WO2022177402A1 - Atf6 as target protein for diagnosing and treating tumor - Google Patents

Atf6 as target protein for diagnosing and treating tumor Download PDF

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WO2022177402A1
WO2022177402A1 PCT/KR2022/002563 KR2022002563W WO2022177402A1 WO 2022177402 A1 WO2022177402 A1 WO 2022177402A1 KR 2022002563 W KR2022002563 W KR 2022002563W WO 2022177402 A1 WO2022177402 A1 WO 2022177402A1
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atf6
cancer
pharmaceutical composition
gastrointestinal stromal
inhibitor
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French (fr)
Korean (ko)
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조수연
권유진
김원규
최요준
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(주)이노셀젠
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    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
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Definitions

  • the present invention relates to a diagnostic composition or a therapeutic pharmaceutical composition for ATF6 overexpressing tumors.
  • Gastrointestinal stromal tumor is caused by mutation of stromal cells present in the muscle layer located in the middle of the wall of the gastrointestinal tract into cancer cells.
  • gastrointestinal stromal tumors are cancers that arise from mesenchymal-derived stromal cells in the muscle layer. It can occur anywhere, and the cause, location, and metastasis pattern are also different from general gastric and digestive cancers.
  • regular check-ups are one of the most important diseases.
  • KIT or PDGFR ⁇ gene mutations are found in most GISTs, and among them, KIT gene mutations account for 75-80% of the time. reported to be common.
  • c-KIT also known as KIT, CD117, or stem cell factor receptor
  • KIT a transmembrane tyrosine kinase protein that acts as a type III receptor.
  • the c-KIT gene located on chromosome 4q11-21 encodes a 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 sub-signaling substances such as PI3K, GRB2, and JAK2, resulting in PI3K-ATK, MAPK-ERK, JAK- Simultaneously activates oncogenic signaling pathways such as STAT signaling.
  • Tyrosine phosphorylation by protein tyrosine kinases is particularly important in cellular signal transduction and is known to mediate signaling for key cellular processes such as proliferation, survival, differentiation, apoptosis, adhesion, invasiveness and migration.
  • the primary c-KIT mutations occur in the juxtamembrane region (JM region) of the protein encoded by exon11, and consist of in-frame deletions or insertions, and missense mutations (ie, V560D).
  • JM region juxtamembrane region
  • missense mutations ie, V560D.
  • the c-KIT exon11 mutation appears as a primary mutation in about 65% of GIST patients, and this JM domain mutation interferes with the self-suppression mechanism of c-KIT kinase, automatically activating the GIST causative kinase and transforming the cell. induce conversion.
  • Other primary GIST-induced c-KIT mutations are known to be located in exon9 (AY501-502 replication/insertion, 8%), exon13 (mutant, 1%) and exon17 (mutant, 1%).
  • c-KIT expression has been demonstrated in studies using Gleevec, which specifically inhibits the tyrosine kinase receptor. Furthermore, a major clinical advance is the GIST, in which Gleevec is generally resistant to conventional chemotherapy. It showed antitumor effect through targeted therapy.
  • Gleevec® a c-KIT inhibitor, although some therapeutic effect can be expected for GIST patients who have metastasized or cannot be operated on, a cure is rare, and about 50% of patients have cancer within 2 years of treatment. Because it recurs, there is a need to develop a new therapeutic agent that can overcome it.
  • GIST patients who relapse during the course of Gleevec® treatment have a much more aggressive activated form of c-KIT mutant protein due to secondary mutation in addition to the primary mutation they had. Secondary mutations are found in the ATP binding pocket (exon13: K642E, V654A; exon14: T670I) and activation loop (exon17: N822K, D816H, D816V, D820A; exon18: A829P), where Gleevec binds to the ATP binding pocket.
  • An object of the present invention is to provide a pharmaceutical composition for preventing or treating gastrointestinal stromal tumor.
  • a composition for diagnosis or prognosis of gastrointestinal stromal tumor comprising a substance measuring the level of ATF6 or its mRNA.
  • composition for diagnosis or prognosis of gastrointestinal stromal tumor according to 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.
  • the pharmaceutical composition of the present invention inhibits the activity of ATF6 overexpressed in gastrointestinal stromal tumors, thereby suppressing the expression of lower chaperone proteins, thereby inducing rapid apoptosis. Accordingly, it is possible to prevent or treat gastrointestinal stromal tumors.
  • composition of the present invention may be administered simultaneously or sequentially with the tyrosine ketase inhibitor to more effectively prevent or treat gastrointestinal stromal tumors.
  • cATF6 (cleaved ATF6, ATF6 activated form) is elevated in GIST, and that it migrates to the nucleus within cells in the GIST cell line by WB and fluorescence immunostaining and by IHC in the GIST patient tissue.
  • ATF6 inhibitors S1Pi, Ceapin A7, Melatonin
  • GIST882 imatinib-sensitive cell line
  • GIST48 imatinib-resistant cell line
  • Cell viability was measured when an inducer (Borteazomib, 17AAG) or imatinib and an ATF6 inhibitor were administered simultaneously, and the ATF6 inhibitor showed excellent GIST inhibitory ability even when treated independently, and ER stress-inducing substances were also independently GIST It is the result of confirming that GIST more efficiently when treated with an ATF6 inhibitor and an ER stress-inducing substance.
  • FIG. 8 is a survival function graph showing the survival probability of a patient group expressing ATF6 and a patient group not expressing ATF6. In the case of a patient group expressing ATF6, it is confirmed that the survival rate is low.
  • ATF6 inhibitor inhibits the activation process in which ATF6 located in the C-terminal region, that is, the cytoplasm, moves into the nucleus by cutting the region connecting the C-terminal region and N-terminal region of ATF6, and inhibits the mechanism of ATF6 activation
  • Serine 1 protease inhibitor S1Pi
  • PF429242 PF429242
  • Ceapin A7 PF429242
  • Melatonin a substance to be used, for example, Serine 1 protease inhibitor (S1Pi), PF429242, Ceapin A7 and Melatonin, but may be, but is not limited thereto.
  • ATF6 overexpressing tumor may be a tumor in which mRNA or protein expression of ATF6 or cleaved ATF6 (cATF6), an activated form of ATF6, is increased compared to normal tissue or normal cells, and ATF6 overexpressing tumor is, for example, breast cancer, cholangiocarcinoma, and colorectal adenocarcinoma , esophageal cancer, glioblastoma multiforme, acute myeloid leukemia, brain low-grade glioma, liver cancer, pancreatic cancer, pheochromocytoma/paraganglioma, sarcoma, skin melanoma, gastric cancer, ovarian cancer, thyroid cancer, thymoma, testicular cancer, rectal cancer, prostate cancer, adrenal cancer Cortical cancer, bladder cancer, lymphoma, mesothelioma, lung cancer, endometrial cancer, cervical cancer, kidney cancer, head and neck cancer, gastrointestinal cancer, meningi
  • prevention refers to any action that inhibits or delays gastrointestinal stromal tumors.
  • treatment refers to any action in which the symptoms of a subject suspected of and having a gastrointestinal stromal tumor are improved or beneficially changed.
  • the pharmaceutical composition of the present invention may be in the form of a capsule, tablet, granule, injection, ointment, powder or beverage.
  • the pharmaceutical composition of the present invention may 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 include an 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 be binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents, flavoring agents, etc., in the case of oral administration, and in the case of injections, buffers, preservatives, analgesics, Solubilizers, isotonic agents, stabilizers, etc. can be mixed and used, and for topical administration, bases, excipients, lubricants, preservatives, etc. can be used.
  • the dosage form of the pharmaceutical composition of the present invention may be prepared in various ways by mixing with a pharmaceutically acceptable carrier, for example, tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., when administered orally. It may be prepared in the form of injections, and in the case of injections, it may be prepared in the form of unit dose ampoules or multiple doses. In addition, the dosage form of the pharmaceutical composition of the present invention may be prepared as a solution, suspension, tablet, capsule, sustained release formulation, and the like.
  • 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, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, filler, anticoagulant, lubricant, wetting agent, flavoring, emulsifying agent or preservative and the like.
  • 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 pharmaceutical composition of the present invention may be administered orally or parenterally, and when administered parenterally, external or intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection may be selected. have.
  • the dosage of the pharmaceutical composition of the present invention varies depending on the condition and weight of the patient, the severity of the disease, the drug form, the route of administration, and the duration, but may be appropriately selected by those skilled in the art.
  • the pharmaceutical composition of the present invention may be administered in an amount of 0.0001 mg to 1000 mg/kg or 0.001 mg to 500 mg/kg per day.
  • Administration of the pharmaceutical composition of the present invention may be administered once a day, may be administered several times divided. The above dosage does not limit the scope of the present invention in any way.
  • composition of the present invention may 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 amplification and transmission of a growth signal by inhibiting tyrosine kinase at the terminal of a growth factor receptor, for example, imatinib, sunitinib, regorafenib, ivakit, etc.
  • a growth factor receptor for example, imatinib, sunitinib, regorafenib, ivakit, etc.
  • the present invention is not limited thereto.
  • 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 may provide an information providing method for diagnosis or prognosis of gastrointestinal stromal tumor, comprising measuring the level of ATF6 or its mRNA in a sample:
  • the sample may be an individual or one isolated from an individual, and the type of the sample may be, for example, tissue, cell, whole blood, plasma, serum, blood, etc., but is not limited thereto.
  • the subject means all animals, such as rats, mice, livestock, including humans, that have or can develop gastrointestinal stromal tumors. As a specific example, it may be a mammal including a human.
  • diagnosis refers to determining the susceptibility of an individual to a particular disease or condition, determining whether an individual currently has a particular disease or disorder, and the prognosis of an individual afflicted with a particular disease or condition. (prognosis), or therametrics (eg, monitoring a subject's condition to provide information about the efficacy of treatment).
  • the expression level of ATF6 mRNA or protein in the sample may be determined.
  • the substance for detecting ATF6 may be, for example, an ATF6-specific primer, probe, or antibody, but is not limited thereto.
  • ATF6 detection can be performed by a method known in the art using the detecting substance, for example, polymerase chain reaction, Western blotting, immunostaining, immunoelectrophoresis, protein immunoprecipitation, enzyme-linked immunoprecipitation assay , may be detected using liquid chromatography, etc., but is not limited thereto.
  • the detecting substance for example, polymerase chain reaction, Western blotting, immunostaining, immunoelectrophoresis, protein immunoprecipitation, enzyme-linked immunoprecipitation assay , may be detected using liquid chromatography, etc., but is not limited thereto.
  • GIST430 and GIST430 (V654A) cell lines were mixed with Iscove's Modified Dulbecco's Medium (IMDM) with 15% supplemented with 15% fetal calf serum and 1% penicillin/streptomycin.
  • the GIST882 cell line was used at Roswell Park Memorial Institute (RPMI). )
  • the GIST48 cell line is an IMDM medium mixed with 15% FBS and 1% penicillin/streptomycin at 37 °C, 5% CO2 condition.
  • RPMI medium mixed with 10% FBS and 1% penicillin/streptomycin was used, and Kasumi-1 cell line was RPMI medium with 20% FBS and 20% FBS.
  • HMC-1 and K562 cell lines were cultured at 37 °C and 5% CO2 by mixing 10% FBS and 1% penicillin/streptomycin in IMDM medium.
  • the cells were lysed using a passive lysis buffer containing a protease inhibitor, and then the lysate was obtained and electrophoresed (premade gel used, Invitrogen) and transferred to a membrane (iBlot2 system). After blocking with nonfat dried milk or 5% BSA solution, the primary antibody and binding at 4 °C overnight. After washing, the secondary antibody is allowed to bind at room temperature for 1 hour, and then the residual antibody is removed by washing sufficiently and the image is analyzed using the LAS4000 system.
  • the antibodies used are c-KIT (DAKO), ATF6 (Abcam), GAPDH ( Trevigen), Phospho-IRE1alpha (Invitrogen), IRE1alpha, phosphor-PERK, PERK, CHOP, HSP70, HSP90, GRP94, BIP, LC3 (Cell signaling).
  • Paraffin-embedded formalin-fixed tissue blocks were sectioned to a thickness of 4- ⁇ m and immunohistochemical staining was performed using a Ventana XT automated stainer.
  • the expression level of ATF6 was quantified by independently reading the nuclear expression level by two experienced pathologists.
  • Each cell line was seeded in a 96-well plate at a concentration of 10 4 cells/well, and after 24 hours, ATF6 inhibitors PF429242, Ceapin A7, and Melatonin were pretreated at each concentration for 12 hours. Afterwards, each ATF6 inhibitor was exchanged with a medium containing 0.1 ⁇ M of Thapsigargin or 1 nM of Bortezomib or 0.5 ⁇ M of 17AAG or 0.1 ⁇ M of Imatinib.
  • MTT [3-(4,5-dimethylthiazol-2-yl-) 2,5-diphenyltetrazolium bromide) assay was performed to measure the number of viable cells after 72 hours of incubation in a 37 °C, 5% CO2 incubator. MTT solution was added to each well to have a final concentration of 0.5 mg/ml in the culture medium, and then incubated at 37 ° C., 5% CO2 incubator for 4 hours. After incubation, the solution was completely removed, and 100 ⁇ l 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) generated by the living cells. The amount of MTT metabolite dissolved in DMSO was measured for absorbance at 570 nm with an ELISA plate reader.
  • mice 7-week-old BALB/c-nude male mice were used, and the xenograft model was created by subcutaneous injection of imatinib-responsive GIST430 cells and imatinib-resistant GIST430-V654A cell lines.
  • 1 x 10 7 cells were diluted in 50 ul IMDM medium, mixed with Matrigel 1:1, and injected into the right flank of a mouse under respiratory anesthesia, the tumor size, weight was measured.
  • mice were randomly selected per group with tumors growing to 70-100 mm 3 or more, and the drug was administered by intraperitoneal injection.
  • mice After 14 to 16 days of drug administration, sacrifices were made at an appropriate time, and the tumor-suppressing efficacy of the administered drug was verified by measuring the mouse weight, tumor size, and tumor weight.
  • the concentration of the drug was 50 mg/kg for imatinib (conversion based on 20 g mouse: 1 mg/20 g mouse), 30 mg/kg for PF-429242 (0.6 mg/20 g mouse), and 1 mg/kg (0.02 mg/20 g for bortezomib), respectively. mice).
  • carcinomas expressing normal KIT 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 in the case of cleaved ATF6 (cATF6), an activated form of ATF6, it was confirmed that the expression was specifically high in GIST cell lines compared to other carcinomas (FIG. 1A).
  • ATF6 is composed of a c-terminal part (endoplasmic reticulum lumen) and an N-terminal part (cytoplasm). After ATF6 moves to the Golgi body, the N-terminal part is cut off and enters the nucleus, resulting in the disruption of genes related to cell survival, death and growth. It is known to prevent cancer cell death. ATF6 can be seen to be activated only when it migrates to the nucleus in the form of cleaved ATF6 (cATF6), and it was confirmed by confocal fluorescence microscopy that ATF6 is located in the nucleus of the GIST cell line (Fig. 1B).
  • cATF6 cleaved ATF6
  • Serine 1 protease (S1P) inhibitor inhibits the cleavage and activation of ATF6, and when it is treated in GIST cells, inhibition of ATF6 activation (inhibition of cATF6 expression) and inhibition of sub-chaperone expression are observed (FIG. 3A).
  • S1Pi Serine 1 protease inhibitor
  • FIG. 3A When TG was treated with 0.1 uM to induce mild ER stress, apoptosis was generally not achieved, but when treated with S1Pi, rapid cell death was observed even under mild ER stress (FIG. 3B).
  • TMTT assay Cell growth evaluation assay was performed using the imatinib-resistant cell line GIST430 (V654A), an imatinib-resistant cell line with additional V654A secondary mutation in GIST430 and GIST430 cells, which are resistant to imatinib.
  • GIST430 V654A
  • ER stress is induced by constraints such as lack of nutrients and oxygen.
  • Thapsigargin (TG) is treated to simulate an ER stress environment similar to in vivo, and in the present invention, when treated (FIG. 6) and In order to check the effect of the ATF6 inhibitor itself, the experiment was divided into untreated cases (FIG. 7).
  • imatinib and ATF6 inhibitors are used as a combination treatment, it will be possible to develop a curative treatment with strong tumor suppression ability at the early stage of treatment by supporting and improving the effect of imatinib treatment.
  • the degree of nuclear expression of ATF6 was analyzed through chemical staining, and as a result of statistical analysis of the disease-free survival period according to the presence or absence of expression, it was confirmed that the stronger the nuclear expression of ATF6, the shorter the disease-free survival period appeared.
  • the combined administration of imatinib, the standard treatment for GIST, and PF-429242, an ATF6 inhibitor, and Bortezomib, an ER stress inducing drug, and PF-429242, an ATF6 inhibitor was compared (FIG. 9).
  • the GIST430 cell line is an imatinib-responsive patient-derived cell line
  • the GIST430-V654A cell line is a cell line derived from an imatinib-resistant patient.

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Abstract

The present invention relates to a pharmaceutical composition for preventing or treating an ATF6-overexpressing tumor comprising an ATF6 inhibitor. The pharmaceutical composition of the present invention may inhibit the activity of ATF6 and induce rapid apoptosis, thereby preventing or treating an ATF6-overexpressing tumor. In addition, the pharmaceutical composition of the present invention may be administered simultaneously or sequentially with a tyrosine kinase inhibitor, thereby more effectively preventing or treating an ATF6-overexpressing tumor.

Description

종양의 진단 및 치료를 위한 표적단백질로서의 ATF6ATF6 as a target protein for the diagnosis and treatment of tumors
본 발명은 ATF6를 과발현하는 종양의 진단용 조성물 또는 치료용 약학 조성물에 관한 것이다.The present invention relates to a diagnostic composition or a therapeutic pharmaceutical composition for ATF6 overexpressing tumors.
위장관 기질 종양 (GIST, Gastrointestinal stromal tumor)은 위장관 벽의 중간에 위치한 근육층에 존재하는 기질세포가 암세포로 변이를 일으켜 발생한다. 일반적으로 위암이나 대장암처럼 소화기에 발생하는 대부분의 암이 상피세포에서 유래하는 것과 달리 위장관 기질 종양은 근육층의 중간엽 유래 기질 세포로부터 생기는 암으로서, 식도, 위, 소장, 결장, 직장 등 위장관의 어디든 발생할 수 있고 원인과 위치, 전이 양상 등이 역시 일반적인 위암 및 소화기암과 다르다. 또한 뚜렷한 증상이 없기 때문에 정기검진이 무엇보다 중요한 질환 중 하나다.Gastrointestinal stromal tumor (GIST) is caused by mutation of stromal cells present in the muscle layer located in the middle of the wall of the gastrointestinal tract into cancer cells. In general, unlike most cancers that occur in the digestive tract, such as gastric or colon cancer, are derived from epithelial cells, gastrointestinal stromal tumors are cancers that arise from mesenchymal-derived stromal cells in the muscle layer. It can occur anywhere, and the cause, location, and metastasis pattern are also different from general gastric and digestive cancers. In addition, since there are no clear symptoms, regular check-ups are one of the most important diseases.
GIST의 주요 병리기전은 KIT 유전자 돌연변이로 인한 c-KIT 단백질의 지속적인 활성화와 PDGFRα 유전자 돌연변이로 알려졌는데, 대부분의 GIST 에서 KIT 또는 PDGFRα 유전자 돌연변이가 발견되고, 이중 KIT 유전자 돌연변이가 75~80%로 매우 흔한 것으로 보고되었다. The main pathological mechanism of GIST is known as the continuous activation of c-KIT protein and PDGFRα gene mutation due to KIT gene mutation. KIT or PDGFRα gene mutations are found in most GISTs, and among them, KIT gene mutations account for 75-80% of the time. reported to be common.
c-KIT (KIT, CD117, 또는 줄기세포인자 수용체로 알려짐)는 III형 수용체로서 작용하는 145 kDa 막관통 티로신 키나아제 단백질이다. 염색체 4q11-21에 위치한 c-KIT 유전자는 줄기세포 인자(SCF, 줄기세포 성장인자, kit 리간드)를 리간드를 가진 c-KIT 수용체 단백질을 암호화한다. c-KIT 수용체는 티로신-단백질 키나아제 활성을 가지며, 리간드인 SCF의 결합은 c-KIT를 자가인산화시키고 PI3K, GRB2, JAK2와 같은 하위신호전달물질과 결합시켜 PI3K-ATK, MAPK-ERK, JAK-STAT 신호전달계와 같은 종양 유발 신호전달계를 동시다발적으로 활성화킨다. 단백질 티로신 키나아제에 의한 티로신 인산화는 세포 신호 전달에서 특히 중요하며, 증식, 생존, 분화, 아폽토시스, 부착, 침습성 및 이동과 같은 주요 세포 과정을 위한 신호전달을 매개할 수 있는 것으로 알려져 있다.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 a 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 sub-signaling substances such as PI3K, GRB2, and JAK2, resulting in PI3K-ATK, MAPK-ERK, JAK- Simultaneously activates oncogenic signaling pathways such as STAT signaling. Tyrosine phosphorylation by protein tyrosine kinases is particularly important in cellular signal transduction and is known to mediate signaling for key cellular processes such as proliferation, survival, differentiation, apoptosis, adhesion, invasiveness and migration.
c-KIT 발현 및 활성의 역할은 급성 백혈병 및 GIST 같은 혈액학적 종양 및 고형 종양에서 연구되어 왔다. 대부분의 GIST는 RTK인 c-KIT(GIST의 75~80%) 또는 PDGFRα(비-c-KIT 돌연변이된 GIST의 8%)를 암호화하는 유전자에서 1차 활성화 돌연변이를 가진다. 특히 80% 정도를 차지하는 대부분의 GIST 환자는 c-KIT 유전자의 기능 획득 돌연변이(gain-of-function mutation)로 인해 c-KIT의 단백질 과발현 및 과인산화를 보인다. 1차 c-KIT 돌연변이 대부분은 exon11에 의해 암호화된 단백질의 막인접 영역(JM region: juxtamembrane region)에서 발생하며, 프레임 내 결실 또는 삽입, 미스센스 돌연변이(즉, V560D)로 이루어진다. c-KIT exon11 돌연변이는 GIST 환자의 약 65%에서 1차 돌연변이로 나타나며, 이러한 JM 도메인 돌연변이는 c-KIT 키나제의 자가억제 기작을 방해하여, GIST의 원인이 되는 키나아제를 자동적으로 활성화시키고 세포의 형질전환을 유발시킨다. 그 외 다른 1차 GIST-유발 c-KIT 돌연변이는 exon9(AY501-502 복제/삽입, 8%),exon13(돌연변이, 1%) 및 exon17(돌연변이, 1%)에 위치하는 것으로 알려져 있다.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 primary activating mutations in the gene encoding the RTKs, c-KIT (75-80% of GISTs) or PDGFRα (8% of non-c-KIT mutated GISTs). In particular, most GIST patients, accounting for about 80%, show c-KIT protein overexpression and hyperphosphorylation due to gain-of-function mutations in the c-KIT gene. Most of the primary c-KIT mutations occur in the juxtamembrane region (JM region) of the protein encoded by exon11, and consist of in-frame deletions or insertions, and missense mutations (ie, V560D). The c-KIT exon11 mutation appears as a primary mutation in about 65% of GIST patients, and this JM domain mutation interferes with the self-suppression mechanism of c-KIT kinase, automatically activating the GIST causative kinase and transforming the cell. induce conversion. Other primary GIST-induced c-KIT mutations are known to be located in exon9 (AY501-502 replication/insertion, 8%), exon13 (mutant, 1%) and exon17 (mutant, 1%).
악성 종양에서 c-KIT 발현의 임상적 중요성은 티로신 키나아제 수용체를 특이적으로 억제하는 Gleevec을 이용한 연구에서 입증되었다 또한, 임상적으로 큰 발전은, Gleevec이 통상적인 화학요법에 일반적으로 내성을 나타내는 GIST에 대한 표적 치료를 통한 항종양 효과를 보인 것이다. 그러나, c-KIT 억제제인 Gleevec®의 경우, 이미 전이가 되었거나 수술이 불가한 GIST 환자에 대해 치료효과를 어느 정도 기대할 수 있지만, 완치는 드물고, 약 50%의 환자는 치료 후 2년 이내에 암이 재발하기 때문에 이를 극복할 수 있는 새로운 치료제 개발이 요구된다.The clinical significance of c-KIT expression in malignant tumors has been demonstrated in studies using Gleevec, which specifically inhibits the tyrosine kinase receptor. Furthermore, a major clinical advance is the GIST, in which Gleevec is generally resistant to conventional chemotherapy. It showed antitumor effect through targeted therapy. However, in the case of Gleevec®, a c-KIT inhibitor, although some therapeutic effect can be expected for GIST patients who have metastasized or cannot be operated on, a cure is rare, and about 50% of patients have cancer within 2 years of treatment. Because it recurs, there is a need to develop a new therapeutic agent that can overcome it.
c-KIT에 존재하는 돌연변이 위치는 Gleevec®mesylate, Novartis Pharm.) 기반 치료에서 임상적으로 매우 중요한데 exon 11번 돌연변이 환자의 경우 초기 Gleevec 약물 반응성이 85% 수준으로 좋지만 exon 9번 돌연변이 환자의 경우 45%, exon 13 및 17번 돌연변이 환자의 경우 거의 약물 반응성을 보이지 않아 돌연변이 위치별 약물 치료 효과가 현저한 차이를 보인다. 이러한 돌연변이 위치별 약물 반응성 차이로 인해 현재 임상에서는 Gleevec 기반 GIST 치료의 한계점을 인정하고 있다. 전술한 것처럼 GIST는 돌연변이가 발생한 exon 위치에 따라서 약물 반응성이 애초에 없거나 또는 약물 치료 과정에서 Gleevec®에 대해 내성을 나타내는 경우가 빈번하다. Gleevec 치료 과정 중 발생하는 약물 내성은 c-KIT 유전자에 발생하는 2차 돌연변이에 의해 유도되며, 2차 돌연변이가 발생했을 경우에 대한 확실한 치료법은 아직 정립되지 않았다. Gleevec® 치료 과정 중 재발한 GIST 환자들은 본래 가지고 있던 1차 돌연변이에 추가하여 2차 돌연변이 발생으로 훨씬 더 공격적인 활성화 형태의 c-KIT 돌연변이 단백질을 갖게된다. 2차 돌연변이는 ATP 결합 포켓 (exon13: K642E, V654A; exon14: T670I) 및 활성화 루프(exon17: N822K, D816H, D816V, D820A; exon18: A829P)에서 발견되는데, Gleevec이 ATP 결합 포켓에 결합하The location of the mutation in c-KIT is very clinically important for treatment based on Gleevec®mesylate, Novartis Pharm.). In patients with exon 11 mutation, the initial Gleevec drug reactivity is 85%, but in patients with exon 9 mutation, 45 %, exon 13 and 17 mutation patients show almost no drug reactivity, showing a significant difference in the drug treatment effect by mutation location. Due to the difference in drug reactivity by mutation site, the limitations of Gleevec-based GIST treatment are currently recognized in clinical practice. As mentioned above, depending on the location of the mutated exon, GIST often has no drug reactivity in the first place or exhibits resistance to Gleevec® during drug treatment. Drug resistance that occurs during Gleevec treatment is induced by secondary mutations in the c-KIT gene, and a definitive treatment for secondary mutations has not yet been established. GIST patients who relapse during the course of Gleevec® treatment have a much more aggressive activated form of c-KIT mutant protein due to secondary mutation in addition to the primary mutation they had. Secondary mutations are found in the ATP binding pocket (exon13: K642E, V654A; exon14: T670I) and activation loop (exon17: N822K, D816H, D816V, D820A; exon18: A829P), where Gleevec binds to the ATP binding pocket.
여 약물 활성을 나타내기 때문에 c-KIT의 이러한 2차 돌연변이체는 약물 내성을 유This secondary mutant of c-KIT induces drug resistance because
도한다.do too
Sutent®(Sunitinib malate, Pfizer)는 다중 RTK의 억제제, 특히 본 맥락에서의 c-KIT의 억제제이며, 2차 돌연변이로 인한 Gleevec 내성 c-KIT (e.g. ATP-결합 포켓 돌연변이체 V654A 및 T670I)을 저해할 수 있는 약제로 개발되었다. 그러나 GIST 환자 중 2차 돌연변이가 exon17에 의해 암호화된 c-KIT 촉매 도메인의 활성화 루프에 위치하는 D816H 및 D816V와 같은 위치에 발생할 경우는 수니티닙(Sunitinib)에 대해서도 내성을 나타내며, 임상에서 수니티닙의 전반적인 약물 반응성은 10% 내외로 큰 성과를 거두지는 못하고 있는 것으로 보고되었다. Sutent® (Sunitinib malate, Pfizer) is an inhibitor of multiple RTKs, especially c-KIT in this context, and inhibits Gleevec resistant c-KIT due to secondary mutations (e.g. ATP-binding pocket mutants V654A and T670I). It has been developed as a drug that can However, in patients with GIST, when secondary mutations occur at the same positions as D816H and D816V located in the activation loop of the c-KIT catalytic domain encoded by exon17, they are also resistant to Sunitinib, It has been reported that the overall drug reactivity of nib is not achieving great results at around 10%.
Gleevec 내성 GIST 환자에게 활용하는 또 따른 multi kinase inhibitor인 레고라페닙(Regorafenib) 역시 치료 반응성을 보이는 환자가 5% 미만에 불과하며, 그마저도 약물 부작용이 매우 심하기 때문에 적극적 활용에 어려움을 겪고 있다. 따라서 수니티닙과 레고라페닙 모두 Gleevec에 내성을 보이는 GIST에 대한 현실적 대안이 아니다.Regorafenib, another multi-kinase inhibitor used for Gleevec-resistant GIST patients, also has less than 5% of patients showing treatment responsiveness, and even that has very severe side effects, so it is difficult to actively use it. Therefore, both sunitinib and regorafenib are not realistic alternatives to Gleevec-resistant GIST.
전술한 것처럼 개별 환자 내에서 발생할 수 있는 복잡한 다중 2차 c-KIT 돌연변이는 Gleevec에 대한 약물 반응성을 저해할 뿐만 아니라, KIT 단백질의 활성화를(키나아제 활성화) 조절하는 방식으로만 GIST를 치료하고자 했던 기존 치료법의 한계를 보여준다. Gleevec은 현재 GIST에서 가장 확실한 치료수단이지만, 1차 돌연변이 발생 위치에 따른 약물 반응성 저하 문제 및 2차 돌연변이로 인한 약물 내성 문제 등의 문제가 존재하는바, 이를 극복할 수 있는 새로운 치료법 개발에 대한 연구 필요성이 강조되고 있다. 최근에는 KIT 단백의 활성화를 조절하기 보다 siRNA, microRNA 등을 이용하여 KIT mRNA나 KIT 단백질의 발현 자체를 직접 조절하거나 KIT의 발현을 조절하는 것으로 알려진 PKC-theta와 같은 인자들을 활용하여 간접적으로 KIT 단백질 발현을 제어하는 방식이 대안으로써 주목받고 있다. 또한 GIST에서 현재까지 알려져 있지 않은 새로운 암발생 및 진행 기전 규명을 통해 완전히 새로운 약물 표적 개발 등의 연구가 수행되고 있으나 현재까지 임상적용할만한 연구 성과는 발표된바 없다.As described above, complex multiple secondary c-KIT mutations that can occur within individual patients not only inhibit drug responsiveness to Gleevec, but also modulate KIT protein activation (kinase activation). It shows the limitations of treatment. Gleevec is currently the most reliable treatment method in GIST, but there are problems such as drug responsiveness lowering according to the location of primary mutation and drug resistance due to secondary mutation. Research on the development of new treatments to overcome these problems need is emphasized. Recently, rather than regulating the activation of KIT protein, directly regulating the expression of KIT mRNA or KIT protein using siRNA or microRNA, or indirectly using factors such as PKC-theta known to regulate KIT expression, KIT protein A method of controlling expression is attracting attention as an alternative. In addition, GIST is conducting research such as the development of completely new drug targets through the identification of new cancer occurrence and progression mechanisms that are not known until now, but no clinically applicable research results have been published so far.
본 발명을 통해 GIST의 새로운 발생 기전을 규명하고 신규 약물 표적 인자를 발굴하여 이를 조절하는 방식을 통해 전술하였던 기존 c-KIT 타겟 치료법의 한계점을 극복하고자 하며, 아울러 Gleevec에 좋은 약물 반응성을 보이는 초기 GIST 환자들 뿐만 아니라 Gleevec에 대해 내성이 발생한 환자들에게 새로운 치료 선택지를 제시하고자 한다. 또한 기존 치료법인 Gleevec과도 병용할 수 있는 치료법을 개발하여 조기의 강력한 암억제능 달성을 통한 근치적 GIST 치료법 개발 근거를 제시하고자 한다.Through the present invention, we seek to overcome the limitations of the existing c-KIT target therapy described above by identifying a new mechanism of GIST development, discovering new drug target factors, and controlling them. We intend to suggest new treatment options for patients who develop resistance to Gleevec as well as patients. In addition, by developing a treatment that can be used in combination with the existing treatment, Gleevec, we intend to present the basis for the development of a curative GIST treatment by achieving strong cancer suppression at an early stage.
본 발명은 위장관 기질종양에 대한 예방 또는 치료용 약학 조성물을 제공하는 것을 목적으로 한다.An object of the present invention is to provide a pharmaceutical composition for preventing or treating gastrointestinal stromal tumor.
본 발명은 위장관 기질종양의 진단 또는 예후예측을 위한 정보 제공방법을 제공하는 것을 목적으로 한다.An object of the present invention is to provide a method for providing information for diagnosis or prognosis of gastrointestinal stromal tumor.
1. ATF6 억제제를 포함하는 ATF6 과발현 종양의 예방 또는 치료용1. For the prevention or treatment of ATF6 overexpressing tumors including ATF6 inhibitors
약학 조성물.pharmaceutical composition.
2. 위 1에 있어서, 상기 ATF6 과발현 종양은 유방암, 담관암, 대장2. The above 1, wherein the ATF6 overexpressing tumor is breast cancer, cholangiocarcinoma, colon
선암, 식도암, 다형성 교모세포종, 급성 골수성 백혈병, 뇌 저등급 신경교종, 간암, 췌장암, 갈색 세포종/부신경 절종, 육종, 피부 흑색종, 위암, 난소암, 갑상선암, 흉선종, 고환암, 직장암, 전립선암, 부신 피질 암, 방광암, 림프종, 중피종, 폐암, 자궁내막암, 자궁경부암, 신장암, 두 경부암, 위장관암, 뇌수막종, 뇌하수체 선종, 청신경초종, 두개인두종, 모낭형 성상세포종, 혈관아 세포종, 미만성 성상세포종, 핍지 세포종, 비정형 뇌수박종, 상의 세포종 및 위장관 기질종양으로 이루어진 군에서 선택되는 적어도 하나인, ATF6 과발현 종양의 예방 또는 치료용 약학 조성물.Adenocarcinoma, esophageal cancer, glioblastoma multiforme, acute myeloid leukemia, brain low-grade glioma, liver cancer, pancreatic cancer, pheochromocytoma/paraganglioma, sarcoma, skin melanoma, gastric cancer, ovarian cancer, thyroid cancer, thymoma, testicular cancer, rectal cancer, prostate cancer, Adrenal 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 A pharmaceutical composition for the prevention or treatment of ATF6 overexpressing tumor, which is at least one selected from the group consisting of celloma, oligodendroblastoma, atypical hydrocephalus, epithelial celloma, and gastrointestinal stromal tumor.
3. 위 1에 있어서, 상기 ATF6 억제제는 PF429242, Ceapin A7 및 Melatonin으로 이루어진 군에서 선택되는 적어도 하나인, ATF6 과발현 종양의 예방 또는 치료용 약학 조성물.3. The pharmaceutical composition of the above 1, wherein the ATF6 inhibitor is at least one selected from the group consisting of PF429242, Ceapin A7 and Melatonin, for preventing or treating ATF6 overexpressing tumor.
4. 위 1에 있어서, 티로신 키나제 억제제를 추가로 포함하는, ATF6 과발현 종양의 예방 또는 치료용 약학 조성물.4. The pharmaceutical composition for the prevention or treatment of ATF6 overexpressing tumors according to the above 1, further comprising a tyrosine kinase inhibitor.
5. 위 4에 있어서, 상기 티로신 키나제 억제제는 이마티닙, 수니티닙, 레고라페닙 및 아이바키트로 이루어진 군에서 선택되는 적어도 하나인, ATF6 과발현 종양의 예방 또는 치료용 약학 조성물.5. The pharmaceutical composition for the prevention or treatment of ATF6 overexpressing tumors according to the above 4, wherein the tyrosine kinase inhibitor is at least one selected from the group consisting of imatinib, sunitinib, regorafenib, and ivakit.
6. 위 4에 있어서, 상기 티로신키나아제 억제제는 ATF6 억제제와 동시에 또는 순차적으로 투여되는, ATF6 과발현 종양의 예방 또는 치료용 약학 조성물.6. The pharmaceutical composition for the prevention or treatment of ATF6 overexpressing tumors according to the above 4, wherein the tyrosine kinase inhibitor is administered simultaneously or sequentially with the ATF6 inhibitor.
7. 시료의 ATF6 또는 그 mRNA의 수준을 측정하는 단계를 포함하는 위장관 기질종양의 진단 또는 예후예측을 위한 정보 제공 방법.7. A method for providing information for diagnosis or prognosis of gastrointestinal stromal tumor, comprising measuring the level of ATF6 or its mRNA in the sample.
8. 위 7에 있어서, 상기 시료는 개체 또는 개체로부터 분리된 것인, 위장관 기질종양의 진단 또는 예후예측을 위한 정보 제공 방법.8. The method of providing information for the diagnosis or prognosis of gastrointestinal stromal tumor according to the above 7, wherein the sample is isolated from an individual or an individual.
9. 위 7에 있어서, 상기 ATF6 또는 그 mRNA의 수준이 정상 대조군의 발현 정도에 비해 높은 경우 위장관 기질종양으로 판단하는 단계를 더 포함하는, 위장관 기질종양의 진단 또는 예후예측을 위한 정보 제공 방법.9. The method of providing information for diagnosis or prognosis of a gastrointestinal stromal tumor according to the above 7, further comprising determining that the ATF6 or its mRNA level is higher than the expression level of the normal control group as a gastrointestinal stromal tumor.
10. 위 7에 있어서, 상기 ATF6 또는 그 mRNA의 수준이 높을수록 위장관 기질종양의 예후가 나쁠 것으로 판단하는 단계를 더 포함하는, 위장관 기질종양의 진단 또는 예후예측을 위한 정보 제공 방법.10. The method of providing information for the diagnosis or prognosis of gastrointestinal stromal tumor according to the above 7, further comprising the step of determining that the higher the level of ATF6 or its mRNA, the worse the prognosis of the gastrointestinal stromal tumor.
11. ATF6 또는 그 mRNA의 수준을 측정하는 물질을 포함하는 위장관 기질종양의 진단 또는 예후예측용 조성물.11. A composition for diagnosis or prognosis of gastrointestinal stromal tumor comprising a substance measuring the level of ATF6 or its mRNA.
12. 위 11에 있어서, 상기 ATF6 또는 그 mRNA의 수준을 측정하는 물질은 ATF6 특이적으로 결합하는 프라이머, 프로브, 항체로 이루어진 군에서 선택되는 것인, 위장관 기질종양의 진단 또는 예후예측용 조성물.12. The composition for diagnosis or prognosis of gastrointestinal stromal tumor according to 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. 시료의 ATF6 또는 그 mRNA의 발현을 감소시키는 물질을 선별하는 단계를 포함하는 위장관 기질종양 치료제 후보물질의 스크리닝 방법.13. 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 the sample.
본 발명의 약학 조성물은 위장관 기질종양에서 과발현된 ATF6의 활성을 억제하여, 하위 샤페론 단백질의 발현을 억제함에 따라 급격한 세포사멸을 유발하는 효과를 나타낼 수 있다. 이에 위장관 기질종양을 예방 또는 치료할 수 있다.The pharmaceutical composition of the present invention inhibits the activity of ATF6 overexpressed in gastrointestinal stromal tumors, thereby suppressing the expression of lower chaperone proteins, thereby inducing rapid apoptosis. Accordingly, it is possible to prevent or treat gastrointestinal stromal tumors.
또한 본 발명의 약학 조성물은 티로신 키타제 억제제와 동시에 또는 순차적으로 투여되어 더 효과적으로 위장관 기질종양을 예방 또는 치료할 수 있다.In addition, the pharmaceutical composition of the present invention may be administered simultaneously or sequentially with the tyrosine ketase inhibitor to more effectively prevent or treat gastrointestinal stromal tumors.
도 1은 GIST에서 cATF6 (cleaved ATF6, ATF6 활성화 형태)의 발현이 높아져 있으며, 세포 내에서도 핵으로 이동해 있음을 WB, 형광 면역염색으로 GIST 세포주에서 확인하고 GIST 환자 조직에서 IHC로 확인한 결과이다.1 shows the results of confirming that the expression of cATF6 (cleaved ATF6, ATF6 activated form) is elevated in GIST, and that it migrates to the nucleus within cells in the GIST cell line by WB and fluorescence immunostaining and by IHC in the GIST patient tissue.
도 2는 TG(Thapsigargin, ER 스트레스 유발)를 농도별로 처리하여 ATF6, cATF6, ER 스트레스 신호전달 관여 인자 (PERK, IRE1α 및 각각의 인산화형태), CHOP (세포사멸 마커) 및 샤페론 단백질 (BIP, HSP90, GRP94, HSP70)의 발현을 단백질 수준에서 확인한 결과이다. 특히, ATF6가 활성화 되어 있는 GIST 세포는 mild한 ER stress가 유도된 경우(TG: 0.1 uM) ER stress를 극복하며 생존할 수 있음을 보여준다. 2 shows TG (Thapsigargin, induces ER stress) by treatment at different concentrations, ATF6, cATF6, ER stress signaling factors (PERK, IRE1α and phosphorylated form of each), CHOP (apoptosis marker) and chaperone proteins (BIP, HSP90) , GRP94, HSP70) is the result of confirming the expression at the protein level. In particular, it shows that GIST cells in which ATF6 is activated can survive by overcoming ER stress when mild ER stress is induced (TG: 0.1 uM).
도 3은 GIST 세포주에 ATF6의 cleavage 및 활성화를 억제하는 S1Pi(Serine 1 protease inhibitor)를 처리하면, 샤페론 단백질 (BIP, HSP90, GRP94, HSP70)의 발현이 억제되고 mild한 ER stress와 함께 유도될 경우에도 급격한 세포 사멸이 유발됨을 확인하였고, 또한 ER 스트레스 유도에 따라 ATF6 관련 샤페론 단백질의 발현이 증가되는 시점이 지연되는 것을 확인하였다3 shows that when a GIST cell line is treated with S1Pi (Serine 1 protease inhibitor) that inhibits the 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 apoptosis was also induced in
도 4는 KIT와 결합하는 단백질을 확인하기 위해 GIST 세포주에서 KIT 항체를이용한 면역침강을 수행한 결과로, ATF6에 의해 조절되는 샤페론인 HSP90 단백질이 KIT와 결합함을 확인하였다. 또한 ATF6를 억제하면 하위 단계인 샤페론인 HSP90의 발현이 억제되고 이를 통해 GIST의 발생원인 중 하나인 돌연변이 KIT도 부수적으로 억제가 가능하여 ER stress 감수성 증가를 통한 항암 효과뿐 아니라 돌연변이 KIT 단백 발현 억제를 통해서도 항암 효과를 유도할 수 있어 ATF6 저해가 여러 경로를 통해 GIST에서 강력한 항암 효과를 유도할 수 있음을 시사한다. 또한 ATF6 저해제 또는 HSP90 저해제 처리시 KIT 단백질의 발현이 감소되는 결과를 확인하였다.4 is a result of performing immunoprecipitation using the KIT antibody in GIST cell line to confirm the protein binding to KIT, it was confirmed that the HSP90 protein, which is a chaperone regulated by ATF6, binds to KIT. In addition, inhibition of ATF6 suppresses the expression of HSP90, a sub-step chaperone, and through this, mutant KIT, one of the causes of GIST, can also be suppressed incidentally. It can also induce anticancer effects, suggesting that ATF6 inhibition can induce strong anticancer effects in GIST through multiple pathways. In addition, it was confirmed that the expression of the KIT protein was reduced when the ATF6 inhibitor or the HSP90 inhibitor was treated.
도 5는 GIST 세포주에 ATF6 억제제 (S1Pi, Ceapin A7, Melatonin)를 처리한 경우, cleaved ATF6 (활성상태의 ATF6)의 발현이 감소한 것을 확인한 결과이다.5 is a result confirming that the expression of cleaved ATF6 (active ATF6) is reduced when the GIST cell line is treated with an ATF6 inhibitor (S1Pi, Ceapin A7, Melatonin).
도 6는 실제 종양 미세 환경을 모사하기 위해 TG를 처리한 GIST 세포주(GIST882: 이마티닙 민감성 세포주; GIST48: 이마티닙 내성 세포주)에 ATF6 억제제(S1Pi, Ceapin A7, Melatonin)를 독립적으로 처리해준 경우와 ER 스트레스 유발 물질 (Borteazomib, 17AAG) 또는 이마티닙과 ATF6 억제제를 동시에 투여한 경우의 세포 생존력 (cell viability)를 측정한 것으로, ATF6 억제제는 독립적으로 처리하여도 우수한 GIST 억제능을 보이고 ER 스트레스 유발 물질 역시 독립적으로 GIST를억제할 수 있으며, ATF6 억제제와 ER 스트레스 유발 물질을 함께 처리한 경우 더 효율적으로 GIST를 억제함을 확인한 결과이다.6 is a case in which ATF6 inhibitors (S1Pi, Ceapin A7, Melatonin) were independently treated and ER stress in a GIST cell line (GIST882: imatinib-sensitive cell line; GIST48: imatinib-resistant cell line) treated with TG to simulate the actual tumor microenvironment. Cell viability was measured when an inducer (Borteazomib, 17AAG) or imatinib and an ATF6 inhibitor were administered simultaneously, and the ATF6 inhibitor showed excellent GIST inhibitory ability even when treated independently, and ER stress-inducing substances were also independently GIST It is the result of confirming that GIST more efficiently when treated with an ATF6 inhibitor and an ER stress-inducing substance.
도 7은 TG를 처리하지 않은 GIST 세포주에 ATF6 억제제 (S1Pi, Ceapin A7, Melatonin)를 독립적으로 처리해준 경우와 ER 스트레스 유발 물질 (Borteazomib, 17AAG) 또는 이마티닙과 ATF6 억제제를 동시에 투여한 경우의 세포 생존력 (cell viability)를 측정한 것으로, ATF6 억제제는 독립적으로 처리하여도 우수한 GIST 억제능을 보이고 ER 스트레스 유발 물질 역시 독립적으로 GIST를 억제할 수 있으며, ATF6 억제제와 ER 스트레스 유발 물질을 함께 처리한 경우 더 효율적으로 GIST를 억제함을 확인한 결과이다.Figure 7 shows the cell viability of GIST cell lines not treated with TG when ATF6 inhibitors (S1Pi, Ceapin A7, Melatonin) were independently treated and when ER stress-inducing substances (Borteazomib, 17AAG) or imatinib and ATF6 inhibitors were simultaneously administered (cell viability) was measured, and ATF6 inhibitor showed excellent GIST inhibitory ability even when treated independently, and ER stress-inducing substances can also independently inhibit GIST. This is the result of confirming that it inhibits GIST.
도 8은 ATF6 발현 환자군과 발현하지 않는 환자군의 생존확률을 나타낸 생존함수 그래프로, ATF6를 발현하는 환자군의 경우, 생존률이 낮음을 확인한 결과이다.8 is a survival function graph showing the survival probability of a patient group expressing ATF6 and a patient group not expressing ATF6. In the case of a patient group expressing ATF6, it is confirmed that the survival rate is low.
도9는 GIST의 표준 치료제인 imatinib 반응성 세포주 GIST430과 imatinib 내성 세포주 GIST430-V654A의 마우스 xenograft 모델에서 1) imatinib과 2) ATF6 저해제인 PF-429242, 3) ER 스트레스 유도 약물인 Bortezomib과 ATF6 저해제 PF-429242의 병용 투여에 따른 종양 억제능을 비교한 결과이다.9 is a mouse xenograft model of the imatinib-responsive cell line GIST430, the standard treatment for GIST, and the imatinib-resistant cell line GIST430-V654A. This is the result of comparing the tumor suppression ability according to the concurrent administration of 429242.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 ATF6 억제제를 포함하는 ATF6 과발현 종양의 예방 또는 치료용 약학 조성물을 제공할 수 있다: The present invention may provide a pharmaceutical composition for preventing or treating an ATF6 overexpressing tumor comprising an ATF6 inhibitor:
ATF6 억제제는 ATF6의 C-terminal 영역과 N-terminal 영역을 연결하는 부위가 절단되어 C-terminal 영역, 즉 세포질 쪽에 위치한 ATF6가 핵 안으로 이동하는 활성화 과정을 억제하는 것으로, ATF6가 활성화 되는 기작을 억제하는 물질로서, 예를 들면 Serine 1 protease 억제제 (S1Pi), PF429242, Ceapin A7 및 Melatonin일 수 있으나, 이에 제한되는 것은 아니다.ATF6 inhibitor inhibits the activation process in which ATF6 located in the C-terminal region, that is, the cytoplasm, moves into the nucleus by cutting the region connecting the C-terminal region and N-terminal region of ATF6, and inhibits the mechanism of ATF6 activation As a substance to be used, for example, Serine 1 protease inhibitor (S1Pi), PF429242, Ceapin A7 and Melatonin, but may be, but is not limited thereto.
ATF6 과발현 종양은 ATF6 또는 ATF6의 활성화 형태인 cleaved ATF6(cATF6)의 mRNA 또는 단백질의 발현이 정상조직 또는 정상세포 대비 증가되어있는 종양일 수 있으며, ATF6 과발현 종양은 예를 들면 유방암, 담관암, 대장선암, 식도암, 다형성 교모세포종, 급성 골수성 백혈병, 뇌 저등급 신경교종, 간암, 췌장암, 갈색 세포종/부신경 절종, 육종, 피부 흑색종, 위암, 난소암, 갑상선암, 흉선종, 고환암, 직장암, 전립선암, 부신 피질 암, 방광암, 림프종, 중피종, 폐암, 자궁내막암, 자궁경부암, 신장암, 두 경부암, 위장관암, 뇌수막종, 뇌하수체선종, 청신경초종, 두개인두종, 모낭형 성상세포종, 혈관아 세포종, 미만성 성상세포종, 핍지 세포종, 비정형 뇌수박종, 상의 세포종 또는 위장관 기질종양일 수 있으나, 이에 제한되는 것은 아니다.ATF6 overexpressing tumor may be a tumor in which mRNA or protein expression of ATF6 or cleaved ATF6 (cATF6), an activated form of ATF6, is increased compared to normal tissue or normal cells, and ATF6 overexpressing tumor is, for example, breast cancer, cholangiocarcinoma, and colorectal adenocarcinoma , esophageal cancer, glioblastoma multiforme, acute myeloid leukemia, brain low-grade glioma, liver cancer, pancreatic cancer, pheochromocytoma/paraganglioma, sarcoma, skin melanoma, gastric cancer, ovarian cancer, thyroid cancer, thymoma, testicular cancer, rectal cancer, prostate cancer, adrenal cancer 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, craniopharyngoma, follicular astrocytoma, hemangioblastoma, diffuse astrocytoma , oligodendroblastoma, atypical hydrocephalus, epithelial celloma, or gastrointestinal stromal tumor, but is not limited thereto.
상기 위장관 기질종양은 위장관 벽의 근육층에 위치한 근육의 수축 이완을 조절하는 세포, 즉 카알세포가 변이를 일으켜 발생하는 희귀질환으로 기스트(GIST, Gastrointestinal stromal tumor)라고 불린다.The gastrointestinal stromal tumor is a rare disease caused by mutations in cells that control contraction and relaxation of muscles located in the muscle layer of the gastrointestinal wall, that is, kall cells, and is called Gastrointestinal stromal tumor (GIST).
용어 "예방"은 위장관 기질종양을 억제시키거나 또는 지연시키는 모든 행위를 말한다.The term "prevention" refers to any action that inhibits or delays gastrointestinal stromal tumors.
용어 "치료"는 위장관 기질종양 의심 및 발병 개체의 증상이 호전되거나 이롭게 변경되는 모든 행위를 말한다.The term "treatment" refers to any action in which the symptoms of a subject suspected of and having a gastrointestinal stromal tumor are improved or beneficially changed.
본 발명 약학 조성물은 캡슐, 정제, 과립, 주사제, 연고제, 분말 또는 음료 형태일 수 있다.The pharmaceutical composition of the present invention may be in the form of a capsule, tablet, granule, injection, ointment, powder or beverage.
본 발명 약학 조성물은 산제, 과립제, 캡슐, 정제, 수성 현탁액 등의 경구형 제형, 외용제, 좌제 및 주사제의 형태로 제형화하여 사용될 수 있다.The pharmaceutical composition of the present invention may 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.
본 발명 약학 조성물은 유효성분을 단독으로 포함하거나, 하나 이상의 약학적으로 허용되는 담체, 부형제 또는 희석제를 더 포함할 수 있다.The pharmaceutical composition of the present invention may include an active ingredient alone, or may further include one or more pharmaceutically acceptable carriers, excipients or diluents.
본 발명 약학 조성물은 약학적으로 허용 가능한 담체를 포함할 수 있다. 약학적으로 허용 가능한 담체는 경구 투여 시에는 결합제, 활탁제, 붕해제, 부형제, 가용화제, 분산제, 안정화제, 현탁화제, 색소, 향료 등일 수 있으며, 주사제의 경우에는 완충제, 보존제, 무통화제, 가용화제, 등장제, 안정화제 등을 혼합하여 사용할 수 있으며, 국소투여용의 경우는 기제, 부형제, 윤활제, 보존제 등을 사용할 수 있다.The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may be binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents, flavoring agents, etc., in the case of oral administration, and in the case of injections, buffers, preservatives, analgesics, Solubilizers, isotonic agents, stabilizers, etc. can be mixed and used, and for topical administration, bases, excipients, lubricants, preservatives, etc. can be used.
본 발명 약학 조성물의 제형은 약학적으로 허용되는 담체와 혼합하여 다양하게 제조될 수 있으며, 예를 들어, 경구 투여시에는 정제, 트로키, 캡슐, 엘릭서(elixir), 서스펜션, 시럽, 웨이퍼 등의 형태로 제조될 수 있으며, 주사제의 경우에는 단위 투약 앰플 또는 다수회 투약 형태로 제조될 수 있다. 또한, 본 발명 약학 조성물의 제형은 용액, 현탁액, 정제, 캡슐, 서방형 제제 등으로 제조될 수 있다.The dosage form of the pharmaceutical composition of the present invention may be prepared in various ways by mixing with a pharmaceutically acceptable carrier, for example, tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., when administered orally. It may be prepared in the form of injections, and in the case of injections, it may be prepared in the form of unit dose ampoules or multiple doses. In addition, the dosage form of the pharmaceutical composition of the present invention may be prepared as a solution, suspension, tablet, capsule, sustained release formulation, and the like.
제제화를 위한 담체, 부형제 및 희석제는 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말디톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로즈, 폴리비닐피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트, 광물유, 충진제, 항응집제, 윤활제, 습윤제, 향료, 유화제 또는 방부제 등일 수 있다.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, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, filler, anticoagulant, lubricant, wetting agent, flavoring, emulsifying agent or preservative and the like.
본 발명 약학 조성물의 투여 경로는 구강, 정맥내, 근육내, 동맥내, 골수내, 경막내, 심장내, 경피, 피하, 복강내, 비강내, 장관, 국소, 설하 또는 직장일 수 있으며, 이에 제한되지 않는다.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
본 발명 본 발명 약학 조성물은 경구 또는 비경구로 투여될 수 있으며, 비경구 투여 시 피부 외용 또는 복강내주사, 직장내주사, 피하주사, 정맥주사, 근육내 주사 또는 흉부내 주사 주입방식이 선택될 수 있다.The pharmaceutical composition of the present invention may be administered orally or parenterally, and when administered parenterally, external or intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection may be selected. have.
본 발명의 약학 조성물의 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다.The dosage of the pharmaceutical composition of the present invention varies depending on the condition and weight of the patient, the severity of the disease, the drug form, the route of administration, and the duration, but may be appropriately selected by those skilled in the art.
예를 들어, 본 발명 약학 조성물은 1일 0.0001 mg 내지 1000mg/kg 또는 0.001mg 내지 500mg/kg으로 투여될 수 있다. 본 발명 약학 조성물의 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.For example, the pharmaceutical composition of the present invention may be administered in an amount of 0.0001 mg to 1000 mg/kg or 0.001 mg to 500 mg/kg per day. Administration of the pharmaceutical composition of the present invention may be administered once a day, may be administered several times divided. The above dosage does not limit the scope of the present invention in any way.
또한 본 발명의 약학 조성물은 티로신 키나제 억제제를 더 포함하는 ATF6 과발현 종양의 예방 또는 치료용 약학 조성물을 제공할 수 있다:In addition, the pharmaceutical composition of the present invention may provide a pharmaceutical composition for preventing or treating ATF6 overexpressing tumors further comprising a tyrosine kinase inhibitor:
상기 티로신 키나제 억제제는 성장인자 수용체 말단의 티로신 키나제(tyrosine kinase)를 억제함으로써 성장신호가 증폭 전달되는 것을 차단하는 물질을 의미하며, 예를 들면 이마티닙, 수니티닙, 레고라페닙, 아이바키트 등일 수 있으나, 이에 제한되는 것은 아니다.The tyrosine kinase inhibitor refers to a substance that blocks amplification and transmission of a growth signal by inhibiting tyrosine kinase at the terminal of a growth factor receptor, for example, imatinib, sunitinib, regorafenib, ivakit, etc. However, the present invention is not limited thereto.
상기 티로신 키나제 억제제는 본 발명의 약학 조성물과 동시에 또는 순차적으로 투여될 수 있으며, 그 투여 순서는 제한되지 않는다.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.
본 발명은 시료의 ATF6 또는 그 mRNA의 수준을 측정하는 단계를 포함하는 위장관 기질종양의 진단 또는 예후예측을 위한 정보 제공 방법을 제공할 수 있다:The present invention may provide an information providing method for diagnosis or prognosis of gastrointestinal stromal tumor, comprising measuring the level of ATF6 or its mRNA in a sample:
상기 시료는 개체 또는 개체로부터 분리된 것일 수 있고, 시료의 종류는 예를 들면 조직, 세포, 전혈, 혈장, 혈청, 혈액 등일 수 있으나, 이에 제한되는 것은 아니다.The sample may be an individual or one isolated from an individual, and the type of the sample may be, for example, tissue, cell, whole blood, plasma, serum, blood, etc., but is not limited thereto.
상기 개체는 위장관 기질종양이 발병하였거나 발병할 수 있는 인간을 포함한 쥐, 생쥐, 가축 등의 모든 동물을 의미한다. 구체적인 예로, 인간을 포함한 포유동물일 수 있다.The subject means all animals, such as rats, mice, livestock, including humans, that have or can develop gastrointestinal stromal tumors. As a specific example, it may be a mammal including a human.
용어 “진단”은 특정 질병 또는 질환에 대한 한 개체의 감수성(susceptibility)을 판정하는 것, 한 개체가 특정 질병 또는 질환을 현재 가지고 있는지 여부를 판정하는 것, 특정 질병 또는 질환에 걸린 한 개체의 예후(prognosis)를 판정하는 것, 또는 테라메트릭스(therametrics) (예컨대, 치료 효능에 대한 정보를 제공하기 위하여 개체의 상태를 모니터링하는 것)을 포함할 수 있다.The term “diagnosis” refers to determining the susceptibility of an individual to a particular disease or condition, determining whether an individual currently has a particular disease or disorder, and the prognosis of an individual afflicted with a particular disease or condition. (prognosis), or therametrics (eg, monitoring a subject's condition to provide information about the efficacy of treatment).
ATF6 mRNA 또는 단백질을 검출하여, 시료 내 ATF6 mRNA 또는 단백질의 발현 수준을 측정할 수 있다. ATF6를 검출하는 물질은 예를 들면 ATF6- 특이적 프라이머, 프로브, 항체일 수 있으나, 이에 제한되는 것은 아니다.By detecting ATF6 mRNA or protein, the expression level of ATF6 mRNA or protein in the sample may be determined. The substance for detecting ATF6 may be, for example, an ATF6-specific primer, probe, or antibody, but is not limited thereto.
ATF6 검출은 상기 검출하는 물질을 이용하여 당 분야에 공지된 방법으로 수행될 수 있으며, 예를 들면 중합효소 연쇄반응, 웨스턴 블로팅, 면역염색, 면역 전기영동, 단백질 면역 침전, 효소 결합 면역 침강 분석법, 액체 크로마토그래피 등을 이용하여 검출할 수 있으나, 이에 제한되는 것은 아니다.ATF6 detection can be performed by a method known in the art using the detecting substance, for example, polymerase chain reaction, Western blotting, immunostaining, immunoelectrophoresis, protein immunoprecipitation, enzyme-linked immunoprecipitation assay , may be detected using liquid chromatography, etc., but is not limited thereto.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 하기 실시예는 본 발명을 예시하는 것으로, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by way of Examples. The following examples illustrate the present invention, but the content of the present invention is not limited to the following examples.
실시예Example
1. 실험 방법1. Experimental method
(1) 세포 배양(1) cell culture
GIST430, GIST430(V654A) 세포주는 Iscove's Modified Dulbecco's Medium (IMDM)에 15%의 supplemented with 15% fetal calf serum과 1%의 penicillin/streptomycin를 혼합한 배지를 이용하였고, GIST882 세포주는 Roswell Park Memorial Institute (RPMI) 배지에 15%의 fetal bovine serum (FBS)와 1%의 penicillin/streptomycin를 혼합한 배지, GIST48 세포주는 IMDM 배지에 15% FBS와 1%의 penicillin/streptomycin를 혼합하여 37 ℃, 5% CO2 조건으로 배양하였다. 대장암 세포주 Colo320DM, DLD-1, LS174T, H69, H128, H209은 RPMI 배지에 10% FBS와 1%의 penicillin/streptomycin를 혼합한 배지를 이용하였고, Kasumi-1 세포주는 RPMI 배지에 20% FBS와 1%의 penicillin/streptomycin를 혼합한 배지, HMC-1, K562세포주는 IMDM 배지에 10% FBS와 1%의 penicillin/streptomycin를 혼합하여 37 ℃, 5% CO2 조건으로 배양하였다.GIST430 and GIST430 (V654A) cell lines were mixed with Iscove's Modified Dulbecco's Medium (IMDM) with 15% supplemented with 15% fetal calf serum and 1% penicillin/streptomycin. The GIST882 cell line was used at Roswell Park Memorial Institute (RPMI). ) In the medium mixed with 15% fetal bovine serum (FBS) and 1% penicillin/streptomycin, the GIST48 cell line is an IMDM medium mixed with 15% FBS and 1% penicillin/streptomycin at 37 °C, 5% CO2 condition. incubated with For colorectal cancer cell lines Colo320DM, DLD-1, LS174T, H69, H128, and H209, RPMI medium mixed with 10% FBS and 1% penicillin/streptomycin was used, and Kasumi-1 cell line was RPMI medium with 20% FBS and 20% FBS. In a medium containing 1% penicillin/streptomycin, HMC-1 and K562 cell lines were cultured at 37 °C and 5% CO2 by mixing 10% FBS and 1% penicillin/streptomycin in IMDM medium.
(2) 웨스턴 블랏(2) Western blot
웨스턴 블랏 수행을 위해 protease 저해제를 포함하는 Passive lysis buffer를 활용하여 세포를 용해시켜준 후 해당 lysate를 얻어 전기 영동을 수행한 후(premade gel 사용, Invitrogen) 멤브레인으로 transfer 해주었다(iBlot2 system). 이후 nonfat dried milk나 5% BSA 용액으로 블로킹해준 후 1차 항체와 4℃로 밤새 결합시켜준다. 워싱 후 2차 항체를 상온에서 1시간 결합시켜준 후 충분히 워싱하여 잔여 항체를 제거하고 LAS4000 시스템을 활용하여 이미지를 분석한다.사용된 항체는 c-KIT (DAKO), ATF6 (Abcam), GAPDH (Trevigen), Phospho-IRE1alpha(Invitrogen), IRE1alpha, phosphor-PERK, PERK, CHOP, HSP70, HSP90, GRP94, BIP, LC3 (Cell signaling)이다.For Western blotting, the cells were lysed using a passive lysis buffer containing a protease inhibitor, and then the lysate was obtained and electrophoresed (premade gel used, Invitrogen) and transferred to a membrane (iBlot2 system). After blocking with nonfat dried milk or 5% BSA solution, the primary antibody and binding at 4 ℃ overnight. After washing, the secondary antibody is allowed to bind at room temperature for 1 hour, and then the residual antibody is removed by washing sufficiently and the image is analyzed using the LAS4000 system. The antibodies used are c-KIT (DAKO), ATF6 (Abcam), GAPDH ( Trevigen), Phospho-IRE1alpha (Invitrogen), IRE1alpha, phosphor-PERK, PERK, CHOP, HSP70, HSP90, GRP94, BIP, LC3 (Cell signaling).
(3) 면역 세포 화학 염색(3) Immunocytochemical staining
이미징 디쉬(imaging dish)에 배양한 세포를 워싱한 후 4% paraformaldehyde 또는 100% 메탄올로 고정시킨다. 이후 0.2% Triton X-100으로 세포막 투과를 유도하고 1차 항체를 4도에서 오버나잇으로 인큐베이션한다. 2차 항체를 상온에서 1시간 인큐베이션한 후 충분히 워싱하고 DAPI (4',6-diamidino-2-phenylindole) 염색하여 공초점 현미경을(Carl zeiss, LSM710) 이용하여 이미징을 수행한다.After washing the cultured cells in an imaging dish, they are fixed with 4% paraformaldehyde or 100% methanol. Thereafter, cell membrane permeation is induced with 0.2% Triton X-100, and the primary antibody is incubated overnight at 4 degrees. After the secondary antibody is incubated for 1 hour at room temperature, it is washed thoroughly, DAPI (4',6-diamidino-2-phenylindole) staining is performed, and imaging is performed using a confocal microscope (Carl zeiss, LSM710).
(4) 면역 조직 화학 염색(4) Immunohistochemical staining
파라핀 포매 포르말린 고정 조직 블록을 4-μm 두께로 섹션하여 Ventana XT 자동화 염색기를 이용하여 면역 조직 화학 염색을 수행하였다. ATF6의 발현 정도는 숙련된 병리의사 2명이 독립적으로 핵발현 정도를 판독하여 정량하였다. Paraffin-embedded formalin-fixed tissue blocks were sectioned to a thickness of 4-μm and immunohistochemical staining was performed using a Ventana XT automated stainer. The expression level of ATF6 was quantified by independently reading the nuclear expression level by two experienced pathologists.
(5) 통계분석(5) Statistical analysis
ATF6의 핵발현에 따른 무병생존기간은 SPSS 소프트웨어를 활용하여 분석하였고 Mann-Whitney tests, Student’s t-tests, Fisher’s exact tests, or chi-square tests가 사용되었다. MTT 어세이 결과의 통계 분석은 one-way analysis of variance (ANOVA) with a post hoc test (Bonferroni) 방법을 통해 분석하였고 데이터는 표준 오차 막대를 포함하여 제시되었다. 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 the MTT assay results was analyzed by one-way analysis of variance (ANOVA) with a post hoc test (Bonferroni), and data were presented including standard error bars.
(6) 세포성장분석 어세이(6) cell growth analysis assay
각 세포주를 96-well plate에 104cells/well이 되도록 seeding 하고 24시간 후 ATF6 저해제인 PF429242, Ceapin A7, Melatonin을 각각의 농도 별로 12시간 전처리 하였다. 이후 각 ATF6 저해제와 Thapsigargin 0.1μM 또는 Bortezomib 1nM 또는 17AAG 0.5μM 또는 Imatinib 0.1μM를 포함한 배지로 교환하였다. 37 ℃, 5% CO2 incubator에서 72시간 배양한 후에 살아있는 세포 수의 측정을 위해 MTT ([3-(4,5-dimethylthiazol-2-yl-) 2,5-diphenyltetrazolium bromide) assay를 수행하였다. MTT 용액을 배양 배지에서 최종 농도 0.5mg/ml가 되도록 각각의 well에 추가한 후 37 ℃, 5% CO2 incubator에서 4시간 배양하였다. 배양 후 용액을 완전히 제거하고 DMSO를 100μl씩 넣어 20분 동안 상온에서 incubation하여 세포를 lysis함과 동시에 살아있는 세포에 의해 생성된 자주색의 MTT metabolite (결정성)을 용해하였다. DMSO에 용해된 MTT metabolite의 양을 ELISA Plate Reader로 570nm에서 흡광도를 측정하였다.Each cell line was seeded in a 96-well plate at a concentration of 10 4 cells/well, and after 24 hours, ATF6 inhibitors PF429242, Ceapin A7, and Melatonin were pretreated at each concentration for 12 hours. Afterwards, each ATF6 inhibitor was exchanged with a medium containing 0.1 μM of Thapsigargin or 1 nM of Bortezomib or 0.5 μM of 17AAG or 0.1 μM of Imatinib. MTT ([3-(4,5-dimethylthiazol-2-yl-) 2,5-diphenyltetrazolium bromide) assay was performed to measure the number of viable cells after 72 hours of incubation in a 37 °C, 5% CO2 incubator. MTT solution was added to each well to have a final concentration of 0.5 mg/ml in the culture medium, and then incubated at 37 ° C., 5% CO2 incubator 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) generated by the living cells. The amount of MTT metabolite dissolved in DMSO was measured for absorbance at 570 nm with an ELISA plate reader.
(7) 동물 실험(7) Animal testing
동물실험에는 7주령의 BALB/c-nude male 마우스를 사용하였으며, 이에 Imatinib 반응성 GIST430 세포와 imatinib 내성 GIST430-V654A 세포주를 피하 주사(subcutaneous injection)을 하여 xenograft 모델을 만들었다. 상기 세포는 1 x 107개를 50 ul IMDM 배지에 희석하고 마트리젤과 1:1로 섞어 호흡 마취한 마우스의 오른쪽 flank에 주사하였으며, 주사한 날 (day 0)으로부터 3일 간격으로 종양 크기, 체중을 측정였다. 이후 2~3주경 종양 크기가 70-100mm3 이상으로 자란 마우스를 그룹당 4~5마리로 임의로 선정한 후, 약물을 복강 주사로 투여하였다. 약물 투여 14~16일 후 적절한 시점에 희생(sacrifice)하였으며, 마우스의 체중, 종양 크기, 종양 무게 등을 측정하여 투여 약물의 종양 억제 효용성을 검증하였다. 약물의 농도는 각각 이마티닙의 경우 50mg/kg (20g 마우스 기준 환산: 1mg/20g 마우스), PF-429242의 경우 30mg/kg (0.6mg/20g 마우스), Bortezomib의 경우 1mg/kg(0.02mg/20g 마우스)로 투여되었다.For animal experiments, 7-week-old BALB/c-nude male mice were used, and the xenograft model was created by subcutaneous injection of imatinib-responsive GIST430 cells and imatinib-resistant GIST430-V654A cell lines. 1 x 10 7 cells were diluted in 50 ul IMDM medium, mixed with Matrigel 1:1, and injected into the right flank of a mouse under respiratory anesthesia, the tumor size, weight was measured. After 2-3 weeks, 4-5 mice were randomly selected per group with tumors growing to 70-100 mm 3 or more, and the drug was administered by intraperitoneal injection. After 14 to 16 days of drug administration, sacrifices were made at an appropriate time, and the tumor-suppressing efficacy of the administered drug was verified by measuring the mouse weight, tumor size, and tumor weight. The concentration of the drug was 50 mg/kg for imatinib (conversion based on 20 g mouse: 1 mg/20 g mouse), 30 mg/kg for PF-429242 (0.6 mg/20 g mouse), and 1 mg/kg (0.02 mg/20 g for bortezomib), respectively. mice).
2. 실험 결과2. Experimental results
(1) GIST에서의 ATF6 과발현 확인(1) Confirmation of ATF6 overexpression in GIST
정상 KIT 또는 돌연변이 KIT을 발현하는 여러 암종 (소세포폐암: H69, H128; 백혈병: ,H209, Kasumi-1, HMC-1; 대장암: K562, LS174T, Colo320DM, DLD-1, GIST: GIST430, GIST882)에서 ATF6는 GIST에서 특이적으로 과발현되지 않았으나, ATF6의 활성화 형태인 cleaved ATF6 (cATF6)의 경우에는 다른 암종에 비하여 GIST세포주에서 발현이 특이적으로 높은 것을 확인할 수 있었다 (도 1A).Several carcinomas expressing normal KIT 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 in the case of cleaved ATF6 (cATF6), an activated form of ATF6, it was confirmed that the expression was specifically high in GIST cell lines compared to other carcinomas (FIG. 1A).
ATF6는 c-terminal 부분 (소포체 내강)과 N-terminal 부분(세포질)으로 구성되는데, ATF6가 골지체로 이동한 후 N-terminal 부분이 잘려 핵 내로 들어가게 되면 세포 생존, 사멸 및 성장에 관련된 유전자를 고절하여 암세포 사멸을 막는 것으로 알려져 있다. ATF6는 cleaved ATF6 (cATF6) 형태로 핵으로 이동하여야 활성화 되어있는 것으로 볼 수 있는데, ATF6는 GIST 세포주의 핵 내에 위치하고 있음을 공초점 형광 현미경 결과로도 확인할 수 있었다 (도 1B)ATF6 is composed of a c-terminal part (endoplasmic reticulum lumen) and an N-terminal part (cytoplasm). After ATF6 moves to the Golgi body, the N-terminal part is cut off and enters the nucleus, resulting in the disruption of genes related to cell survival, death and growth. It is known to prevent cancer cell death. ATF6 can be seen to be activated only when it migrates to the nucleus in the form of cleaved ATF6 (cATF6), and it was confirmed by confocal fluorescence microscopy that ATF6 is located in the nucleus of the GIST cell line (Fig. 1B).
GIST 환자에서도 ATF6가 핵 내 분포하며 과발현되어 있음을 조직 면역 염색 결과를 통해서도 확인할 수 있었다 (도 1C).It was also confirmed through tissue immunostaining results that ATF6 was distributed in the nucleus and overexpressed in GIST patients (FIG. 1C).
(2) GIST 세포주에서 ER stress 에 따른 세포 사멸 관련 단백질 및 샤페론 단백질의 발현 확인(2) Confirmation of expression of apoptosis-related proteins and chaperone proteins according to ER stress in GIST cell lines
Thapsigargin (TG, ER stress inducer)를 처리하여 mild to strong ER stress를 유도하였을 때 0.1 uM의 mild ER stress의 경우 세포 사멸이 크게 증가하지 않지만 strong ER stress인 5 uM의 경우 세포 사멸이 크게 증가하였다 (도 2A).When mild to strong ER stress was induced by treatment with thapsigargin (TG, ER stress inducer), apoptosis was not significantly increased in the case of mild ER stress of 0.1 uM, but apoptosis was significantly increased in the case of strong ER stress of 5 uM ( Figure 2A).
Strong ER stress를 유도할 경우, ATF6 및 cATF6의 발현이 감소하며, cell death marker인 CHOP이 증가한 것을 확인할 수 있었는데, 이는 GIST 세포가 strong ER stress는 견뎌낼 수 없음을 의미한다. 반면 ER stress의 다른 pathway이며, 세포 사멸에 관여하는 것으로 알려진 PERK, IRE1a의 활성화형 (p-PERK, p-IRE1a) 경우 발현이 증가하여 세포 사멸을 유도하는 것을 알 수 있었다(도2B). When strong ER stress was induced, the expression of ATF6 and cATF6 was decreased, and it was confirmed that CHOP, a cell death marker, was increased, which means that GIST cells cannot withstand strong ER stress. On the other hand, it was found that the expression of PERK and IRE1a activated (p-PERK, p-IRE1a), which is another pathway of ER stress and is known to be involved in apoptosis, was increased to induce apoptosis (Fig. 2B).
mild ER stress를 유도할 경우, ATF6, cATF6의 발현이 초반에는 조금 감소하지만 결국 mild ER stress 정도는 24시간이 지나면 극복을 하고 세포 사멸 마커인 CHOP역시 증가하다 감소하는 양상을 보였다. 이러한 결과는 ATF6가 활성화될 경우에는 암세포가 ER stress에 의해 사멸되는 것을 억제할 수 있음을 의미한다. 특히 ATF6에 의해 BIP, HSP90, GRP94, 등의 chaperone 단백질의 발현을 증가시켜 암세포 사멸을 막기위한 방어기전을 (잘못접힘 단백질 반응 기전, unfolded protein response) 작동시킨다는 사실도 확인할 수 있었다 (도 2 C).When mild ER stress was induced, the expression of ATF6 and cATF6 decreased slightly at the beginning, but eventually the mild ER stress level was overcome after 24 hours, and CHOP, an apoptosis marker, also increased and decreased. These results mean that when ATF6 is activated, it can inhibit the death of cancer cells by ER stress. In particular, it was also confirmed that ATF6 increases the expression of chaperone proteins such as BIP, HSP90, GRP94, and activates a defense mechanism to prevent cancer cell death (unfolded protein response) (Fig. 2C). .
(3) ATF6 활성화 억제에 따른 ER stress및 잘못접힘 단백질 반응 기전에 미치는 효과 확인(3) Confirmation of effect on ER stress and misfolded protein response mechanism according to ATF6 activation inhibition
Serine 1 protease (S1P) inhibitor (S1Pi)는 ATF6의 cleavage 및 활성화를 억제하는데, 이를 GIST 세포에 처리해주면 ATF6 활성화 억제(cATF6 발현 억제), 하위 chaperone 발현 억제가 관찰된다 (도 3A). TG를 0.1 uM로 처리하여 mild ER stress를 유발한 경우에는 일반적으로 세포 사멸이 거의 이루어지지 않지만, S1Pi를 같이 처리할 경우에는 mild ER stress에서도 급격한 세포 사멸이 나타남을 확인하였다 (도 3B). Serine 1 protease (S1P) inhibitor (S1Pi) inhibits the cleavage and activation of ATF6, and when it is treated in GIST cells, inhibition of ATF6 activation (inhibition of cATF6 expression) and inhibition of sub-chaperone expression are observed (FIG. 3A). When TG was treated with 0.1 uM to induce mild ER stress, apoptosis was generally not achieved, but when treated with S1Pi, rapid cell death was observed even under mild ER stress (FIG. 3B).
TG 와 함께 SP1i 를 처리할 경우 ER stress 유도에 따른 BIP, GRP94, HSP90 등의 ATF6 pathway dependent chaperone의 발현 증가 타이밍이 많이 delay되는 것을 볼 수 있는데, 이는 ATF6 pathway를 억제하면 암세포가 ER stress에 적절히 대응하지 못하여 방어기전이 빠르게 작동하지 못해 세포사멸에 이르게 됨을 의미한다 (도 3C).When SP1i was treated together with TG, the timing of increase in expression of ATF6 pathway-dependent chaperones such as BIP, GRP94, and HSP90 following ER stress induction was greatly delayed. This means that the defense mechanism does not work quickly, leading to apoptosis (FIG. 3C).
(4) ATF6 억제에 따른 HSP90 발현 및 KIT 발현 억제(4) Suppression of HSP90 expression and KIT expression according to ATF6 inhibition
ATF6에 의해 조절되는 샤페론 단백질 중 GIST 발생에 중요한 역할을 수행하는 KIT와 결합하는 샤페론 단백질을 확인하기 위해 GIST 세포주에 KIT 항체를 이용하여 면역침강법을 수행한 결과, ATF6에 의해 조절되는 chaperone인 HSP90가 돌연변이 KIT 단백질과 결합하고 있음을 확인할 수 있었다 (도4). 이러한 결과는 ATF6를 억제하여 하위 신호전달체계에 위치한 샤페론의 발현이 억제되면, GIST의 발생원인 중 하나인 돌연변이 KIT 단백 역시 부수적으로 억제가 가능하여 강력한 항암 효과를 유도할 수 있음을 간접적으로 보여주는 결과이다. 실제로 ATF6를 억제하고, 그에 따른 HSP90 및 KIT의 발현수준 변화를 확인해본 결과, KIT 및 HSP90의 발현이 억제되었음을 확인할 수 있었다. 또한, HSP90의 저해제인 17AAG를 처리하여 HSP90을 억제한 경우에도 KIT 단백질의 발현이 감소되었음을 확인하였는데 (도 5), 이러한 결과들를 종합해보면 HSP90를 조절하는 ATF6를 저해할 경우, HSP90가 억제되고 그에 따라 KIT 단백질의 발현까지 억제되는 것이 타당하다는 결론에 도달하게 된다.Among the chaperone proteins regulated by ATF6, to identify chaperone proteins that bind to KIT, which play an important role in GIST generation, immunoprecipitation was performed on GIST cell lines using KIT antibody. As a result, HSP90, a chaperone regulated by ATF6, was performed. It was confirmed that the mutant KIT protein was bound (Fig. 4). These results indirectly show that when the expression of a chaperone located in the lower signaling system is suppressed by suppressing ATF6, the mutant KIT protein, one of the causes of GIST, can also be suppressed incidentally, thereby inducing a strong anticancer effect. to be. In fact, it was confirmed that the expression of KIT and HSP90 was suppressed as a result of inhibiting ATF6 and checking the change in the expression level of HSP90 and KIT accordingly. 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 (FIG. 5). Taken together, these results show that when ATF6, which regulates HSP90, is inhibited, HSP90 is inhibited and thus Accordingly, we arrive at the conclusion that it is appropriate to suppress the expression of KIT protein.
(5) GIST 세포주에서의 ATF6 억제제 및 이마티닙의 병용투여 효과 확인(5) Confirmation of co-administration effect of ATF6 inhibitor and imatinib in GIST cell line
Imatinib에 저항성을 보이는 세포주인 GIST430과 GIST430 세포에서 추가로 V654A 2차 돌연변이가 생긴 imatinib 내성 세포주인 GISTS430(V654A)를 이용하여 세포 성장 평가 어세이 (MTT 어세이) 수행하였다. 종양 미세환경은 영양분 및 산소 부족과 같은 제약에 따라 ER stress가 유도되는데, Thapsigargin (TG)는 in vivo와 유사한 ER stress 환경을 모사하기 위해 처리하는 것으로, 본 발명에서는 처리한 경우 (도 6)와 ATF6 억제제 자체의 효과를 확인하기 위해 처리하지 않은 경우(도 7)로 나누어 실험하였다.Cell growth evaluation assay (MTT assay) was performed using the imatinib-resistant cell line GIST430 (V654A), an imatinib-resistant cell line with additional V654A secondary mutation in GIST430 and GIST430 cells, which are resistant to imatinib. In the tumor microenvironment, ER stress is induced by constraints such as lack of nutrients and oxygen. Thapsigargin (TG) is treated to simulate an ER stress environment similar to in vivo, and in the present invention, when treated (FIG. 6) and In order to check the effect of the ATF6 inhibitor itself, the experiment was divided into untreated cases (FIG. 7).
결과적으로 TG를 처리하거나 처리하지 않은 두 경우 모두 ATF6 저해제(PF429242, CeapinA7, Melatonin) 처리시 강력하게 세포사멸이 유도됨을 확인할 수 있었고, ER stress 유도 물질(Bortezomib, 17AAG)만을 투여하였을 경우에도 강력한 세포사멸이 발생하였으며, ATF6 저해제와 ER stress 유도 물질을 동시에 투여하였을 경우 강력한 종양 억제능 시너지를 확인하였다 (도 6 및 도 7). 특히 이러한 결과는 이마티닙에 대해 반응성을 보이거나 내성을 보이는 세포주에서 모두 확인한 것으로, ATF6를 저해하는 치료 접근법이 이마티닙에 대해 내성을 보이는 환자군에게 새로운 치료 옵션이 될 수 있음을 보여주는 것이며, 병용치료의 측면에서는As a result, in both cases treated with or without TG, it was confirmed that apoptosis was strongly induced when ATF6 inhibitors (PF429242, CeapinA7, Melatonin) were treated. Death occurred, and when an ATF6 inhibitor and an ER stress-inducing substance were administered at the same time, a strong tumor suppressive synergy was confirmed ( FIGS. 6 and 7 ). In particular, these results were confirmed in all cell lines showing responsiveness to or resistance to imatinib, showing that a therapeutic approach that inhibits ATF6 can be a new treatment option for the patient group resistant to imatinib. in
Imatinib과 ATF6 저해제를 병용치료로 사용하면 imatinib 치료 효과를 보조 및 개선하여 치료 초기에 강력한 종양 억제능을 통해 근치적 치료법 개발이 가능할 것이다.If imatinib and ATF6 inhibitors are used as a combination treatment, it will be possible to develop a curative treatment with strong tumor suppression ability at the early stage of treatment by supporting and improving the effect of imatinib treatment.
(6) ATF6 발현과 생존확률과의 상관관계 확인(6) Confirmation of correlation between ATF6 expression and survival probability
GIST 환자 50명로부터 얻은 조직을 ATF6 항체를 활용하여 면역 조직Immune tissue obtained from 50 GIST patients using ATF6 antibody
화학 염색을 통해 ATF6의 핵발현 정도를 분석하였고 발현 유무에 따른 무병생존기간을 통계 분석한 결과 ATF6의 핵발현이 강할수록 무병생존기간이 짧아지는 경향성이 나타남을 확인하였다.The degree of nuclear expression of ATF6 was analyzed through chemical staining, and as a result of statistical analysis of the disease-free survival period according to the presence or absence of expression, it was confirmed that the stronger the nuclear expression of ATF6, the shorter the disease-free survival period appeared.
(7) GIST 마우스 모델에서의 효과 확인(7) Confirmation of effect in GIST mouse model
GIST의 표준 치료제인 imatinib과 ATF6 저해제인 PF-429242, 그리고 ER 스트레스 유도 약물인 Bortezomib과 ATF6 저해제 PF-429242의 병용 투여를 비교하였다 (도 9). GIST430 세포주는 imatinib 반응성 환자 유래 세포주이며, GIST430-V654A 세포주는 imatinib 내성 환자 유래 세포주이며, 각 세포주를 마우스 모델에 피하주사(subcutaneous injection)하여 xenograft 모델을 제작하였다. 상기 마우스 모델에 각각의 약물을 투여한 결과, GIST430 세포 xenograft 모델의 경우 imatinib과 ATF6 저해제인 PF-429242, 그리고 ER 스트레스 유도 약물인 Bortezomib과 ATF6 저해제 PF-429242의 병용 투여 모든 경우에서 우수한 종양 억제능이 관찰되었으나, imatinib 내성 세포주의 경우 imatinib은 종양 억제능이 나타나지 않았다. 반면 PF-429242를 처리한 경우에는 imatinib 보다는 우수한 종양 억제능을 보였고, 더 나아가 Bortezomib과 PF-429242를 병용 처리한 경우에는 매우 우수한 종양 억제능을 나타내었다. 이러한 결과는 imatinib을 대체할 수 있는 새로운 종양 억제 요법을 제시할 수 있을 것으로 보인다.The combined administration of imatinib, the standard treatment for GIST, and PF-429242, an ATF6 inhibitor, and Bortezomib, an ER stress inducing drug, and PF-429242, an ATF6 inhibitor was compared (FIG. 9). The GIST430 cell line is an imatinib-responsive patient-derived cell line, and the GIST430-V654A cell line is a cell line derived from an imatinib-resistant patient. As a result of administering each drug to the mouse model, in the case of the GIST430 cell xenograft model, excellent tumor suppression ability was obtained in all cases of co-administration of imatinib and PF-429242, an ATF6 inhibitor, and Bortezomib, an ER stress-inducing drug, and PF-429242, an ATF6 inhibitor. However, in the case of imatinib-resistant cell lines, imatinib did not show tumor suppression activity. On the other hand, when PF-429242 was treated, it showed superior tumor suppression ability than imatinib. These results seem to suggest a new tumor suppressor therapy that can replace imatinib.

Claims (13)

  1. ATF6 억제제를 포함하는 ATF6 과발현 종양의 예방 또는 치료용 약학 조성물.A pharmaceutical composition for preventing or treating ATF6 overexpressing tumors comprising an ATF6 inhibitor.
  2. 청구항 1에 있어서 , 상기 ATF6 과발현 종양은 유방암, 담관암, 대장 선암, 식도암, 다형성 교모세포종, 급성 골수성 백혈병, 뇌 저등급 신경교종, 간암, 췌장암, 갈색 세포종/부신경 절종, 육종, 피부 흑색종, 위암, 난소암, 갑상선암, 흉선종, 고환암, 직장암, 전립선암, 부신 피질 암, 방광암, 림프종, 중피종, 폐암, 자궁내막암, 자궁경부암, 신장암, 두 경부암, 위장관암, 뇌수막종, 뇌하수체 선종, 청신경초종, 두개인두종, 모낭형 성상세포종, 혈관아 세포종, 미만성 성상세포종, 핍지 세포종, 비정형 뇌수박종, 상의 세포종 및 위장관 기질종양으로 이루어진 군에서 선택되는 적어도 하나인, ATF6 과발현 종양의 예방 또는 치료용 약학 조성물.The method according to 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, gastric cancer , ovarian cancer, thyroid cancer, thymoma, testicular cancer, rectal cancer, prostate cancer, adrenal 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 , At least one selected from the group consisting of craniopharyngoma, follicular astrocytoma, hemangioblastoma, diffuse astrocytoma, oligodendroblastoma, atypical hydrocephalus, epithelial celloma and gastrointestinal stromal tumor, pharmaceutical composition for preventing or treating ATF6 overexpressing tumor .
  3. 청구항 1에 있어서, 상기 ATF6 억제제는 PF429242, Ceapin A7 및 Melatonin으로 이루어진 군에서 선택되는 적어도 하나인, ATF6 과발현 종양의 예방 또는 치료용 약학 조성물.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.
  4. 청구항 1에 있어서, 티로신 키나제 억제제를 더 포함하는, ATF6 과발현 종양의 예방 또는 치료용 약학 조성물.The pharmaceutical composition for preventing or treating ATF6 overexpressing tumors according to claim 1, further comprising a tyrosine kinase inhibitor.
  5. 청구항 4에 있어서, 상기 티로신 키나제 억제제는 이마티닙, 수니티닙, 레고라페닙, 아이바키트, 아파티닙, 악시티닙, 보스티닙, 카보잔티닙, 세툭시맙, 세리티닙, 크리조티닙, 다브라페닙, 다사티닙, 에를로티닙, 에버롤리무스, 제피티닙, 이브루티닙, 이델라리십, 라파티닙, 렌바티닙, 닐로티닙, 닌테다닙, 파조파닙, 포나티닙, 팔보시클립, 럭솔리티닙, 소라페닙, 트라스투주맙, 트라메티닙, 토파시티닙, 반데타닙 및 베무라페닙으로 이루어진 군에서 선택되는 적어도 하나인, ATF6 과발현 종양의 예방 또는 치료용 약학 조성물.5. The method of claim 4, wherein the tyrosine kinase inhibitor is imatinib, sunitinib, regorafenib, ivakit, afatinib, axitinib, vostinib, caboxantinib, cetuximab, ceritinib, crizotinib, Dabrafenib, dasatinib, erlotinib, everolimus, gefitinib, ibrutinib, idelarisib, lapatinib, lenvatinib, nilotinib, nintedanib, pazopanib, ponatinib, palvo At least one selected from the group consisting of ciclib, luxolitinib, sorafenib, trastuzumab, trametinib, tofacitinib, vandetanib, and vemurafenib, a pharmaceutical composition for preventing or treating ATF6 overexpressing tumors.
  6. 청구항 4에 있어서, 상기 티로신 키나제 억제제는 ATF6 억제제와 동시에 또는 순차적으로 투여되는, ATF6 과발현 종양의 예방 또는 치료용 약학 조성물.The pharmaceutical composition of claim 4, wherein the tyrosine kinase inhibitor is administered simultaneously or sequentially with the ATF6 inhibitor.
  7. 시료의 ATF6 또는 그 mRNA의 수준을 측정하는 단계를 포함하는 위장관 기질종양의 진단 또는 예후예측을 위한 정보 제공 방법.A method for providing information for diagnosis or prognosis of a gastrointestinal stromal tumor comprising measuring the level of ATF6 or its mRNA in a sample.
  8. 청구항 7에 있어서, 상기 시료는 개체 또는 개체로부터 분리된 것인, 위장관 기질종양의 진단 또는 예후예측을 위한 정보 제공 방법.The method according to claim 7, wherein the sample is an individual or isolated from the individual, information providing method for diagnosis or prognosis of gastrointestinal stromal tumor.
  9. 청구항 7에 있어서, 상기 ATF6 또는 그 mRNA의 수준이 정상 대조군의 발현 정도에 비해 높은 경우 위장관 기질종양으로 판단하는 단계를 더 포함하는, 위장관 기질종양의 진단 또는 예후예측을 위한 정보 제공 방법.The method of claim 7 , further comprising determining a gastrointestinal stromal tumor when the level of ATF6 or its mRNA is higher than that of a normal control group.
  10. 청구항 7에 있어서, 상기 ATF6 또는 그 mRNA의 수준이 높을수록 위장관 기질종양의 예후가 나쁠 것으로 판단하는 단계를 더 포함하는, 위장관 기질종양의 진단 또는 예후예측을 위한 정보 제공 방법.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.
  11. ATF6 또는 그 mRNA의 수준을 측정하는 물질을 포함하는 위장관 기질종양의 진단 또는 예후예측용 조성물.A composition for diagnosis or prognosis of gastrointestinal stromal tumor comprising a substance measuring the level of ATF6 or its mRNA.
  12. 청구항 11에 있어서, 상기 ATF6 또는 그 mRNA의 수준을 측정하는 물질은 ATF6 특이적으로 결합하는 프라이머, 프로브, 항체로 이루어진 군에서 선택되는 것인, 위장관 기질종양의 진단 또는 예후예측용 조성물.The composition for diagnosis or 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.
  13. 시료의 ATF6 또는 그 mRNA의 발현을 감소시키는 물질을 선별하는 단계를 포함하는 위장관 기질종양 치료제 후보물질의 스크리닝 방법.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.
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