without artificial gene knockout; widely used ovarian, uterine, gastric and biliary tract cancer cell lines; and newly established ovarian patient derived cells (PDCs). Based on results herein, it was demonstrated that ARID1A-mutated tumors without ARID1A protein expression, but not those retaining ARID1A expression, respond well to GSH inhibitor- or GSH metabolic pathway inhibitor-targeted therapy. Thus, immunohistochemical assessment of ARID1A protein will be a useful diagnostic tool for detecting and/or selecting patients suitable for GSH inhibitor- or GSH metabolic pathway inhibitor-targeted therapy.
The following examples are set forth so as to provide a person skilled in the art with a complete disclosure and description of how the methods claimed wherein are made and evaluated, and are intended to purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as the invention.
To investigate cellular vulnerabilities caused by ARID1A deficiency, we examined the differential sensitivities of parental ARID1A-wild-type (ARID1A-WT) and ARID1A-knockout (ARID1A-KO) HCT116 colon cancer cells to 334 inhibitors whose targets have been elucidated. A drug sensitivity screen identified PRIMA-1 (APR-017) (
To explore the sensitivity of ARID1A-KO cells to APR-246, genome-wide expression profiling was conducted. A pathway analysis of 6,429 genes whose expression levels in ARID1A-KO cells, but not in ARID1A-WT cells, increased or decreased by more than 2-fold upon APR-246 treatment was performed to identify the pathways responsible for the sensitivity of ARID1A-KO cells to ARP-246 (
APR-246 is converted to the Michael acceptor methylene quinuclidinone (MQ), which inhibits activity of the antioxidant metabolite GSH and the antioxidant regulator thioredoxin reductase (TrxR) by reacting with their thiols (See, Peng et al., Cell Death Dis 4, e881, 2013; Tessoulin et al., Blood 124, 1626-1636, 2014). Covalent binding of MQ decreased the level of GSH and inhibited TrxR activity, thereby shifting the intracellular balance between ROS generation and antioxidation toward an increase in ROS levels (
The decreased GSH level and increased ROS level after APR-246 treatment were only evident in ARID1A-deficient human ovarian and other cancer cells (
MQ derived from APR-246 and PRIMA-1 also binds covalently to cysteine residues in TrxR (See, Bykov et al., Front Oncol 6, 21, 2016). In fact, ARID1A-KO cells and ARID1A-deficient cells were more sensitive to the TrxR inhibitor auranofin than ARID1A-proficient cells (
GSH is synthesized from cysteine, glutamate, and glycine via the actions of multiple metabolic factors (
Among the three GSH synthesis pathway enzymes listed above, we focused on GCLC because it is a rate-limiting enzyme (i.e., possibly a druggable target) in GSH synthesis and its transient knockdown markedly inhibited the growth of ARID1A-deficient cancer cells (
To determine whether GCLC is a druggable target, we examined the sensitivities of four ARID1A-proficient and six ARID1A-deficient cancer cell lines to the GCLC inhibitor BSO. ARID1A-deficient cancer cell lines were selectively sensitive to BSO (
ARID1A-deficient cancer cells were sensitive to GSH inhibition compared with ARID1A-proficient cells. We therefore hypothesized that ARID1A regulates transcription of genes encoding components of the GSH synthesis pathway. To investigate this, a genome-wide expression analysis of a panel of ARID1A-proficient and ARID1A-deficient cancer cells was performed. In total, 343 genes whose expression levels were consistently more than 1.5-fold lower in ARID1A-deficient cancer cells were identified (
SLC7A11 encodes a subunit of the cystine/glutamate transporter XCT. Cystine is taken up into cells through the XCT transporter and is then metabolized into two molecules of cysteine, which is essential for GSH synthesis. To investigate the effects of downregulation of SLC7A11 caused by ARID1A deficiency, we screened metabolites whose levels were decreased in the three pairs of ARID1A-proficient and ARID1A-deficient cells using gas chromatography/mass spectrometry (GCMS), which is able to detect 475 metabolites (including cysteine, glutamate, and glycine, but not GSH) (
The decrease in GSH, increase in ROS, and decrease in viability in APR-246-treated ARID1A-deficient cancer cells were completely suppressed by co-treatment with the cystine compensator cystine dimethyl ester (CC-DME), a cell-permeable version of cystine (See, Steinherz et al., Proc Natl Acad Sci USA 79, 4446-4450, 1982) (
To further address the clinical relevance of this hypothesis, we examined expression of ARID1A and SLC7A11 by immunohistochemical staining of tumor specimens surgically obtained from 11 ovarian cancer patients (
Patient-derived cancer cells (PDCs) are often serve as a tool to obtain proof-of-concept for cancer therapy. Thus, PDCs from four other ovarian cancer patients, three cases lacked ARID1A whereas the remaining case retained ARID1A expression, were cultured (
Next, APR-246 was tested for its ability to suppress the growth of OCCC tumor xenografts in vivo. After the tumor formation, mice were treated with APR-246 or vehicle. APR-246 treatment significantly suppressed the growth of ARID1A-deficient TOV21G OCCC xenografts, but not that of ARID1A-proficient RMG-I OCCC xenografts (
To further validate GCLC as a therapeutic target, the effects of GCLC depletion were assessed in an ARID1A-deficient OCCC tumor xenograft model. TOV21G OCCC cells carrying non-targeting (shNT) or GCLC-targeting (shGCLC) shRNAs were injected into mice. GCLC expression in TOV21G-shGCLC cells was conditionally reduced by Dox (
Of >100 patient-derived cells (PDCs) obtained from the ascites of 65 patients with diffuse-type gastric cancer, we selected 13 cell types (NSC-4X1a, -7C, -14C, -20C, -22C, -34C, -48CA, -58C, -64C, -65C, -67C, -68C, and -70C) showing adherent cell growth and lower dispersion in the drug-sensitivity test than floating cells. ARID1A protein expression was investigated by immunoblot analysis. Eight PDCs were selected for further analysis based on whole exome data. Of these eight PDCs, four (NSC-7C, -58C, -65C, and -67C) lacked ARID1A protein expression (ARID1A-deficient: ARID1A-) and four (NSC-48C, -64C, -68C, and -70C) retained ARID1A protein expression (ARID1A-proficient: ARID1A+) (
We next examined the sensitivity of ARID1A-deficient gastric cancer cells to GSH inhibitors. The IC50 values for the GSH inhibitor APR-246 were markedly lower in ARID1A-deficient PDCs than in ARID1A-proficient PDCs (
Next, we investigated whether low expression of the SLC7A11 protein in ARID1A-deficient gastric cancers is associated with decreased SLC7A11 transcription. SLC7A11 mRNA levels were lower in ARID1A-deficient than in ARID1A-proficient PDCs (
APR-246 inhibits GSH activity by reacting with thiol groups (See, B. Tessoulin et al., Blood 124 (2014) 1626-1636). Therefore, we next examined whether APR-246 preferentially inhibits GSH in ARID1A-deficient cancer cells. APR-246 treatment markedly decreased GSH levels in ARID1A-deficient PDCs and not in ARID1A-proficient PDCs (
We next examined whether the vulnerability of ARID1A-deficient cancer cells is related to cysteine shortage and consequent GSH shortage. The APR-246-induced GSH decrease, ROS increase, and cell death in ARID1A-deficient cancer cells were markedly suppressed by co-treatment with the cystine compensator cystine dimethyl ester (CC-DME) or the GSH compensator glutathione monoethyl ester (GSH-MEE), cell-permeable versions of cystine and GSH, respectively, suggesting that these cell-permeable metabolites were able to compensate for impairment of cystine uptake due to diminished SLC7A11 expression (
We examined the sensitivity of ARID1A-deficient uterine cancer cells to GSH inhibitors. The IC50 values for the GSH inhibitor APR-246 were markedly lower in three ARID1A-deficient cell lines than in two ARID1A-proficient cell lines (
We examined the sensitivity of ARID1A-deficient bile duct cancer cells to GSH inhibitors. The IC50 values for the GSH inhibitor APR-246 were markedly lower in two ARID1A-deficient cell lines than in two ARID1A-proficient cell lines (
We examined the sensitivity of various SWI/SNF complexes (SMARCA2 (BRM), SMARCA4 (BRG1, BAF190), SMARCB1 (BAF47, hSNF5, INI1), PBRM1) deficient cancer cells to GSH inhibitors. The IC50 values for the GSH inhibitor APR-246 were markedly lower in these SWI/SNF complexes deficient cell lines than in wild-type cell lines (
SWI/SNF complexes except for ARID1A (i.e, SMARCA2, SMARCA4, SMARCB1, PBRM1) -deficient cancer cells were similarly sensitive to GSH inhibition compared with these SWI/SNF complexes proficient cells. To investigate this, expression of SLC7A11 mRNA was lower in these SWI/SNF complexes-deficient cancer cells than in various SWI/SNF complexes proficient cancer cells (
Similar to Example 4, the basal GSH level was lower in these SWI/SNF complexes-deficient cells than in these SWI/SNF complexes proficient cells (
Tumor samples and ascites were obtained from four ovarian cancer patients who underwent surgery or cell-free and concentrated ascites reinfusion therapy at the National Cancer Center Hospital or Kaname-cho Hospital (Tokyo, Japan) and were cultured in vitro. This protocol was approved by the Institutional Review Board of the National Cancer Center (Tokyo, Japan), and informed consent was obtained from the patients. To establish CCCO219 and CCC1216 PDCs, patient ascites (24 ml) were diluted in two volumes of PBS containing 2 mM EDTA, layered over 15 ml Ficoll-Paque PLUS (GE Healthcare), and centrifuged at 2200 rpm for 30 min. The interphase mononuclear layer was transferred to a fresh conical tube and washed twice with PBS containing 2 mM EDTA. Epithelial cells were labeled magnetically with microbeads conjugated to a monoclonal human epithelial antigen-125 antibody (EasySep Human EpCAM Positive Selection Kit, STEMCELL Technologies). Epithelial antigen-125-positive cells were collected by magnetic selection and cultured in DMEM/F-12 supplemented with 10% FBS. To establish NOVC-1C and NOVC-4C PDCs, whole ascetic cells were pelleted by centrifugation at 1500 rpm for 5 min at room temperature and then incubated in hemolysis buffer (0.75% NH4Cl and 17 mM Tris-HCl, pH 7.65) for 10 min. After centrifugation, pellets were washed with PBS and cultured in RPMI 1640 containing 10% FBS for 1 week. Thereafter, the culture medium was replaced with DMEM containing 10% FBS to remove lymphocytes, and cells were cultured for another week. Adherent cells were cultured in RPMI 1640 containing 10% FBS for several weeks, exchanging the medium once per week, until multiple colonies appeared. If needed, cultured cells were treated repeatedly with 0.05% trypsin-EDTA for a short duration to remove fibroblasts or other cell types such as mesothelial cells. The culture was passaged when colonies became dense. The ARID1A expression status was confirmed by immunoblot analysis.
All mouse experiments were approved by the National Cancer Center (NCC) animal Ethical Committee. Cells were counted and re-suspended in a 1:1 mixture of 100 μl culture medium and 100 μl Matrigel (BD Biosciences) on ice. Thereafter, cells (RMG-I: 2×106 cells/mouse; TOV21G: 2×105 cells/mouse for ARP-246 treatment and 1×106 cells/mouse for BSO treatment; OVISE: 2×106 cells/mouse; TOV21G-shNT: 2×105 cells/mouse; and TOV21G-shGCLC: 2×105 cells/mouse) were injected subcutaneously into the flank of 6-week-old female BALB/c-nu/nu mice (CLEA and Charles River). In the subcutaneous model, once the tumors were palpable (about 3-14 days after implantation), mice were randomly divided into two groups. In the drug treatment group, mice were injected intraperitoneally with either PBS or compounds [APR-246 (50 mg/kg), PRIMA-1 (25 mg/kg), or BSO (750 mg/kg)] once daily for 12-14 days. In the doxycycline (Dox) treatment study, TOV21G-shNT cells and TOV21G-shGCLC cells were injected into the flanks of 6-week-old female BALB/c-nu/nu mice. Once the tumors were palpable (13 days after implantation), mice were randomly divided into two groups and fed a diet containing Dox (625 ppm) or a control diet. In other experiments, TOV21G-shNT cells and TOV21G-shGCLC cells were treated with Dox (0.5 μg/ml) for 4 days and then injected into the flank of 6-week-old female BALB/c-nu/nu mice. The mice were then fed a diet containing Dox (625 ppm) or a control diet. Tumor growth was measured every several days using calipers. The volume of implanted tumors was calculated using the formula V=L×W2/2, where V is volume (mm3), L is the largest diameter (mm), and W is the smallest diameter (mm). At the end of the experiment, mice were sacrificed in accordance with standard protocols.
Cells were maintained in a humidified incubator containing 5% CO2 at 37° C. in DMEM/F-12 (Wako) supplemented with 10% fetal bovine serum (FBS; Gibco/Life Technologies), 2 μmol/l glutamine, 100 U/ml penicillin, and 100 μg/mL streptomycin (Wako). TOV21G, HEC1A, H2228, H2122, H1819, H1299, H522, and H1703 cells were obtained from the American Type Culture Collection (ATCC). RMG-I, OVISE, HEC-265, HEC-151, KKU-100, KKU-055, Ishikawa, JHUEM-2, HuCCA-1, KP-4, JMU-RTK-2, G401, G402 and KMRC-1 cells were obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank. JHUEM-2, SSP25, PC9, and HS-ES-1 cells were obtained from the Riken Cell Bank (RCB). 2008 and A2780 cells were provided by Drs. S. B. Howell and E. Reed. ARID1A-KO (Q456X/Q456X) and parental HCT116 cells were purchased from Horizon Discovery. HCC-44 cells were gifted from Gazdar, A. F. RCC-MF cells were obtained from Cell Lines Service. The cell lines were authenticated by verifying alterations of multiple cancer-related genes via sequencing. Cells were used for functional experiments after less than 3 months of passaging post-receipt. All cell lines tested negative for mycoplasma by MycoAlert (Lonza).
The shRNA-expressing lentiviral vectors pGIPZ (shNT, OHS4346; shp53, RHS4430-200289946) (Open Biosystems) and pTRIPZ (shNT, OHS5832; shGCLC #3, RHS4946_200777182), the cDNA-expressing lentiviral vectors (pLOC-GCLC, OHS5897_202616616; pLOC-SLC7A11, OHS5898_219582558; pLOC-NRF2, OHS5900-202624558) (all from ThermoFisher Scientific), (pLenti-puro-ARID1A, #39478) (Addgene), and packaging plasmids (psPAX2: #12260 and pMD2.G: #12259) (Addgene) were used for constitutive expression of shRNA or cDNAs. To generate virus, 293LTV cells were transfected with lentiviral plasmids and packaging plasmids using Lipofectamine 3000 (Invitrogen/ThermoFisher Scientific). On the following day, the medium was replaced with fresh growth medium and lentivirus-containing supernatants were harvested and concentrated by centrifugation. To establish cells infected with viral constructs, cells were transduced with lentiviral vectors and then incubated for 7-14 days in growth medium containing 2 μg/ml puromycin (Sigma-Aldrich) or 20 μg/ml blasticidin (Wako).
Tumor samples and ascites were obtained from patients with diffuse-type gastric cancer who underwent surgery or cell-free and concentrated ascites reinfusion therapy at the National Cancer Center Hospital or Kanamecho Hospital (Tokyo, Japan) and were cultured in vitro. The study protocol was approved by the Institutional Review Board of the National Cancer Center (Tokyo, Japan), and written informed consent was obtained from the patients. Whole ascetic cells were pelleted by centrifugation at 1500 rpm for 5 min at room temperature and then incubated in hemolysis buffer (0.75% NH4Cl and 17 mM Tris-HCl, pH 7.65) for 10 min. After centrifugation, pellets were washed with PBS and cultured in RPMI 1640 containing 10% FBS for 1 week, after which the culture medium was replaced with DMEM containing 10% FBS to remove lymphocytes. Cells were cultured for an additional week. Adherent cells were cultured in RPMI 1640 containing 10% FBS for several weeks with weekly medium exchanges until the appearance of multiple colonies. When necessary, cultured cells were treated repeatedly with 0.05% trypsin-EDTA for a short duration to remove fibroblasts or other cell types such as mesothelial cells. The culture was passaged when colonies became dense.
Method
ARID1A-WT and ARID1A-KO HCT116 cancer cells were used for screening assays. Cells were seeded in 96-well plates, incubated for 24 hrs, and then treated with the drug at a concentration of 0.1, 1, or 10 μM [SCADS Inhibitor Kit, including 334 compounds (Table S1)]. Cell viability was assessed after 5 days using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). Luminescence was measured using an Envision Multi-label plate reader (PerkinElmer). The luminescence reading was used to determine the cell viability relative to that of cells treated with solvent (DMSO). Candidate compounds were considered if viability of ARID1A-KO cells was less than 40% that of ARID1A-WT cells was more than 80%.
Cell viability was examined by measuring the cellular ATP level using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). To measure cell viability after siRNA-mediated knockdown, cell lines were transfected with siRNAs (25 nM) using Lipofectamine RNAiMAX. After 48 hrs, cells were trypsinized and repeatedly transfected with siRNAs (25 nM) using Lipofectamine RNAiMAX. Cells were trypsinized after a further 48 hrs, counted, and reseeded at the specified density in 96-well plates. To measure cell viability after drug treatment, cells were trypsinized, counted, reseeded at the specified density in 96-well plates, and exposed to the indicated concentrations of drugs. Cell viability was measured using the CellTiter-Glo Luminescent Cell Viability Assay. Luminescence was measured using an Envision Multi-label plate reader (PerkinElmer).
The effect of drug treatment on cancer cell survival was evaluated in a colony formation assay. Cells were trypsinized, counted, reseeded at the specified density in 12-well plates, exposed to the indicated concentrations of drugs for 10-14 days, and fixed for 10 min in 50% (v/v) methanol containing 0.01% (w/v) crystal violet. Images were taken on LAS-3000 Imaging System (Fujifilm) and colonies were counted using Multi Gauge software.
The Annexin V-FITC/PI Apoptosis Detection Kit (Sigma-Aldrich) was used to detect apoptotic cells. Briefly, the cell pellet was suspended in 1× binding buffer and then incubated with Annexin V-FITC and PI in the dark for 10 min. Fluorescence was analyzed on a Guava flow cytometer (Millipore). Data were analyzed using GuavaSoft software (v. 2.7). Relative ratios of the Annexin V-positive fraction in treated samples were normalized against untreated samples.
GSH, ROS and apoptosis were detected using the GSH/GSSG-Glo Assay (Promega) and/or the GSH-Glo Assay (Promega), the ROS-Glo Assay (Promega), and the Caspase-Glo 3/7 Assay, respectively. To measure levels of GSH, ROS and apoptosis after drug treatment, cells were trypsinized, counted, reseeded at the specified density in 96-well plates and exposed to the indicated concentrations of drugs. After 16-48 hrs, luminescence was measured using an Envision Multi-label plate reader (PerkinElmer). To measure levels of GSH and ROS after siRNA-mediated knockdown, cell lines were transfected with siRNAs (25 nM) using Lipofectamine RNAiMAX. After 48 hrs, cells were trypsinized and transfected repeatedly with siRNAs (25 nM) using Lipofectamine RNAiMAX. Cells were trypsinized after a further 48 hrs, counted, and reseeded at the specified density in 96-well plates. After 72-120 hrs, luminescence was measured using an Envision Multi-label plate reader (PerkinElmer). Cell viability was also measured using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). Caspase-3/7, GSH, and ROS levels were normalized against cell viability. The GSH/GSSG ratio was calculated as the GSH-GSSG signal divided by the GSSG/2 signal. Relative signal ratios in treated samples were normalized against untreated samples.
GSH was detected using the GSH-Glo Assay (Promega). Tumor samples derived from xenografts were weighed and washed with PBS. The tumor samples were mixed with 50 μl of PBS and homogenized using a Mini Cordless Grinder (Funakoshi). PBS (950 μl) was added to homogenized tumor samples and centrifuged at 4° C. for 10 min at 15,000 rpm. Tumor extract and 2×GSH-Glo Reagent (25 μl of each) were mixed in white 96-well plates (Greiner) and incubated for 30 min at room temperature. Luciferin Detection Reagent (50 μl) was added and the samples were incubated for 15 min at room temperature. Luminescence was measured using an Envision Multi-label plate reader (PerkinElmer). GSH signal intensities per 1 mg of tumor sample were calculated. Relative GSH ratios were normalized against untreated samples (without APR-246 or Dox).
TrxR activity including TrxR1, TrxR2 and TrxR3 was measured using the Thioredoxin Reductase Assay Kit (Abcam). Cells were trypsinized, counted, reseeded at the specified density in 10 cm dishes and exposed to the indicated concentrations of APR-246. After 24 hrs, cells were washed with cold PBS and lysed with buffer containing a proteinase inhibitor. After centrifugation, the supernatant was supplemented with a TrxR inhibitor and incubated for 20 min at 25° C. Absorbance was measured using an Envision Multi-label plate reader (PerkinElmer). Relative TrxR ratios were normalized against untreated samples.
Antibody array analysis was conducted using the Human Cell Stress Array (R&D Systems). For whole-cell extraction, 1×107 cells were harvested, washed with PBS, lysed in Lysis Buffer 6 supplemented with a proteinase inhibitor cocktail and a phosphatase inhibitor cocktail (Active Motif), incubated for 30 min on ice, and centrifuged at 4° C. for 10 min at 15,000 rpm. Whole-cell lysates (1 ml) were mixed with 0.5 ml of Array Buffer 4 and 20 μl of reconstituted Detection Antibody Cocktail for 1 hr at room temperature. These samples were added to membranes blocked with Array Buffer 4. After incubation overnight at 4° C., the membranes were washed twice with 1× Wash Buffer and rinsed with distilled water and then dried. Diluted streptavidin-HRP (2 ml) was added and the membrane was incubated for 30 min at room temperature and then was washed with 1× Wash Buffer. Chemi Reagent Mix was applied evenly to the membrane and incubated for 1 min. Chemiluminescence signals were measured using LAS-3000 Imaging System (Fujifilm). Signal intensities were measured using Multi Gauge software. The ratios of signal intensities in cells treated with 40 μM APR-246 for 24 hrs were calculated relative to the corresponding intensities in untreated cells.
Total RNA was extracted using the Qiagen RNeasy kit. The integrity of extracted RNA was confirmed by NanoDrop spectrophotometry (NanoDrop Technologies). Total RNA was reverse-transcribed using the Agilent Low Input Quick Amp Labeling Kit (Agilent Technologies). cDNA was hybridized for 16 hrs at 65° C. on duplicate Agilent microarrays (SurePrint G3 Human Gene Expression 8×60K Ver. 1.0, G4851: 42405 probes) using the Gene Expression Hybridization Kit (Agilent Technologies). After the arrays were washed using the Gene Expression Wash Pack (Agilent Technologies), data were extracted using an Agilent scanner. The arrays were analyzed initially using Feature Extraction software (Agilent Technologies). A quantitative signal and qualitative detection call were generated for each sample and transcript.
Data files were subsequently analyzed, normalized, and compared using GeneSpring GX12.6 (Agilent Technologies). Raw expression data of 42,545 probe sets on SurePrint G3 Human Gene Expression arrays were processed and log 2-transformed. Expression data for each sample were normalized against median expression levels in the control condition. Genes were grouped according to fold changes. All raw microarray data files have been deposited in the Gene Expression Omnibus (GEO: GSE122925 and GSE122926).
mRNA was extracted and cDNA was synthesized using the SuperPrep (Registered Trademark) Cell Lysis & RT Kit for qPCR (TOYOBO). Aliquots of cDNA were subjected to quantitative PCR using the SuperPrep/THUNDERBIRD Probe qPCR Set (TOYOBO) and TaqMan Gene Expression Assays (Life Technologies). The following gene-specific primer/probe sets were used: NOXA (PMAIP1) (Hs00560402 ml), NRF2 (NFE2L2) (Hs00975961 g_1), GCLC (Hs00155249 ml), GSS (Hs00609286_m1), and SLC7A11 (Hs00921938_m1). PCR was performed in an ABI StepOnePlus Real-Time PCR System (Life Technologies) under the following conditions: denaturation at 95° C. for 15 s, followed by annealing and extension at 60° C. for 30 s (40 cycles). For each sample, the mRNA levels of target genes were normalized against levels of GAPDH mRNA. The target/GAPDH ratios were then normalized against those in control samples using the 2-ΔΔCt method.
For whole-cell extraction, 5×105 cells were harvested, washed with PBS, lysed in NETN420 buffer [20 mM Tris-HCl (pH 7.5), 420 mM NaCl, 0.5% NP-40, and 1 mM EDTA] supplemented with a proteinase inhibitor cocktail and a phosphatase inhibitor cocktail (Active Motif), incubated for 30 min on ice, and centrifuged at 4° C. for 10 min at 15,000 rpm. The soluble fractions of whole-cell lysates were mixed with SDS sample buffer. For cell extraction including the membrane fraction to detect SLC7A11, cells were harvested, washed with PBS, lysed in M-PER Mammalian Protein Extraction Regent Buffer (ThermoFisher Scientific) supplemented with a proteinase inhibitor cocktail and a phosphatase inhibitor cocktail (Active Motif), incubated for 10 min on ice, and centrifuged at 4° C. for 10 min at 15,000 rpm. The soluble fractions were mixed with SDS sample buffer. Tumor samples derived from xenografts were weighed and washed with PBS. The tumor samples (10 mg) were mixed with 50 μl of NETN420 buffer supplemented with a proteinase inhibitor cocktail and a phosphatase inhibitor cocktail (Active Motif) and homogenized using a Mini Cordless Grinder (Funakoshi). The homogenized tumor samples were diluted in an additional 450 μl of NETN420 buffer, incubated for 30 min on ice, and centrifuged at 4° C. for 10 min at 15,000 rpm. The soluble fractions of whole-cell lysates were mixed with SDS sample buffer. Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and immunoblotted with the indicated antibodies. β-actin was used as a loading control. Membranes were blocked overnight at 4° C. or for 1 hr at 25° C. with PVDF Blocking Reagent for Can Get Signal (TOYOBO) and then probed with Can Get Signal Solution 1 (TOYOBO) containing primary antibodies. After washing with TBS containing 0.1% Tween 20, the membranes were incubated with TBS containing 0.1% Tween 20, 1% BSA, and horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies, and visualized using Western Lightning ECL Pro (Perkin Elmer). Chemiluminescence signals were measured using LAS-3000 Imaging System (Fujifilm). Signal intensities were measured using Multi Gauge software. The protein levels of GCLC were normalized against the levels of (β-actin. The GCLC/(β-actin ratios were then normalized against those in control samples without Dox treatment. Data are expressed as the mean±SEM (n=6). The following antibodies were used for immunoblotting: GCLC (Abcam, ab190685), NRF2 (Abcam, ab62352), p53 (Calbiochem, OP43), β-actin (CST, 4790), ARID1A (CST, 12354), BRG1 (CST, 49360), SLC7A11 (CST, 12691), NOXA (CST, 14766), p21 (CST, 2947), JNK (Santa Cruz Biotechnology, sc-7345), and phospho-JNK (Santa Cruz Biotechnology, sc-6254).
1×106 cells were harvested 24 hrs after seeding and treated with 1% formaldehyde for 10 min at room temperature to crosslink proteins to DNA. Glycine (0.125 M) was added to stop the crosslinking process. ChIP assays were performed using the ChIP-IT Express Enzymatic kit (Active Motif) and antibodies against ARID1A (CST, 12354), BRG1 (CST, 49360), NRF2 (CST, 12721) or RNAPII (Active Motif, 39097). Purified DNA was subjected to quantitative PCR using the SuperPrep/THUNDERBIRD SYBR qPCR Set (TOYOBO) and the following primer pairs: SLC7A11-1182-1099-F (5′-TCAGAAGCTTATTTAATGGTGCG-3′) and SLC7A11-1182-1099-R (5′-GTGGTTTTGGATTCAGTGAGAAG-3′); SLC7A11-297-241-F (5′-CAGCTTTTGTTGCTCACTACG-3′) and SLC7A11-297-241-R (5′-TCGGAACAGACCTTCCCAG-3′); SLC7A11-14_79-F (5′-GAGGAAGCTGAGCTGGTTTG-3′) and SLC7A11-14_79-R (5′-GCATCGTGCTCTCAATTCTC-3′); SLC7A11_71_190-F (5′-GCACGATGCATACACAGGTG-3′) and SLC7A11_71_190-R (5′-CCTCTGCTTTCAGACTGTCT-3′); and SLC7A11_972_1070-F (5′-CGGAGTGTTCAGCAGAAGTC-3′) and SLC7A11_972_1070-R (5′-GAGGTGACAAGCACATGAAC-3′). The PCR conditions were as follows: denaturation at 95° C. for 15 s, followed by annealing and extension at 60° C. for 60 s (45 cycles). PCR was performed on an ABI StepOnePlus Real-Time PCR System (Life Technologies). Protein enrichment was expressed as a percentage of input.
Metabolites were extracted from 2×106 cells. Culture medium was removed, and cells were washed twice with 5% mannitol solution (8 ml and then 4 ml) and then treated with 800 μl methanol and 150 μl Milli-Q water containing 5 μg 2-isopropylmalic acid as an internal control. The metabolite extract was transferred to a microfuge tube and dried using a Spin Dryer (TAITEC). Derivatization in the solid phase was conducted as described below. The solid phase cartridge Presh-SPE AOS was supplied by AiSTI SCIENCE (Wakayama). Cell extract was mixed with 200 μl Milli-Q water and 800 μl acetonitrile and incubated at 37° C. for 30 min. After centrifugation at 14,000 rpm for 5 min at 4° C. the supernatant was transferred to a new tube. The derivatization conditions were 3 min of methoximation with 5 μl of >5% methoxyamine solution and 10 min of trimethylsilylation with 25 μl N-methyl-N-trimethylsilyl-trifluoroacetamide. Derivatized analytes were effectively eluted with 100 μl n-hexane, and 1.0 μl of the derivatized solution was injected into the gas chromatograph/mass spectrometer GCMS-TQ8050 (Shimadzu).
Metabolome analysis was performed on a GCMS-TQ8050 equipped with a BPX-5 capillary column (internal diameter: 30 m×0.25 mm; film thickness: 0.25 μm; SEG, Victoria). Parameter setting was described previously (See, Nishiumi et al., Oncotarget 8, 17115-17126, 2017). During GCMS-TQ8050 analysis, the inlet temperature was kept at 250° C. and helium was used as a carrier gas at a constant flow rate of 39.0 cm per sec. The injector split ratio was set to 1:10. The GC column temperature was programmed to remain at 60° C. for 2 min and then to rise from 60° C. to 330° C. at a rate of 15° C. per min, before being kept at 330° C. for 3 min. The total GC run time was 23 min. The transfer-line and ion-source temperatures were 280° C. and 200° C., respectively. The ionization voltage was 70 eV. Argon gas was used as a collision-induced dissociation gas. Metabolites were detected using the Smart Metabolites Database (Shimadzu), which contains the relevant MRM method file and data regarding the GC analytical conditions, MRM parameters, and retention index employed for metabolite measurements. The Automatic Adjustment of Retention Time (AART) function of GCMS solution software (Shimadzu) and a standard alkane series mixture (C7 to C33) were used to correct the retention time. Peaks were identified automatically and confirmed manually based on the specific precursor and product ions and the retention time. Relative cysteine ratios were normalized against ARID1A-proficient cells.
Targeted sequencing was conducted using 1.0 μg DNA extracted from cultured cancer cells. Targeted genome capture was performed using the Agilent SureSelect kit NCC Oncopanel (931196). Sequencing was performed on the Illumina NextSeq platform using 150 bp paired-end reads (Illumina). Basic alignment and sequence quality control were conducted using the Picard and Firehose pipelines. Reads were aligned against the reference human genome from the UCSC human genome 19 (hg19) using the Burrows-Wheeler Aligner Multi-Vision software package. Duplicate reads were generated during PCR; therefore, paired-end reads that aligned to the same genomic positions were removed using SAMtools. Somatic single-nucleotide variants were called by the MuTect program, which applies a Bayesian classifier to allow detection of somatic mutations with low allele frequencies. Somatic insertion/deletion mutations (indels) were called using the GATK Somatic IndelDetector (http://archive.broadinstitute.org/cancer/cga/indelocator).
Eleven patients were diagnosed with ovarian cancer and underwent surgery at the National Cancer Center Hospital (NCCH), Tokyo, Japan, or at the Jikei University Hospital (JUH), Tokyo, Japan. None of the 11 patients had received any pre-surgical treatment. This study was approved by the Institutional Review Board of the National Cancer Center (Tokyo, Japan) and Jikei University, and informed consent was obtained from the patients. Ovarian tumors were diagnosed in accordance with the International Federation of Gynecology and Obstetrics (FIGO) guidelines and classified according to the World Health Organization (WHO) classification system. ARID1A mutations were determined by target and whole-exome sequencing, as described previously (See, Kanke et al., Oncotarget 9, 6228-6237, 2018).
Formalin-fixed, paraffin-embedded ovarian cancer clinical specimens and TOV21G xenografts were deparaffinized and representative whole 4-μm-thick sections were analyzed by IHC. TOV21G-shGCLC xenografts were embedded in OTC compound (25608-930; Tissue-Tek) and stored at −80° C. The samples were removed from the freezer and equilibrated at −20° C. for approximately 15 minutes before sectioning. Tissue sections (6 μm thick) were placed on positively charged slides, dried, and fixed for 15 minutes at room temperature in 3% formaldehyde, followed by 5 minutes in methanol at −20° C. After fixation, representative sections were analyzed by IHC. Tissue sections were stained using antibodies against Ki-67 (MIB-1) (GA62661-2, 1:100 dilution; Dako), cleaved caspase-3 (5A1E) (9664, 1:200 dilution; CST), cleaved PARP (D64E10) (5625, 1:100 dilution; CST), NOXA (114C307) (ab13654, 1:2000 dilution; Abcam), 8-hydroxy-2′-deoxyguanosine (N45.1) (ab48508, 1:500 dilution; Abcam), ARID1A (HPA005456, 1:2000 dilution; Sigma-Aldrich), and anti-SLC7A11 (xCT) (ab175186, 1:400 dilution; Abcam). All IHC staining was performed using a Dako autostainer Link48 (Dako).
Immunohistochemical staining for 8-OHdG was further evaluated by a semiquantitative approach used to assign a histological score (H-score) to tumor samples (See, Hirsch et al., J Clin Oncol 21, 3798-3807, 2003). First, membrane staining intensity (0, 1+, 2+, or 3+) was determined for each cell in a fixed field. The H-score was assigned using the following formula: [1×(% cells 1+)+2×(% cells 2+)+3×(% cells 3+)]. The final score, ranging from 0 to 300, gives more relative weight to higher-intensity membrane staining in a given tumor sample. The percentage of NOXA-, cleaved caspase-3-, cleaved PARP- and Ki67-positive cells (of the total number of cells) in each slide were counted.
Exome libraries were generated using the Agilent SureSelect XT (Agilent, Palo Alto, Calif.) according to the manufacturer's protocol. Prior to sequencing, exome libraries were analyzed using the Agilent BioAnalyzer 2200 with the High Sensitivity D1000 (Agilent) and QuantStudio bFlex (Thermo Fisher Scientific, Rockford, Ill.) with KAPA Library Quantification Kit (IIlumina, San Diego, Calif.). All libraries were sequenced using the Illumina HiSeq 2000/2500 system with TruSeq SBS Kit v3-HS (200-cycles) reagents (Illumina). Sequence data were mapped to the Genome Reference Consortium GRCh37 assembly using BWA-MEM. Somatic variant calling was conducted using GATK4 Mutect2.
Six-week-old female CAnN.Cg-Foxn1nu/CrlCrlj (BALB/c-nu/nu) mice (Charles River Laboratories Japan were bred at room temperature with a 12 h light/dark daily cycle. The mice were maintained under specific pathogen-free conditions and were provided sterile food, water, and cages. Approximately 5×106 cancer cells were suspended with 100 μl phosphate-buffered saline and were injected subcutaneously into mice using a 26.5-gauge needle. All experiments were conducted in accordance with the ethical guidelines of the International Association for the Study of Pain and were approved by the Committee for Ethics in Animal Experimentation of the National Cancer Center. Specimens fixed in formalin and embedded in paraffin were cut into 8 μm sections, which were dewaxed and dehydrated for routine hematoxylin and eosin staining.
Quantification and Statistical Analysis
Statistical analyses were performed using Microsoft Excel. Data are expressed as the mean±SD or mean±SEM, as indicated in the figure legends. The sample size (n) is indicated in the figure legends and represents biological replicates. Statistical significance was evaluated using the two-tailed Student's t-test. Statistically significant differences are indicated by asterisks as follows: *p<0.05, **p<0.01, and ***p<0.001.
It is possible to treat SWI/SNF complex-deficient cancer by administering a glutathione (GSH) metabolic pathway inhibitor, according to the disclosure of the present invention.
This application claims the benefit of the priority under 35 U.S.C. § 119(e) to U.S. Provisional Application Ser. No. 62/785,458 filed on Dec. 27, 2018 and U.S. Provisional Application Ser. No. 62/902,480 filed on Sep. 19, 2019, the disclosure of each one are incorporated herein by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2019/051342 | 12/16/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62785458 | Dec 2018 | US | |
| 62902480 | Sep 2019 | US |
