KR20240035958A - ATF6 as a target protein for diagnosis or treatment of tumor - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating ATF6-overexpressing tumors, including an ATF6 inhibitor. The pharmaceutical composition of the present invention can prevent or treat ATF6-overexpressing tumors by inhibiting the activity of ATF6 and causing rapid cell death. Additionally, the pharmaceutical composition of the present invention can be administered simultaneously or sequentially with a tyrosine kinase inhibitor to more effectively prevent or treat ATF6-overexpressing tumors.

Description

ATF6 as a target protein for diagnosis or treatment of tumor}

The present invention relates to a composition for diagnosis or treatment of tumors overexpressing ATF6.

Gastrointestinal stromal tumor (GIST) occurs when stromal cells present in the muscle layer located in the middle of the wall of the gastrointestinal tract mutate into cancer cells. In general, unlike most cancers that occur in the digestive tract, such as stomach cancer or colon cancer, which originate from epithelial cells, gastrointestinal stromal tumors are cancers that arise from mesenchymal-derived stromal cells of the muscle layer and form gastrointestinal tract tumors such as the esophagus, stomach, small intestine, colon, and rectum. It can occur anywhere, and its cause, location, and metastasis pattern are also different from general stomach cancer and digestive cancer. It is also one of the diseases for which regular checkups are most important because it has no clear symptoms.

The main pathological mechanism of GIST is known to be continuous activation of c-KIT protein due to KIT gene mutation and PDGFRα gene mutation. KIT or PDGFRα gene mutation is found in most GISTs, and among them, KIT gene mutation is very high at 75-80%. reported to be common.

c-KIT (also known as KIT, CD117, or stem cell factor receptor) is a 145 kDa transmembrane tyrosine kinase protein that acts as a type III receptor. The c-KIT gene, located on chromosome 4q11-21, encodes the c-KIT receptor protein with a ligand for stem cell factor (SCF, stem cell growth factor, kit ligand). The c-KIT receptor has tyrosine-protein kinase activity, and binding of the ligand SCF autophosphorylates c-KIT and binds to downstream signaling substances such as PI3K, GRB2, and JAK2, resulting in PI3K-ATK, MAPK-ERK, JAK- It simultaneously activates tumor-inducing signaling systems such as the STAT signaling system. It is known that tyrosine phosphorylation by protein tyrosine kinases is particularly important in cell signaling and can mediate signaling for key cellular processes such as proliferation, survival, differentiation, apoptosis, adhesion, invasiveness, and migration.

The role of c-KIT expression and activity has been studied in hematological and solid tumors such as acute leukemia and GIST. Most GISTs have a primary activating mutation in the gene encoding the RTK, c-KIT (75–80% of GISTs) or PDGFRα (8% of non-c-KIT mutated GISTs). In particular, most GIST patients, accounting for approximately 80%, show protein overexpression and hyperphosphorylation of c-KIT due to a gain-of-function mutation in the c-KIT gene. Most primary c-KIT mutations occur in the juxtamembrane region (JM region) of the protein encoded by exon 11 and consist of in-frame deletions or insertions or missense mutations (i.e. V560D). c-KIT exon11 mutation occurs as a primary mutation in approximately 65% of GIST patients, and this JM domain mutation interferes with the autoinhibition mechanism of c-KIT kinase, automatically activating the kinase causing GIST and causing cell transformation. It causes conversion. Other primary GIST-causing c-KIT mutations are known to be located in exon9 (AY501-502 duplication/insertion, 8%), exon13 (mutation, 1%), and exon17 (mutation, 1%).

The clinical importance of c-KIT expression in malignant tumors has been demonstrated in studies using Gleevec, which specifically inhibits tyrosine kinase receptors. Additionally, a major clinical advance has been the study of Gleevec in GISTs that are generally resistant to conventional chemotherapy. It showed an anti-tumor effect through targeted treatment. However, in the case of Gleevec®, a c-KIT inhibitor, some therapeutic effect can be expected for patients with GIST that has already metastasized or is inoperable, but cure is rare, and approximately 50% of patients develop cancer within 2 years after treatment. Because it recurs, the development of new treatments to overcome it is required.

The mutation site in c-KIT is clinically very important in Gleevec®mesylate (Novartis Pharm.)-based treatment. For patients with exon 11 mutations, the initial Gleevec drug responsiveness is good at 85%, but for patients with exon 9 mutations, it is 45%. %, patients with exon 13 and exon 17 mutations show almost no drug responsiveness, showing significant differences in drug treatment effects depending on the mutation location. Due to these differences in drug responsiveness depending on the mutation location, the limitations of Gleevec-based GIST treatment are currently recognized in clinical practice. As mentioned above, depending on the exon location where the mutation occurred, GIST often does not have drug responsiveness in the first place or shows resistance to Gleevec® during drug treatment. Drug resistance that develops during Gleevec treatment is induced by secondary mutations that occur in the c-KIT gene, and a definitive treatment for secondary mutations has not yet been established. GIST patients who relapse during Gleevec® treatment develop a secondary mutation in addition to the original primary mutation, resulting in a much more aggressive activated form of the c-KIT mutant protein. Secondary mutations are found in the ATP binding pocket (exon13: K642E, V654A; exon14: T670I) and activation loop (exon17: N822K, D816H, D816V, D820A; exon18: A829P), which prevents Gleevec from binding to the ATP binding pocket and causing drug activity. Because these secondary mutants of c-KIT induce drug resistance.

Sutent® (Sunitinib malate, Pfizer) is an inhibitor of multiple RTKs, especially c-KIT in this context, and inhibits Gleevec resistance c-KIT due to secondary mutations (e.g. ATP-binding pocket mutants V654A and T670I). It was developed as a drug that can However, among GIST patients, when secondary mutations occur at positions such as D816H and D816V, which are located in the activation loop of the c-KIT catalytic domain encoded by exon 17, resistance to Sunitinib is shown, and Suniti is used in clinical practice. It has been reported that the overall drug reactivity of the nib is around 10%, which is not showing great results.

Regorafenib, another multi-kinase inhibitor used for Gleevec-resistant GIST patients, also has less than 5% of patients showing treatment response, and even that has severe side effects, making active use difficult. Therefore, neither sunitinib nor regorafenib are realistic alternatives for GISTs resistant to Gleevec.

As mentioned above, the complex multiple secondary c-KIT mutations that can occur within an individual patient not only impair drug responsiveness to Gleevec, but also inhibit the existing method of treating GIST only by controlling the activation of the KIT protein (kinase activation). It shows the limitations of treatment. Gleevec is currently the most reliable treatment method for GIST, but there are problems such as reduced drug responsiveness depending on the location of the primary mutation and drug resistance due to secondary mutations, so research is being conducted on developing new treatments that can overcome these problems. The need is being emphasized. Recently, rather than controlling the activation of the KIT protein, the expression of KIT mRNA or KIT protein itself has been directly controlled using siRNA, microRNA, etc., or indirectly by using factors such as PKC-theta, which are known to regulate the expression of KIT, to control the KIT protein. Methods that control expression are attracting attention as an alternative. In addition, research is being conducted on the development of completely new drug targets by identifying new mechanisms of cancer occurrence and progression that are not yet known in GIST, but no research results suitable for clinical application have been published to date.

Through the present invention, we aim to overcome the limitations of the existing c-KIT target therapy described above by identifying a new mechanism of development of GIST and discovering and controlling new drug targeting factors. In addition, we aim to treat early GIST that shows good drug responsiveness to Gleevec. We aim to offer new treatment options not only to patients but also to patients who have developed resistance to Gleevec. In addition, we aim to develop a treatment that can be used in combination with the existing treatment, Gleevec, and provide a basis for the development of a curative GIST treatment by achieving early and strong cancer suppression.

Korean Patent Publication No. 10-2020-0115607

The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating gastrointestinal stromal tumors.

The purpose of the present invention is to provide a method of providing information for diagnosis or prognosis of gastrointestinal stromal tumors.

1. A pharmaceutical composition for preventing or treating ATF6-overexpressing tumors containing an ATF6 inhibitor.

2. In 1 above, the tumor overexpressing ATF6 is breast cancer, cholangiocarcinoma, colon adenocarcinoma, esophageal cancer, glioblastoma multiforme, acute myeloid leukemia, brain low-grade glioma, liver cancer, pancreatic cancer, pheochromocytoma/paraganglioma, sarcoma, and skin melanoma. , stomach cancer, ovarian cancer, thyroid cancer, thymoma, testicular cancer, rectal cancer, prostate cancer, adrenal cortex cancer, bladder cancer, lymphoma, mesothelioma, lung cancer, endometrial cancer, cervix cancer, kidney cancer, head and neck cancer, gastrointestinal cancer, meningioma, pituitary adenoma, For the prevention or treatment of ATF6-overexpressing tumor, which is at least one selected from the group consisting of acoustic schwannoma, craniopharyngioma, follicular astrocytoma, hemangioblastoma, diffuse astrocytoma, oligodendrogytoma, atypical medullary tumor, ependymoma, and gastrointestinal stromal tumor. Pharmaceutical composition.

3. The pharmaceutical composition for preventing or treating ATF6-overexpressing tumors according to 1 above, wherein the ATF6 inhibitor is at least one selected from the group consisting of PF429242, Ceapin A7, and Melatonin.

4. The pharmaceutical composition for preventing or treating ATF6-overexpressing tumors according to 1 above, further comprising a tyrosine kinase inhibitor.

5. The pharmaceutical composition for preventing or treating ATF6-overexpressing tumors according to item 4 above, wherein the tyrosine kinase inhibitor is at least one selected from the group consisting of imatinib, sunitinib, regorafenib, and ivakit.

6. The pharmaceutical composition for preventing or treating ATF6-overexpressing tumors according to item 4 above, wherein the tyrosine kinase inhibitor is administered simultaneously or sequentially with the ATF6 inhibitor.

7. A method of providing information for diagnosis or prognosis of gastrointestinal stromal tumor, including the step of measuring the level of ATF6 or its mRNA in a sample.

8. The method of providing information for diagnosis or prognosis of gastrointestinal stromal tumor according to item 7 above, wherein the sample is an individual or isolated from an individual.

9. The method of 7 above, further comprising determining that it is a gastrointestinal stromal tumor when the level of ATF6 or its mRNA is higher than the expression level of the normal control group.

10. The method of 7 above, further comprising determining that the higher the level of ATF6 or its mRNA, the worse the prognosis of the gastrointestinal stromal tumor.

11. A composition for diagnosing or predicting prognosis of gastrointestinal stromal tumor containing a substance that measures the level of ATF6 or its mRNA.

12. The composition of 11 above, wherein the substance for measuring the level of ATF6 or its mRNA is selected from the group consisting of primers, probes, and antibodies that specifically bind to ATF6.

13. A screening method for a gastrointestinal stromal tumor treatment candidate comprising the step of selecting a substance that reduces the expression of ATF6 or its mRNA in a sample.

The pharmaceutical composition of the present invention can suppress the activity of ATF6 overexpressed in gastrointestinal stromal tumors and induce rapid cell death by suppressing the expression of downstream chaperone proteins. Accordingly, gastrointestinal stromal tumors can be prevented or treated.

Additionally, the pharmaceutical composition of the present invention can be administered simultaneously or sequentially with a tyrosine kitase inhibitor to more effectively prevent or treat gastrointestinal stromal tumors.

Figure 1 shows the results confirmed in GIST cell lines by WB and fluorescent immunostaining and by IHC in GIST patient tissue, showing that the expression of cATF6 (cleaved ATF6, activated form of ATF6) is elevated in GIST and has moved to the nucleus within cells.
Figure 2 shows ATF6, cATF6, factors involved in ER stress signaling (PERK, IRE1α and their respective phosphorylated forms), CHOP (apoptosis marker), and chaperone proteins (BIP, HSP90) by treating TG (Thapsigargin, which causes ER stress) at different concentrations. , GRP94, and HSP70) expression was confirmed at the protein level. In particular, GIST cells with activated ATF6 can survive by overcoming ER stress when mild ER stress is induced (TG: 0.1 uM).
Figure 3 shows that when GIST cell lines are treated with S1Pi (Serine 1 protease inhibitor), which inhibits cleavage and activation of ATF6, the expression of chaperone proteins (BIP, HSP90, GRP94, HSP70) is suppressed and induced with mild ER stress. It was confirmed that rapid cell death was induced, and the time at which the expression of ATF6-related chaperone protein was increased was confirmed to be delayed due to induction of ER stress.
Figure 4 shows the results of immunoprecipitation using a KIT antibody in GIST cell lines to identify the protein that binds to KIT. It was confirmed that HSP90 protein, a chaperone regulated by ATF6, binds to KIT. In addition, inhibiting ATF6 inhibits the expression of HSP90, a downstream chaperone, and this can concomitantly inhibit mutant KIT, which is one of the causes of GIST. This not only has an anticancer effect through increased sensitivity to ER stress, but also inhibits expression of mutant KIT protein. It can also induce an anticancer effect, suggesting that ATF6 inhibition can induce a strong anticancer effect in GIST through multiple pathways. In addition, it was confirmed that the expression of KIT protein was reduced when treated with ATF6 inhibitor or HSP90 inhibitor.
Figure 5 shows the results confirming that the expression of cleaved ATF6 (active ATF6) was reduced when GIST cell lines were treated with ATF6 inhibitors (S1Pi, Ceapin A7, Melatonin).
Figure 6 shows the case of ATF6 inhibitor (S1Pi, Ceapin A7, Melatonin) independently treated with TG-treated GIST cell lines (GIST882: imatinib-sensitive cell line; GIST48: imatinib-resistant cell line) to simulate the actual tumor microenvironment and ER stress. Cell viability is measured when an inducing agent (Borteazomib, 17AAG) or imatinib and an ATF6 inhibitor are administered simultaneously. The ATF6 inhibitor shows excellent GIST inhibition ability even when treated independently, and the ER stress-inducing agent also independently inhibits GIST. This result confirms that GIST can be inhibited more efficiently when treated with an ATF6 inhibitor and an ER stress-inducing substance.
Figure 7 shows cell viability when GIST cell lines without TG were independently treated with ATF6 inhibitors (S1Pi, Ceapin A7, Melatonin) and when ER stress-inducing substances (Borteazomib, 17AAG) or imatinib and ATF6 inhibitors were simultaneously administered. As a measure of cell viability, ATF6 inhibitors show excellent GIST inhibition ability even when treated independently, and ER stress-inducing substances can also independently inhibit GIST, and are more efficient when ATF6 inhibitors and ER stress-inducing substances are treated together. This is a result confirming that GIST is suppressed.
Figure 8 is a survival function graph showing the survival probability of the patient group expressing ATF6 and the patient group not expressing ATF6. This result confirms that the survival rate is low in the patient group expressing ATF6.

Hereinafter, the present invention will be described in detail.

The present invention can provide a pharmaceutical composition for preventing or treating ATF6-overexpressing tumors comprising an ATF6 inhibitor:

ATF6 inhibitor inhibits the activation process in which ATF6 located in the C-terminal region, i.e. the cytoplasm, moves into the nucleus by cutting the region connecting the C-terminal region and N-terminal region of ATF6, thus inhibiting the mechanism by which ATF6 is activated. Examples of the substance may be Serine 1 protease inhibitor (S1Pi), PF429242, Ceapin A7, and Melatonin, but are not limited thereto.

ATF6-overexpressing tumors may be tumors in which the mRNA or protein expression of ATF6 or cleaved ATF6 (cATF6), an activated form of ATF6, is increased compared to normal tissues or normal cells. ATF6-overexpressing tumors include, for example, breast cancer, cholangiocarcinoma, and colon adenocarcinoma. , esophageal cancer, glioblastoma multiforme, acute myeloid leukemia, brain low-grade glioma, liver cancer, pancreatic cancer, pheochromocytoma/paraganglioma, sarcoma, skin melanoma, stomach cancer, ovarian cancer, thyroid cancer, thymoma, testicular cancer, rectal cancer, prostate cancer, adrenal gland. Cortical cancer, bladder cancer, lymphoma, mesothelioma, lung cancer, endometrial cancer, cervical cancer, kidney cancer, head and neck cancer, gastrointestinal cancer, meningioma, pituitary adenoma, acoustic schwannoma, craniopharyngioma, follicular astrocytoma, hemangioblastoma, diffuse astrocytoma , oligodendrogytoma, atypical melanoma, ependymoma, or gastrointestinal stromal tumor, but is not limited thereto.

The gastrointestinal stromal tumor is a rare disease caused by mutations in cells that regulate muscle contraction and relaxation located in the muscle layer of the gastrointestinal tract wall, that is, CAR cells, and is called GIST (Gastrointestinal stromal tumor).

The term “prevention” refers to any action that inhibits or delays gastrointestinal stromal tumors.

The term “treatment” refers to any action that improves or beneficially changes the symptoms of a suspected or affected gastrointestinal stromal tumor.

The pharmaceutical composition of the present invention may be in the form of capsules, tablets, granules, injections, ointments, powders or beverages.

The pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, capsules, tablets, and aqueous suspensions, external preparations, suppositories, and injections.

The pharmaceutical composition of the present invention may contain the active ingredient alone, or may further include one or more pharmaceutically acceptable carriers, excipients, or diluents.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavorings, etc. for oral administration, and buffers, preservatives, analgesics, etc. for injections. Solubilizers, isotonic agents, stabilizers, etc. can be mixed and used, and for topical administration, bases, excipients, lubricants, preservatives, etc. can be used.

The formulation of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with a pharmaceutically acceptable carrier. For example, for oral administration, it can be used in tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. It can be manufactured in the form of an injection, and in the case of an injection, it can be manufactured in the form of unit dosage ampoules or multiple dosage forms. Additionally, the dosage form of the pharmaceutical composition of the present invention may be prepared as a solution, suspension, tablet, capsule, sustained-release preparation, etc.

Carriers, excipients and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, It may be microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, filler, anti-coagulant, lubricant, wetting agent, fragrance, emulsifier or preservative.

The route of administration of the pharmaceutical composition of the present invention may be oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal. Not limited.

The present invention The pharmaceutical composition of the present invention can be administered orally or parenterally, and when administered parenterally, the injection method may be selected for external application through the skin or intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection. there is.

The dosage of the pharmaceutical composition of the present invention varies depending on the patient's condition and weight, degree of disease, drug form, administration route and period, but can be appropriately selected by a person skilled in the art.

For example, the pharmaceutical composition of the present invention may be administered at 0.0001 mg to 1000 mg/kg or 0.001 mg to 500 mg/kg per day. The pharmaceutical composition of the present invention may be administered once a day or may be administered several times. The above dosage does not limit the scope of the present invention in any way.

In addition, the pharmaceutical composition of the present invention can provide a pharmaceutical composition for preventing or treating ATF6-overexpressing tumors, further comprising a tyrosine kinase inhibitor:

The tyrosine kinase inhibitor refers to a substance that blocks the amplification and transmission of growth signals by inhibiting the tyrosine kinase at the terminal of the growth factor receptor. For example, it may be imatinib, sunitinib, regorafenib, ivakit, etc. However, it is not limited to this.

The tyrosine kinase inhibitor may be administered simultaneously or sequentially with the pharmaceutical composition of the present invention, and the order of administration is not limited.

The present invention can provide a method of providing information for diagnosis or prognosis of gastrointestinal stromal tumor, including the step of measuring the level of ATF6 or its mRNA in a sample:

The sample may be an individual or separated from an individual, and the type of sample may be, for example, tissue, cell, whole blood, plasma, serum, blood, etc., but is not limited thereto.

The above entity refers to all animals such as rats, mice, and livestock, including humans, that have or may develop gastrointestinal stromal tumors. As a specific example, it may be a mammal, including humans.

The term “diagnosis” refers to determining an individual's susceptibility to a specific disease or condition, determining whether an individual currently has a specific disease or condition, and prognosis of an individual suffering from a specific disease or condition. determining prognosis, or therametrics (e.g., monitoring the condition of an individual to provide information about treatment efficacy).

By detecting ATF6 mRNA or protein, the expression level of ATF6 mRNA or protein in the sample can be measured. Substances that detect ATF6 may be, for example, ATF6-specific primers, probes, or antibodies, but are not limited thereto.

ATF6 detection can be performed by methods known in the art using the above-described detecting substances, for example, polymerase chain reaction, Western blotting, immunostaining, immunoelectrophoresis, protein immunoprecipitation, and enzyme-linked immunoprecipitation assay. , can be detected using liquid chromatography, etc., but is not limited thereto.

Hereinafter, the present invention will be described in detail by examples. The following examples illustrate the present invention, and the content of the present invention is not limited to the following examples.

Example

1. Experimental method

(1) Cell culture

For GIST430 and GIST430(V654A) cell lines, Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 15% fetal calf serum and 1% penicillin/streptomycin was used, and for GIST882 cell lines, Roswell Park Memorial Institute (RPMI) was used. ) Medium mixed with 15% fetal bovine serum (FBS) and 1% penicillin/streptomycin, GIST48 cell line mixed with 15% FBS and 1% penicillin/streptomycin in IMDM medium at 37°C, 5% CO ₂ It was cultured under these conditions. Colon cancer cell lines Colo320DM, DLD-1, LS174T, H69, H128, and H209 used RPMI medium mixed with 10% FBS and 1% penicillin/streptomycin, and Kasumi-1 cell line used RPMI medium mixed with 20% FBS and 1% penicillin/streptomycin. 1% penicillin/streptomycin mixed medium, HMC-1, and K562 cell lines were cultured in IMDM medium mixed with 10% FBS and 1% penicillin/streptomycin at 37°C and 5% CO ₂ conditions.

(2) Western blot

To perform Western blotting, cells were lysed using a passive lysis buffer containing protease inhibitors, and the lysate was obtained and subjected to electrophoresis (using premade gel, Invitrogen) and then transferred to a membrane (iBlot2 system). Afterwards, block with nonfat dried milk or 5% BSA solution and bind with primary antibody at 4°C overnight. After washing, the secondary antibody is allowed to bind at room temperature for 1 hour, then washed sufficiently to remove any remaining antibodies, and the image is analyzed using the LAS4000 system. Antibodies used were c-KIT (DAKO), ATF6 (Abcam), GAPDH (Trevigen), Phospho-IRE1alpha (Invitrogen), IRE1alpha, phosphor-PERK, PERK, CHOP, HSP70, HSP90, GRP94, BIP, LC3 (Cell signaling )am.

(3) Immunocytochemical staining

Cells cultured in an imaging dish are washed and fixed with 4% paraformaldehyde or 100% methanol. Afterwards, cell membrane permeation is induced with 0.2% Triton X-100 and the primary antibody is incubated overnight at 4 degrees. After incubating the secondary antibody at room temperature for 1 hour, wash thoroughly, stain with DAPI (4',6-diamidino-2-phenylindole), and perform imaging using a confocal microscope (Carl Zeiss, LSM710).

(4) Immunohistochemical staining

Paraffin-embedded formalin-fixed tissue blocks were sectioned at 4-μm thickness and immunohistochemical staining was performed using a Ventana XT automated stainer. The expression level of ATF6 was quantified by two experienced pathologists independently reading the nuclear expression level.

(5) Statistical analysis

Disease-free survival according to nuclear expression of ATF6 was analyzed using SPSS software, and Mann-Whitney tests, Student's t-tests, Fisher's exact tests, or chi-square tests were used. Statistical analysis of MTT assay results was performed using one-way analysis of variance (ANOVA) with a post hoc test (Bonferroni) method, and data were presented including standard error bars.

(6) Cell growth analysis assay

Each cell line was seeded in a 96-well plate at 10 ⁴ cells/well, and 24 hours later, it was pretreated with ATF6 inhibitors PF429242, Ceapin A7, and Melatonin at each concentration for 12 hours. Afterwards, the medium was replaced with a medium containing each ATF6 inhibitor and Thapsigargin 0.1 μM, Bortezomib 1 nM, 17AAG 0.5 μM, or Imatinib 0.1 μM. After culturing in a 5% CO ₂ incubator at 37°C for 72 hours, MTT ([3-(4,5-dimethylthiazol-2-yl-) 2,5-diphenyltetrazolium bromide) assay was performed to measure the number of viable cells. The MTT solution was added to each well to a final concentration of 0.5 mg/ml in the culture medium, and then cultured in a 5% CO ₂ incubator at 37°C for 4 hours. After incubation, the solution was completely removed, and 100 μl of DMSO was added and incubated at room temperature for 20 minutes to lyse the cells and at the same time dissolve the purple MTT metabolite (crystalline) produced by living cells. The amount of MTT metabolite dissolved in DMSO was measured by absorbance at 570 nm using an ELISA Plate Reader.

2. Experimental results

(1) Confirmation of ATF6 overexpression in GIST

In several carcinomas expressing normal or mutant KIT (small cell lung cancer: H69, H128; leukemia: H209, Kasumi-1, HMC-1; colorectal cancer: K562, LS174T, Colo320DM, DLD-1, GIST: GIST430, GIST882) ATF6 was not specifically overexpressed in GIST, but cleaved ATF6 (cATF6), an activated form of ATF6, was found to have a specific higher expression in GIST cell lines compared to other carcinomas (Figure 1A).

ATF6 is composed of a c-terminal part (endoplasmic reticulum lumen) and an N-terminal part (cytoplasm). After ATF6 moves to the Golgi apparatus, the N-terminal part is cut and enters the nucleus, where it knocks down genes related to cell survival, death, and growth. It is known to prevent cancer cell death. ATF6 can be considered active only when it moves to the nucleus in the form of cleaved ATF6 (cATF6), and it was also confirmed by confocal fluorescence microscopy that ATF6 is located within the nucleus of the GIST cell line (Figure 1B)

It was also confirmed through tissue immunostaining results that ATF6 was distributed and overexpressed in the nucleus in GIST patients (Figure 1C).

(2) Confirmation of expression of apoptosis-related proteins and chaperone proteins due to ER stress in GIST cell lines

When mild to strong ER stress was induced by treatment with Thapsigargin (TG, ER stress inducer), cell death did not significantly increase in the case of 0.1 uM of mild ER stress, but cell death increased significantly in the case of 5 uM of strong ER stress ( Figure 2A).

When strong ER stress was induced, the expression of ATF6 and cATF6 was decreased, and CHOP, a cell death marker, was confirmed to increase, which means that GIST cells cannot withstand strong ER stress. On the other hand, the expression of the activated form of PERK and IRE1a (p-PERK, p-IRE1a), which is another pathway of ER stress and is known to be involved in cell death, was found to increase and induce cell death (Figure 2B).

When mild ER stress was induced, the expression of ATF6 and cATF6 decreased slightly in the beginning, but eventually the mild ER stress was overcome after 24 hours, and CHOP, a cell death marker, also increased and then decreased. These results mean that when ATF6 is activated, it can inhibit cancer cell death caused by ER stress. In particular, it was confirmed that ATF6 activates a defense mechanism (unfolded protein response) to prevent cancer cell death by increasing the expression of chaperone proteins such as BIP, HSP90, and GRP94 (Figure 2C) .

(3) Confirmation of the effect on ER stress and misfolded protein response mechanisms due to inhibition of ATF6 activation

Serine 1 protease (S1P) inhibitor (S1Pi) inhibits the cleavage and activation of ATF6. When treated with GIST cells, inhibition of ATF6 activation (inhibition of cATF6 expression) and expression of downstream chaperones are observed (Figure 3A).

When mild ER stress was induced by treating with TG at 0.1 uM, little cell death generally occurred, but when treated with S1Pi, rapid cell death occurred even under mild ER stress (Figure 3B).

When SP1i is treated with TG, the timing of the increase in expression of ATF6 pathway dependent chaperones such as BIP, GRP94, and HSP90 due to ER stress induction is significantly delayed. This means that inhibiting the ATF6 pathway allows cancer cells to respond appropriately to ER stress. This means that the defense mechanism cannot operate quickly, leading to cell death (Figure 3C).

(4) Inhibition of HSP90 expression and KIT expression following ATF6 inhibition

To identify chaperone proteins that bind to KIT, which plays an important role in GIST development among the chaperone proteins regulated by ATF6, immunoprecipitation was performed using a KIT antibody on GIST cell lines. As a result, HSP90, a chaperone regulated by ATF6, was found. It was confirmed that was binding to the mutant KIT protein (Figure 4). These results indirectly show that when the expression of chaperones located in the downstream signaling system is suppressed by inhibiting ATF6, the mutant KIT protein, which is one of the causes of GIST, can also be suppressed concomitantly, leading to a strong anticancer effect. am. In fact, when ATF6 was suppressed and the resulting changes in the expression levels of HSP90 and KIT were checked, it was confirmed that the expression of KIT and HSP90 was suppressed. In addition, it was confirmed that the expression of KIT protein was reduced even when HSP90 was inhibited by treatment with 17AAG, an inhibitor of HSP90 (Figure 5). Considering these results, when ATF6, which regulates HSP90, is inhibited, HSP90 is inhibited and Accordingly, it is concluded that it is reasonable to suppress the expression of KIT protein.

(5) Confirmation of the effect of combined administration of ATF6 inhibitor and imatinib in GIST cell lines

A cell growth evaluation assay (MTT assay) was performed using GIST430, a cell line resistant to imatinib, and GISTS430 (V654A), an imatinib-resistant cell line with an additional V654A secondary mutation in GIST430 cells.

In the tumor microenvironment, ER stress is induced depending on constraints such as lack of nutrients and oxygen. Thapsigargin (TG) is treated to simulate an ER stress environment similar to that in vivo. In the present invention, when treated (FIG. 6), To confirm the effect of the ATF6 inhibitor itself, the experiment was divided into the untreated case (Figure 7).

As a result, it was confirmed that treatment with ATF6 inhibitors (PF429242, CeapinA7, Melatonin) strongly induced apoptosis in both cases with or without TG treatment, and even when only ER stress-inducing substances (Bortezomib, 17AAG) were administered, strong cell death was observed. Death occurred, and when the ATF6 inhibitor and ER stress-inducing substance were administered simultaneously, strong tumor suppressive synergy was confirmed (Figures 6 and 7). In particular, these results were confirmed in both cell lines that were either responsive or resistant to imatinib, showing that a treatment approach that inhibits ATF6 could be a new treatment option for patient groups that are resistant to imatinib, and is an aspect of combination therapy. Using imatinib and an ATF6 inhibitor as a combination treatment will assist and improve the effect of imatinib treatment, making it possible to develop a curative treatment through strong tumor suppression in the early stages of treatment.

(6) Confirmation of correlation between ATF6 expression and survival probability

Tissues obtained from 50 GIST patients were analyzed for the degree of nuclear expression of ATF6 through immunohistochemical staining using an ATF6 antibody, and statistical analysis of disease-free survival according to the presence or absence of expression showed that the stronger the nuclear expression of ATF6, the longer the disease-free survival. It was confirmed that there was a tendency for it to become shorter.

Claims (13)

A pharmaceutical composition for preventing or treating ATF6-overexpressing tumors, comprising an ATF6 inhibitor. The method of claim 1, wherein the ATF6-overexpressing tumor is breast cancer, cholangiocarcinoma, colorectal adenocarcinoma, esophageal cancer, glioblastoma multiforme, acute myeloid leukemia, brain low-grade glioma, liver cancer, pancreatic cancer, pheochromocytoma/paraganglioma, sarcoma, skin melanoma, and stomach cancer. , ovarian cancer, thyroid cancer, thymoma, testicular cancer, rectal cancer, prostate cancer, adrenal cortex cancer, bladder cancer, lymphoma, mesothelioma, lung cancer, endometrial cancer, cervical cancer, kidney cancer, head and neck cancer, gastrointestinal cancer, meningioma, pituitary adenoma, acoustic neuroma. A pharmaceutical composition for preventing or treating ATF6-overexpressing tumors, which is at least one selected from the group consisting of craniopharyngioma, follicular astrocytoma, hemangioblastoma, diffuse astrocytoma, oligodendrogytoma, atypical melanoma, ependymoma, and gastrointestinal stromal tumor. . The pharmaceutical composition for preventing or treating ATF6-overexpressing tumors according to claim 1, wherein the ATF6 inhibitor is at least one selected from the group consisting of PF429242, Ceapin A7, and Melatonin. The pharmaceutical composition for preventing or treating ATF6-overexpressing tumors according to claim 1, further comprising a tyrosine kinase inhibitor. The method of claim 4, wherein the tyrosine kinase inhibitor is imatinib, sunitinib, regorafenib, ivakit, afatinib, axitinib, bostinib, cabozantinib, cetuximab, ceritinib, crizotinib, Dabrafenib, dasatinib, erlotinib, everolimus, gefitinib, ibrutinib, idelalisib, lapatinib, lenvatinib, nilotinib, nintedanib, pazopanib, ponatinib, palvo A pharmaceutical composition for preventing or treating ATF6-overexpressing tumors, which is at least one selected from the group consisting of ciclib, ruxolitinib, sorafenib, trastuzumab, trametinib, tofacitinib, vandetanib, and vemurafenib. The pharmaceutical composition for preventing or treating ATF6-overexpressing tumors according to claim 4, wherein the tyrosine kinase inhibitor is administered simultaneously or sequentially with the ATF6 inhibitor. A method of providing information for diagnosis or prognosis of gastrointestinal stromal tumor, comprising measuring the level of ATF6 or its mRNA in a sample. The method of claim 7, wherein the sample is an individual or isolated from an individual. The method of claim 7, further comprising determining that the gastrointestinal stromal tumor is a gastrointestinal stromal tumor when the level of ATF6 or its mRNA is higher than the expression level of a normal control group. The method of claim 7, further comprising determining that the higher the level of ATF6 or its mRNA, the worse the prognosis of the gastrointestinal stromal tumor. A composition for diagnosing or predicting prognosis of gastrointestinal stromal tumor, comprising a substance that measures the level of ATF6 or its mRNA. The composition for diagnosing or predicting prognosis of gastrointestinal stromal tumor according to claim 11, wherein the substance for measuring the level of ATF6 or its mRNA is selected from the group consisting of primers, probes, and antibodies that specifically bind to ATF6. A screening method for a candidate substance for treating gastrointestinal stromal tumor, comprising the step of selecting a substance that reduces the expression of ATF6 or its mRNA in a sample.