KR20230084059A - A composition for preventing or treating biliary tract cancer - Google Patents

Abstract

The composition according to the present invention selects patients with overexpression of the PUM1-TRAF3 fusion protein or the gene encoding the same, and has genetic characteristics suitable for drugs including NF-κB inhibitor drugs, particularly QNZ or pharmaceutically acceptable salts thereof. By selecting a subject and administering the drug to the selected subject, the treatment effect of cholangiocarcinoma can be maximized.

Description

Composition for preventing or treating biliary tract cancer {A composition for preventing or treating biliary tract cancer}

The present invention relates to a composition for preventing or treating bile duct cancer, and can provide a method for maximizing drug effects by simultaneously diagnosing a patient's genetic characteristics.

The bile duct is a tube that carries bile produced in the liver to the duodenum. Among the extrahepatic bile ducts, the sac that temporarily stores and concentrates bile is called the gallbladder, and the bile duct and the gallbladder are collectively called the biliary tract. Biliary Tract Cancer (BTC), also called cholangiocarcinoma, is a malignant tumor arising from the epithelium of the bile duct. Bile duct cancer is largely classified into extrahepatic cholangiocarcinoma, intrahepatic cholangiocarcinoma, and gallbladder cancer. It is one of the incurable cancers that are very difficult to treat due to its low survival rate. As described above, since cholangiocarcinoma is discovered after a significant progression of the cancer and has a poor prognosis and is difficult to treat, early diagnosis and treatment of cholangiocarcinoma are urgently needed.

Early diagnosis of bile duct cancer is very difficult as no characteristic symptoms appear until it has progressed significantly, and curative resection (removal of all areas where cancer exists or is likely to exist as much as possible) is very difficult as it invades nearby major organs at the time of diagnosis. There are cases where this is not possible. The treatment method is selected in consideration of the size, location, stage of the cancer, age and health condition of the patient, etc., and one or several methods are used in combination.

In particular, biliary tract cancer (BTC) is one of the most lethal malignancies with a 5-year survival rate of less than 20%. To date, gemcitabine is known to be most effective in biliary tract cancer. Combination therapy is being performed, but there is a limitation that it is not effective in all patients with cholangiocarcinoma.

Accordingly, the inventors of the present invention completed the present invention by discovering a new therapeutic target drug capable of precisely providing treatment according to the genetic characteristics of patients with bile duct cancer.

One object of the present invention is to provide a composition for determining the suitability of a therapeutic agent for cholangiocarcinoma.

Another object of the present invention is to provide a kit for determining the suitability of a therapeutic agent for bile duct cancer.

Another object of the present invention is to provide a method for providing information on the suitability of a therapeutic agent for cholangiocarcinoma.

Another object of the present invention is to provide a diagnostic device that provides information on the suitability of a therapeutic agent for cholangiocarcinoma.

Another object of the present invention is to provide a composition for preventing, improving or treating bile duct cancer.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

Hereinafter, various embodiments described herein are described with reference to the drawings. In the following description, numerous specific details are set forth, such as specific forms, compositions and processes, etc., in order to provide a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or with other known methods and forms. In other instances, well known processes and manufacturing techniques have not been described in specific detail in order not to unnecessarily obscure the present invention. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, form, composition or characteristic described in connection with the embodiment is included in one or more embodiments of the invention. Thus, the appearances of "in one embodiment" or "an embodiment" in various places throughout this specification do not necessarily refer to the same embodiment of the invention. Additionally, particular features, forms, compositions, or properties may be combined in one or more embodiments in any suitable way.

Unless there is a specific definition within the specification, all scientific and technical terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention belongs.

According to one embodiment of the present invention, it relates to a composition for determining the suitability of a cancer treatment agent.

In the present invention, the composition for discrimination may include an agent for measuring the expression level of a pumilio RNA binding family member 1-TNF receptor associated factor 3 (PUM1-TRAF3) fusion protein or a gene encoding the same.

In the present invention, the fusion protein or the fusion gene encoding the same is caused by a kind of genetic mutation, and the fusion gene is a type of genetic mutation, which is generated when two genes existing at different positions are arranged side by side. When the fusion gene occurs, it produces a chimeric protein that affects normal cell proliferation. That is, the fusion gene forms a chimera protein, and the fusion protein causes a modified activity different from that of the existing protein and acts as a principle to affect cell growth. A fusion gene arranged side by side causes a problem in that the later gene is affected by the promoter of the preceding gene and is expressed abnormally. In the history of cancer research, the fusion gene was first reported in CML, which is the famous Philadelphia chromosome. As the genetic loci and functions of chromosomes were identified, they became targets for drug treatment, and in 2001, the drug imatinib received FDA approval and was widely used. In addition, fusion genes have been continuously discovered in B cell lymphoma and T cell neoplasm, and with the recent development of deep sequencing technology, there are now close to 10,000 tumors. Associated fusion genes have been reported.

The PUM1-TRAF3 fusion protein of the present invention is a PUF (pumilio RNA binding family member 1) that plays a certain role in cell division, differentiation and development, PUM1 (pumilio RNA binding family member 1) and the TNF receptor (TNFR) superfamily It is a protein in which some sites of TRAF3 (TNF receptor associated factor 3), which is associated with members of and mediates signal transduction therefrom, exist in a fused form.

The PUM1-TRAF3 fusion protein of the present invention has a 3'-end truncated PUM1 (pumilio RNA binding family member 1) gene fragment sequence on the 5' side, and TRAF3 (TNF receptor associated factor 3) gene fragment sequence on the 3' side. It may include a PUM1-TRAF3 fusion protein encoded by a PUM1-TRAF3 fusion gene having a fragment sequence on the 3' side, the 3'-end is truncated on the 5' side and exon 14 is deleted PUM1 (pumilio RNA binding family member 1) may include a PUM1-TRAF3 fusion protein encoded by a PUM1-TRAF3 fusion gene having a fragment sequence of a gene and having a 3' fragment sequence of a TRAF3 (TNF receptor associated factor 3) gene on the 3' side. However, it is not limited thereto.

In the present invention, the amino acid sequence of the PUM1-TRAF3 fusion protein may include at least one of the sequences represented by SEQ ID NO: 1 or SEQ ID NO: 2, more preferably the sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2 It may include all, but is not limited thereto.

In the present invention, the nucleic acid base sequence encoding the PUM1-TRAF3 fusion protein may include at least one of the sequences represented by SEQ ID NO: 3 or SEQ ID NO: 4, more preferably SEQ ID NO: 3 and SEQ ID NO: 4. It may include all of the sequences shown, but is not limited thereto.

The agent for measuring the level of the PUM1-TRAF3 fusion protein of the present invention consists of an antibody, oligopeptide, ligand, PNA (peptide nucleic acid) and aptamer that specifically bind to the PUM1-TRAF3 fusion protein. It may include at least one selected from the group, but is not limited thereto.

The "antibody" of the present invention refers to a protein molecule capable of specifically binding to the PUM1-TRAF3 fusion protein, and the form of the antibody is not particularly limited, but polyclonal antibody, monoclonal antibody, or antigen binding. If it has, it may be included even if it is part of an antibody, and all types of immunoglobulin antibodies may be included. In addition, special antibodies such as humanized antibodies may be included, and the antibodies include functional fragments of antibody molecules as well as complete forms having two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen-binding function, and may include Fab, F(ab'), F(ab') 2, Fv, etc., but is not limited thereto.

In the present invention, the "oligopeptide" is a peptide composed of 2 to 20 amino acids and may include a dipeptide, tripeptide, tetrapeptide and pentapeptide, but is not limited thereto.

In the present invention, the "PNA (Peptide Nucleic Acid)" refers to an artificially synthesized polymer similar to DNA or RNA, and was first introduced in 1991 by Professors Nielsen, Egholm, Berg and Buchardt of the University of Copenhagen, Denmark. Whereas DNA has a phosphate-ribose backbone, PNA has a repeated N-(2-aminoethyl)-glycine backbone linked by peptide bonds, which greatly increases the binding force and stability to DNA or RNA, and is thus used in molecular biology. , diagnostic assays and antisense therapies. PNA is described by Nielsen PE, Egholm M, Berg RH, Buchardt O (December 1991). "Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide". Science 254 (5037): 1497-1500.

The "aptamer" of the present invention refers to a single-stranded oligonucleotide, and refers to a nucleic acid molecule having binding activity to the PUM1-TRAF3 fusion protein. The aptamer may have various three-dimensional structures according to its base sequence, and may have high affinity for a specific substance, such as an antigen-antibody reaction. An aptamer can inhibit the activity of a given target molecule by binding to the given target molecule. The aptamer may be RNA, DNA, modified nucleic acid, or a mixture thereof, and may be linear or cyclic in shape.

The agent for measuring the expression level of the gene encoding the PUM1-TRAF3 fusion protein of the present invention is one selected from the group consisting of primers, probes and antisense nucleotides that specifically bind to the gene encoding the PUM1-TRAF3 fusion protein It may include the above, but is not limited thereto.

In the present invention, the "primer" is a fragment that recognizes a target gene sequence, and includes a forward and reverse primer pair, preferably a primer pair that provides an analysis result having specificity and sensitivity. High specificity can be imparted when the nucleic acid sequence of the primer is a sequence that is inconsistent with the non-target sequence present in the sample, so that the primer amplifies only the target gene sequence containing a complementary primer binding site and does not cause non-specific amplification. .

In the present invention, the "probe" means a substance that can specifically bind to a target substance to be detected in a sample, and means a substance that can specifically confirm the presence of a target substance in a sample through the binding. The type of probe is not limited as a material commonly used in the art, but preferably may be peptide nucleic acid (PNA), locked nucleic acid (LNA), peptide, polypeptide, protein, RNA or DNA, and most preferably Most likely it is PNA. More specifically, the probe is a biomaterial, including one derived from or similar to a living organism or manufactured in vitro, for example, enzymes, proteins, antibodies, microorganisms, animal and plant cells and organs, nerve cells, DNA, and It may be RNA, DNA includes cDNA, genomic DNA, and oligonucleotides, RNA includes genomic RNA, mRNA, and oligonucleotides, and examples of proteins may include antibodies, antigens, enzymes, peptides, and the like.

In the present invention, the "LNA (Locked nucleic acids)" means a nucleic acid analog containing a 2'-O, 4'-C methylene bridge [J Weiler, J Hunziker and J Hall Gene Therapy (2006) 13, 496.502 ]. LNA nucleosides contain the common nucleic acid bases of DNA and RNA, and can base pair according to the Watson-Crick base pairing rules. However, due to molecular 'locking' due to the methylene bridge, the LNA does not form an ideal shape in the Watson-Crick bond. When LNAs are included in DNA or RNA oligonucleotides, they can more rapidly pair with complementary nucleotide chains to increase the stability of the double helix.

In the present invention, the "antisense" refers to a sequence of nucleotide bases in which an antisense oligomer is hybridized with a target sequence in RNA by Watson-Crick base pairing, allowing the formation of a heteroduplex of mRNA and RNA oligomers in the target sequence, and It refers to an oligomer having an inter-subunit backbone. Oligomers may have exact sequence complementarity or near complementarity to the target sequence.

Since the information of the PUM1-TRAF3 fusion protein according to the present invention or the gene encoding them is known, those skilled in the art can easily design primers, probes or antisense nucleotides that specifically bind to the gene encoding the protein based on this information. You will be able to.

The composition for discrimination of the present invention compares the expression level of the PUM1-TRAF3 fusion protein or the gene encoding it measured in a biological sample isolated from a target object with a control group to determine whether the expression level is increased or decreased, thereby treating cancer suitability can be diagnosed.

In the present invention, the "determination" means to confirm the presence or characteristics of a pathological state, and more specifically, to determine the suitability of a therapeutic agent by confirming the existence or characteristics of a pathological state, in particular, accompanies for application to customized treatment Companion Diagnostics refers to pre-selecting target patients for the purpose of diagnosis and determining whether or not there is a high possibility that a specific treatment will have a good treatment effect.

In the present invention, the companion diagnostics is a test that selects a subject for a target therapeutic agent in advance, and is performed by examining the genetic characteristics of the subject, more specifically, the level of expression of a gene or protein, the presence or absence of a specific gene, etc. It can be. This can provide essential information for effective use of the target drug, and can monitor the response during future treatment by predicting or treating the subject's drug reactivity, drug sensitivity, and the possibility of drug side effects through accompanying diagnosis. there is. Recently, in the field of anticancer drugs, companion diagnostics for personalized treatment are in the limelight.

In the case of using the composition of the present invention, by pre-selecting the patient's drug reactivity before prescribing a cancer treatment, the efficiency of cancer treatment can be increased or the side effects of the treatment can be reduced, and furthermore, the economic effect of preventing unnecessary medical expenses is also achieved. can be expected

B 억제제 또는 NIK 억제제일 수 있으나, 암과 관련된 질환의 예방 또는 치료 효과가 예견되는 약물이라면 이에 제한되는 것은 아니다.In the present invention, the cancer therapeutic agent refers to a candidate drug that inhibits proliferation of cancer cells, invasion or metastasis of cancer cells, and prevents or treats cancer-related diseases even if it corresponds to drugs for preventing or treating diseases other than cancer. It may be included if it corresponds to a drug with a predicted effect, preferably a drug that inhibits nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) or NF-κB-induced kinase ( A drug that inhibits NF-κB-inducing kinase; NIK)

It may be a B inhibitor or a NIK inhibitor, but is not limited thereto as long as it is a drug that is expected to have a preventive or therapeutic effect on cancer-related diseases.

In the present invention, the NF-κB inhibitor is QNZ or a pharmaceutically acceptable salt thereof; MG132 or a pharmaceutically acceptable salt thereof; At least one selected from the group consisting of pyrrolidinedithiocarbamate ammonium or a pharmaceutically acceptable salt thereof and caffeic acid phenethyl ester or a pharmaceutically acceptable salt thereof; It can be done, but is not limited thereto.

In the present invention, the QNZ is 4-N-[2-(4-phenoxyphenyl)ethyl]-1,2-dihydroquinazoline-4,6-diamine represented by Formula 1 (4-N-[2 -(4-Phenoxyphenyl)ethyl]-1,2-dihydroquinazoline-4,6-diamine).

[Formula 1]

In the present invention, the MG132 is benzyl[(2S)-4-methyl-1-{[(2S)-4-methyl-1-{[(2S)-4-methyl-1-oxopentane represented by Formula 2 -2-yl]amino}-1-oxopentan-2-yl]amino}-1-oxopentan-2-yl]carbamate (Benzyl [(2S)-4-methyl-1-{[(2S)- 4-methyl-1-{[(2S)-4-methyl-1-oxopentan-2-yl]amino}-1-oxopentan-2-yl]amino}-1-oxopentan-2-yl]carbamate) there is.

[Formula 2]

In the present invention, the pyrrolidinedithiocarbamate ammonium may be pyrrolidine-1-carbodithioate represented by Formula 3 below.

[Formula 3]

In the present invention, the caffeic acid phenethyl ester is 2-phenylethyl (E) -3- (3,4-dihydroxyphenyl) prop-2-inoate represented by the following formula (4) -3-(3,4-dihydroxyphenyl)prop-2-enoate).

[Formula 4]

In the present invention, the pharmaceutically acceptable salts may include acid or base addition salts, and stereochemical isomers thereof. For example, the compounds may be in the form of addition salts of organic or inorganic acids. Salts have desirable effects on patients when administered to patients, and include any salts that retain the activity of their parent compound, but may not be particularly limited thereto. These salts include inorganic and organic salts such as acetic acid, nitric acid, aspartic acid, sulfonic acid, sulfuric acid, maleic acid, glutamic acid, formic acid, succinic acid, phosphoric acid, phthalic acid, tannic acid, tartaric acid, hydrobromic acid, propionic acid, benzenesulfonic acid, Benzoic acid, stearic acid, lactic acid, bicarboxylic acid, bisulfuric acid, bitartaric acid, oxalic acid, butyric acid, calcium idete, carbonic acid, chlorobenzoic acid, citric acid, idetic acid, toluenesulfonic acid, fumaric acid, gluseptic acid, ecilin Acid, pamoic acid, gluconic acid, methyl nitric acid, malonic acid, hydrochloric acid, hydroiodoic acid, hydroxynaphtholic acid, isethionic acid, lactobionic acid, mandelic acid, mucoic acid, napsylic acid, muconic acid, p -It may include salts of nitromethanesulfonic acid, hexamic acid, pantothenic acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, salicylic acid, sulfamic acid, sulfanilic acid, methanesulfonic acid, and the like. Addition salts of bases include salts of alkali metals or alkaline earth metals, such as salts of ammonium, lithium, sodium, potassium, magnesium, calcium and the like; salts with organic bases such as benzathine, N-methyl-D-glucamine, hydrabamine and the like; and salts with amino acids such as arginine, lysine, and the like. Also, these salts can be converted to the free form by treatment with an appropriate base or acid.

In the present invention, the NIK inhibitor is B022 or a pharmaceutically acceptable salt thereof; XT2 or a pharmaceutically acceptable salt thereof; And NIK SMI1 or a pharmaceutically acceptable salt thereof; may include at least one selected from the group consisting of, but is not limited thereto.

In the present invention, B022 is 4-[1-(2-amino-5-chloropyrimidin-4-yl)-2,3-dihydroindol-6-yl]-2-(1 represented by Formula 5) ,3-thiazol-2-yl)but-3-yn-2-ol (4-[1-(2-amino-5-chloropyrimidin-4-yl)-2,3-dihydroindol-6-yl]- 2-(1,3-thiazol-2-yl)but-3-yn-2-ol).

[Formula 5]

In the present invention, the XT2 is (R) -4- (3- (2-amino-5H-pyrrolo [3,2-d] pyrimidin-7-yl) -4-fluorophenyl represented by the following formula (6) )-2-(thiazol-2-yl)but-3-yn-2-ol ((R)-4-(3-(2-Amino-5H-pyrrolo[3,2-d]pyrimidin-7- yl)-4-fluorophenyl)-2-(thiazol-2-yl)but-3-yn-2-ol).

[Formula 6]

In the present invention, the NIK SMI1 is 6-[3-[2-[(3R)-3-hydroxy-1-methyl-2-oxopyrrolidin-3-yl]ethynyl]phenyl represented by Formula 7 below ]-4-methoxypyridine-2-carboxamide (6-[3-[2-[(3R)-3-hydroxy-1-methyl-2-oxopyrrolidin-3-yl]ethynyl]phenyl]-4- methoxypyridine-2-carboxamide).

[Formula 7]

In the present invention, the pharmaceutically acceptable salts may include acid or base addition salts, and stereochemical isomers thereof. For example, the compounds may be in the form of addition salts of organic or inorganic acids. Salts have desirable effects on patients when administered to patients, and include any salts that retain the activity of their parent compound, but may not be particularly limited thereto. These salts include inorganic and organic salts such as acetic acid, nitric acid, aspartic acid, sulfonic acid, sulfuric acid, maleic acid, glutamic acid, formic acid, succinic acid, phosphoric acid, phthalic acid, tannic acid, tartaric acid, hydrobromic acid, propionic acid, benzenesulfonic acid, Benzoic acid, stearic acid, lactic acid, bicarboxylic acid, bisulfuric acid, bitartaric acid, oxalic acid, butyric acid, calcium idete, carbonic acid, chlorobenzoic acid, citric acid, idetic acid, toluenesulfonic acid, fumaric acid, gluseptic acid, ecilin Acid, pamoic acid, gluconic acid, methyl nitric acid, malonic acid, hydrochloric acid, hydroiodoic acid, hydroxynaphtholic acid, isethionic acid, lactobionic acid, mandelic acid, mucoic acid, napsylic acid, muconic acid, p -It may include salts of nitromethanesulfonic acid, hexamic acid, pantothenic acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, salicylic acid, sulfamic acid, sulfanilic acid, methanesulfonic acid, and the like. Addition salts of bases include salts of alkali metals or alkaline earth metals, such as salts of ammonium, lithium, sodium, potassium, magnesium, calcium and the like; salts with organic bases such as benzathine, N-methyl-D-glucamine, hydrabamine and the like; and salts with amino acids such as arginine, lysine, and the like. Also, these salts can be converted to the free form by treatment with an appropriate base or acid.

In the present invention, the "subject" is a mammal including humans, for example, humans, rats, mice, guinea pigs, hamsters, rabbits, monkeys, dogs, cats, cows, horses, pigs, sheep and goats. It may be selected from, but may be preferably a human, but is not limited thereto.

In the present invention, cancer refers to or refers to a physiological condition typically characterized by unregulated cell growth in mammals. In the present invention, the cancer is biliary tract cancer, gastric cancer, thyroid cancer, parathyroid cancer, ovarian cancer, colon cancer, pancreatic cancer, liver cancer, breast cancer, cervical cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, biliary tract cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, blood cancer, bladder cancer, kidney cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, rectal cancer, brain tumor, perianal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer , endocrine adenocarcinoma, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, CNS central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma and pituitary adenoma It may be at least one selected from the group consisting of, but preferably may be cholangiocarcinoma, but is not limited thereto.

In the present invention, the cholangiocarcinoma is also referred to as cholangiocarcinoma, and is a tumor arising from the epithelium of the bile duct, and is divided into intrahepatic cholangiocarcinoma and extrahepatic cholangiocarcinoma according to its location. In particular, in the case of intrahepatic cholangiocarcinoma, it is distinguished from hepatocellular carcinoma, which we commonly call liver cancer. In general, cholangiocarcinoma occurs frequently in the age group of 60 years or older, and is known to occur 1.3 times more often in men than in women. Since biliary obstruction progresses slowly, when cancer is clinically diagnosed, 70 to 80% of cancers are already quite advanced. .

For the purpose of the present invention, the composition for determining the suitability of the cancer therapeutic agent is non-canonical induced by expression of a PUM1-TRAF3 fusion gene by using a PUM1-TRAF3 fusion marker as a new target marker related to cancer, particularly cholangiocarcinoma, preferably cholangiocarcinoma. It is effective in selecting subjects with genetic characteristics capable of maximizing the therapeutic effect of cholangiocarcinoma because it can determine the suitability of a therapeutic agent with a mechanism that suppresses the activation of NF-κB signaling (non-canonical NF-κB signaling).

According to another embodiment of the present invention, it relates to a kit for determining the suitability of the cancer therapeutic agent of the present invention.

The kit of the present invention can be predicted to be suitable for cancer treatment when the PUM1-TRAF3 fusion protein or the gene encoding it is present at a higher level in a subject using the composition of the present invention than a control group. Preferably, when the expression level of the PUM1-TRAF3 fusion protein or the gene encoding it is present at a higher level than the control group, nuclear factor kappa-light-chain-enhancer of activated B cells; NF-κB ) inhibitor or a candidate drug corresponding to an NF-κB-inducing kinase (NIK) inhibitor can be predicted to have therapeutic suitability.

In the present invention, the "control group" is the expression level of the corresponding biomarker protein or the gene encoding the protein in a normal control group, or the average expression level of the corresponding marker protein or the gene encoding the same in a biological sample derived from a patient with a biliary tract disease. value or median value, or may be the average value or median value of the expression level of the corresponding marker protein or gene encoding it in biological samples derived from cancer patients, preferably from patients with other carcinomas other than cholangiocarcinoma, but are not limited thereto.

The kit of the present invention predicts that the target subject has suitability for cancer treatment when the expression level of the PUM1-TRAF3 fusion protein or the gene encoding it is higher than that of a normal control in the target subject using the composition of the present invention. can

In the kit for discrimination of the present invention, the description of the PUM1-TRAF3 fusion protein or the gene encoding it, cancer, cancer treatment, NF-κB inhibitor, NIK inhibitor, etc. is the same as that described in the composition for discrimination, so excessive omitted to avoid complexity.

The kit of the present invention may be an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a Rapid kit, or a Multiple Reaction Monitoring (MRM) kit, but is not limited thereto.

The kit of the present invention may further include one or more other component compositions, solutions or devices suitable for the assay method. For example, in the present invention, the kit may further include essential elements necessary for carrying out the reverse transcription polymerase reaction. The reverse transcription polymerase reaction kit contains a pair of primers specific for a gene encoding a marker protein. The primer is a nucleotide having a sequence specific to the nucleic acid sequence of the gene, and may have a length of about 7 bp to 50 bp, more preferably about 10 bp to 30 bp. In addition, a primer specific for the nucleic acid sequence of the control gene may be included. Other reverse transcription polymerase reaction kits contain a test tube or other suitable container, reaction buffer (with varying pH and magnesium concentration), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase and RNase inhibitor DEPC. -Water (DEPC-water), sterilized water, etc. may be included.

In addition, the kit for discrimination of the present invention may include essential elements required to perform DNA chip. The DNA chip kit may include a substrate to which cDNA or oligonucleotides corresponding to genes or fragments thereof are attached, and reagents, reagents, enzymes, and the like for producing fluorescently labeled probes. In addition, the substrate may include a cDNA or oligonucleotide corresponding to a control gene or a fragment thereof.

In addition, the kit for discrimination of the present invention may include essential elements necessary for performing ELISA. ELISA kits contain antibodies specific for the protein. An antibody is an antibody that has high specificity and affinity for a marker protein and little cross-reactivity to other proteins, and is a monoclonal antibody, polyclonal antibody, or recombinant antibody. Additionally, the ELISA kit may include antibodies specific for a control protein. Other ELISA kits include reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (eg, conjugated with antibodies) and substrates thereof or those capable of binding the antibody. may contain other substances and the like.

In the kit for discrimination of the present invention, the fixture for the antigen-antibody binding reaction includes a nitrocellulose film, a PVDF film, a well plate made of polyvinyl resin or polystyrene resin, and a glass slide. Glass or the like may be used, but is not limited thereto.

In addition, the marker of the secondary antibody in the kit for discrimination of the present invention is preferably a conventional coloring agent that reacts with color, and HRP (horseradish peroxidase), basic dephosphorylation enzyme (alkaline phosphatase), colloid gold, FITC (Poly L-lysine-fluorscein isothiocyanate), RITC (rhodamine-B-isothiocyanate), and other fluorescent substances (fluorescein) and dyes (dyes) may be used, but are not limited thereto no.

In addition, it is preferable to use a chromogenic substrate for inducing color development in the kit for discrimination of the present invention according to a label that produces a color reaction, such as TMB (3,3',5,5'-tetramethyl bezidine), ABTS [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)], OPD (o-phenylenediamine), etc. can be used. At this time, it is more preferable that the chromogenic substrate is provided in a state dissolved in a buffer solution (0.1 M NaAc, pH 5.5). A chromogenic substrate such as TMB is degraded by HRP used as a marker for the secondary antibody conjugate to generate chromogenic deposits, and the presence or absence of the marker proteins is detected by visually checking the degree of chromogenic deposits.

In the kit for discrimination of the present invention, the washing solution preferably includes a phosphate buffer solution, NaCl and Tween 20, and a buffer solution (PBST) composed of 0.02 M phosphate buffer solution, 0.13 M NaCl, and 0.05% Tween 20 this is more preferable The washing solution is washed 3 to 6 times by reacting the secondary antibody with the antigen-antibody conjugate after the antigen-antibody binding reaction, and then adding an appropriate amount to the stationary body. As the solution for stopping the reaction, a sulfuric acid solution (H ₂ SO ₄ ) may be preferably used.

According to another embodiment of the present invention, it relates to a method for providing information on suitability for cancer treatment.

The method of the present invention includes measuring the expression level of a PUM1-TRAF3 (pumilio RNA binding family member 1-TNF receptor associated factor 3) fusion protein or a gene encoding the same in a biological sample isolated from a subject of interest. .

The method of the present invention may be for screening whether a biological sample isolated from a subject of interest is suitable for a cancer treatment.

In the present invention, the "object of interest" refers to mammals including humans, such as humans, rats, mice, guinea pigs, hamsters, rabbits, monkeys, dogs, cats, cows, horses, pigs, sheep and goats. It may be selected from the group consisting of, and may preferably be a human, but is not limited thereto.

In the present invention, the "human" may refer to a person who has developed cancer or is suspected of having a high possibility of developing cancer, and may mean a patient who needs or is expected to receive appropriate treatment for cancer disease, preferably requires appropriate treatment for biliary tract cancer or It may be an expected patient, but is not limited thereto.

The "biological sample" of the present invention refers to any material, biological fluid, tissue, or cell obtained from or derived from a patient with a cancer disease or a subject suspected of having a cancer disease, for example, whole blood Whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, blood including plasma and serum, sputum, tears ), mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, pelvis Pelvic fluids, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid ), nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, organ secretions, cell, cell extract Or cerebrospinal fluid (cerebrospinal fluid) may be included, but is not limited thereto.

The step of measuring the level of the fusion protein of the present invention includes protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry) using the composition of the present invention. ) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radioimmunodiffusion method, Oukteroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, 2 dimensional electrophoretic analysis, liquid chromatography-mass spectrometry (LC-MS), LC-MS/MS (liquid chromatography-Mass Spectrometry/ Mass Spectrometry), western blotting or ELISA (enzyme linked immunosorbentassay) It may be performed by, but is not limited thereto.

The step of measuring the expression level of the gene encoding the fusion protein of the present invention is reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time reverse transcription using the composition of the present invention It may be performed by polymerase reaction (Real-time RT-PCR), RNase protection assay (RPA), Northern blotting, or DNA chip, but is not limited thereto.

According to the method of the present invention, when the expression level of the PUM1-TRAF3 fusion protein or the gene encoding the same measured in the biological sample is higher than that of the normal control group, it can be predicted that the subject has suitability for cancer treatment. More preferably, when the expression level of the PUM1-TRAF3 fusion protein or the gene encoding it is higher than that of the normal control, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) inhibition It can be predicted that there is suitability for the treatment of bile duct cancer by drugs or drugs that inhibit NF-κB-inducing kinase (NIK).

In the present invention, the cancer is biliary tract cancer, gastric cancer, thyroid cancer, parathyroid cancer, ovarian cancer, colon cancer, pancreatic cancer, liver cancer, breast cancer, cervical cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, biliary tract cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, blood cancer, bladder cancer, kidney cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, rectal cancer, brain tumor, perianal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer , endocrine adenocarcinoma, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, CNS central nervous system tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma and pituitary adenoma It may be at least one selected from the group consisting of, but preferably may be cholangiocarcinoma, but is not limited thereto.

In the method for providing information on the suitability of the cancer treatment of the present invention, PUM1-TRAF3 fusion protein or a gene encoding it, an agent for measuring expression level, cancer, cancer treatment, NF-κB inhibitor, NIK inhibitor, control, etc. The description is the same as that described in the composition for discrimination, and is omitted to avoid excessive complexity of the present specification.

According to another embodiment of the present invention, it relates to a diagnostic device for determining the suitability of a cancer treatment.

The measurement unit of the diagnostic device of the present invention can measure the expression level of a PUM1-TRAF3 (pumilio RNA binding family member 1-TNF receptor associated factor 3) fusion protein or a gene encoding it in a biological sample obtained from a subject of interest. .

In the present invention, the object of interest may be selected from the group consisting of humans, rats, mice, guinea pigs, hamsters, rabbits, monkeys, dogs, cats, cows, horses, pigs, sheep and goats, and is preferably a human. It may be, but is not limited thereto.

In the present invention, the biological sample is whole blood, leukocytes, peripheral blood mononuclear cells, leukocyte buffy coat, plasma, serum, sputum , tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings), ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid ), nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, organ secretions, cell, cell extract And it may be at least one selected from the group consisting of cerebrospinal fluid, etc., but is not limited thereto.

The agent used in the measuring unit of the diagnostic device of the present invention may be an agent that measures the expression level of the PUM1-TRAF3 fusion protein or the gene encoding it. More specifically, it may include one or more selected from the group consisting of an antibody, an oligopeptide, a ligand, a peptide nucleic acid (PNA), and an aptamer that specifically binds to the fusion protein, and the fusion gene It may include at least one selected from the group consisting of a primer, a probe, and an antisense nucleotide that specifically binds.

In the measurement unit of the present invention, the suitability of a cancer therapeutic agent, more preferably, the suitability of treating cholangiocarcinoma, can be predicted by checking the expression level of the fusion protein or fusion gene using the agent.

The diagnostic device of the present invention adds a detection unit that predicts and outputs the suitability of the cancer treatment agent for the target individual, more preferably, the suitability for treatment of cholangiocarcinoma, from the expression level of the fusion protein or fusion gene obtained from the measurement unit. can be further included.

In the present invention, the detection unit generates and classifies information on the suitability of a cancer therapeutic agent according to the category of the expression level of the fusion protein or fusion gene obtained by the measurement unit, thereby discriminating suitability for treatment of cancer, more preferably, biliary tract cancer. can be diagnosed

In one example of the present invention, when the expression level of the PUM1-TRAF3 fusion protein or the gene encoding the PUM1-TRAF3 fusion protein measured by the measurement unit is higher than that of the normal control unit, the detection unit predicts that the target individual has high suitability for cancer treatment and outputs the result. can do. More preferably, when the expression level of the PUM1-TRAF3 fusion protein or the gene encoding it is higher than that of the normal control, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) inhibition It can be predicted that there is suitability for the treatment of bile duct cancer by drugs or drugs that inhibit NF-κB-inducing kinase (NIK).

In the diagnostic device of the present invention, descriptions of PUM1-TRAF3, cancer treatment, NF-κB inhibitor, NIK inhibitor, cholangiocarcinoma, control, etc. are the same as described above, and are omitted to avoid excessive complexity in the present specification.

According to another embodiment of the present invention, it relates to a composition for preventing, improving or treating cancer.

The composition of the present invention is effective as a nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) inhibitor or an NF-κB-inducing kinase (NIK) inhibitor It may include as a component, the NF-κB inhibitor is QNZ or a pharmaceutically acceptable salt thereof; MG132 or a pharmaceutically acceptable salt thereof; At least one selected from the group consisting of pyrrolidinedithiocarbamate ammonium or a pharmaceutically acceptable salt thereof and caffeic acid phenethyl ester or a pharmaceutically acceptable salt thereof; The NIK inhibitor may be B022 or a pharmaceutically acceptable salt thereof; XT2 or a pharmaceutically acceptable salt thereof; And NIK SMI1 or a pharmaceutically acceptable salt thereof; may include at least one selected from the group consisting of, but is not limited thereto.

In one embodiment of the present invention, the QNZ is 4-N-[2-(4-phenoxyphenyl)ethyl]-1,2-dihydroquinazoline-4,6-diamine (4-N-[2-( 4-Phenoxyphenyl)ethyl] -1,2-dihydroquinazoline-4,6-diamine) and may be a compound represented by Formula 1 below.

[Formula 1]

In another embodiment of the present invention, the MG132 is benzyl[(2S)-4-methyl-1-{[(2S)-4-methyl-1-{[(2S)-4-methyl-1-oxopentane-2 -yl]amino}-1-oxopentan-2-yl]amino}-1-oxopentan-2-yl]carbamate (Benzyl [(2S)-4-methyl-1-{[(2S)-4- methyl-1-{[(2S)-4-methyl-1-oxopentan-2-yl]amino}-1-oxopentan-2-yl]amino}-1-oxopentan-2-yl]carbamate) to Formula 2 It may be a compound represented by

[Formula 2]

In another embodiment of the present invention, the pyrrolidinedithiocarbamate ammonium is pyrrolidine-1-carbodithioate and may be a compound represented by Formula 3 below. .

[Formula 3]

In another embodiment of the present invention, the caffeic acid phenethyl ester is 2-phenylethyl (E) -3- (3,4-dihydroxyphenyl) prop-2-inoate (2-phenylethyl (E)- 3-(3,4-dihydroxyphenyl)prop-2-enoate) may be a compound represented by Formula 4 below.

[Formula 4]

In another embodiment of the present invention, the B022 is 4-[1-(2-amino-5-chloropyrimidin-4-yl)-2,3-dihydroindol-6-yl]-2-(1, 3-thiazol-2-yl)but-3-yn-2-ol (4-[1-(2-amino-5-chloropyrimidin-4-yl)-2,3-dihydroindol-6-yl]-2 -(1,3-thiazol-2-yl)but-3-yn-2-ol) may be a compound represented by Formula 5 below.

[Formula 5]

In another embodiment of the present invention, the XT2 is (R)-4-(3-(2-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-4-fluorophenyl) -2-(Thiazol-2-yl)but-3-yn-2-ol ((R)-4-(3-(2-Amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl )-4-fluorophenyl)-2-(thiazol-2-yl)but-3-yn-2-ol) and may be a compound represented by Formula 6 below.

[Formula 6]

In another embodiment of the present invention, the NIK SMI1 is 6-[3-[2-[(3R)-3-hydroxy-1-methyl-2-oxopyrrolidin-3-yl]ethynyl]phenyl] -4-methoxypyridine-2-carboxamide (6-[3-[2-[(3R)-3-hydroxy-1-methyl-2-oxopyrrolidin-3-yl]ethynyl]phenyl]-4-methoxypyridine -2-carboxamide) may be a compound represented by Formula 7 below.

[Formula 7]

The compounds represented by Chemical Formulas 1 to 7 or pharmaceutically acceptable salts thereof of the present invention can very effectively inhibit the growth, metastasis and invasion of cancer cells. The composition of the present invention can be used very effectively for preventing, improving or treating cancer by inhibiting uncontrolled growth of cancer cells or reducing metastasis and invasiveness of cancer cells, and more preferably preventing or improving biliary tract cancer. Or it can be used very effectively for treatment.

The pharmaceutically acceptable salts of the present invention may include acid or base addition salts, and stereochemical isomers thereof. For example, the compounds may be in the form of addition salts of organic or inorganic acids. Salts, when administered to a patient, have desirable effects on the patient, and include, but are not particularly limited to, any salts that retain the activity of their parent compound. These salts include inorganic and organic salts such as acetic acid, nitric acid, aspartic acid, sulfonic acid, sulfuric acid, maleic acid, glutamic acid, formic acid, succinic acid, phosphoric acid, phthalic acid, tannic acid, tartaric acid, hydrobromic acid, propionic acid, benzenesulfonic acid, Benzoic acid, stearic acid, lactic acid, bicarboxylic acid, bisulfuric acid, bitartaric acid, oxalic acid, butyric acid, calcium idete, carbonic acid, chlorobenzoic acid, citric acid, idetic acid, toluenesulfonic acid, fumaric acid, gluseptic acid, ecilin Acid, pamoic acid, gluconic acid, methyl nitric acid, malonic acid, hydrochloric acid, hydroiodoic acid, hydroxynaphtholic acid, isethionic acid, lactobionic acid, mandelic acid, mucoic acid, napsylic acid, muconic acid, p -It may include salts of nitromethanesulfonic acid, hexamic acid, pantothenic acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, salicylic acid, sulfamic acid, sulfanilic acid, methanesulfonic acid, and the like. Addition salts of bases include salts of alkali metals or alkaline earth metals, such as salts of ammonium, lithium, sodium, potassium, magnesium, calcium and the like; salts with organic bases such as benzathine, N-methyl-D-glucamine, hydrabamine and the like; and salts with amino acids such as arginine, lysine, and the like. Also, these salts can be converted to the free form by treatment with an appropriate base or acid.

The disease to be prevented, improved or treated in the composition of the present invention may be a cancer that has occurred or is likely to develop in a subject of interest, preferably bile duct cancer, specifically a PUM1-TRAF3 fusion protein, It may be cholangiocarcinoma that has been or is likely to be developed in individuals whose genes encoding these are highly expressed.

In the present invention, the "object of interest" refers to mammals including humans, such as humans, rats, mice, guinea pigs, hamsters, rabbits, monkeys, dogs, cats, cows, horses, pigs, sheep and goats. It may be selected from the group consisting of, and may preferably be a human, but is not limited thereto.

In the present invention, the "human" may refer to a patient who has or is suspected of having cancer and requires or is expected to receive appropriate treatment for cancer, preferably bile duct cancer, but is not limited thereto.

In the present invention, the disease to be prevented or treated, and the "cancer" refers to biliary tract cancer, gastric cancer, thyroid cancer, parathyroid cancer, ovarian cancer, colon cancer, pancreatic cancer, liver cancer, breast cancer, cervical cancer, lung cancer, non-small cell lung cancer, Prostate cancer, gallbladder cancer, biliary tract cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, blood cancer, bladder cancer, kidney cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, rectal cancer, brain tumor, perianal cancer, fallopian tube carcinoma, uterus Endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteric cancer, renal cell carcinoma, renal pelvic carcinoma, CNS central nervous system tumor, primary It may be CNS lymphoma, spinal cord tumor, brainstem glioma or pituitary adenoma, preferably bile duct cancer, but is not limited thereto.

The "prevention" of the present invention can be included without limitation as long as it is an act of blocking, suppressing or delaying symptoms caused by uncontrolled growth, metastasis, invasion, etc. of cancer cells using the composition of the present invention. there is.

The "improvement" of the present invention may include without limitation any action that improves or benefits symptoms caused by uncontrolled growth, metastasis, invasion, etc. of cancer cells by using the composition of the present invention.

The "treatment" of the present invention can be included without limitation as long as symptoms caused by uncontrolled growth, metastasis, invasion, etc. of cancer cells are improved or beneficial by using the composition of the present invention.

The composition for preventing, improving or treating cancer of the present invention may be used as a pharmaceutical composition or a food composition, but is not limited thereto.

In the present invention, the pharmaceutical composition may be in the form of capsules, tablets, granules, injections, ointments, powders or beverages, and the pharmaceutical composition may be intended for humans.

The pharmaceutical composition of the present invention is not limited to these, but is formulated according to conventional methods into oral formulations such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories and sterile injection solutions. can 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, pigments, flavors, etc. for oral administration, and buffers, preservatives, and painless agents for injections. A topical, solubilizing agent, isotonic agent, stabilizer, etc. may be mixed and used, and in the case of topical administration, a base, excipient, lubricant, preservative, etc. may be used. The formulation of the pharmaceutical composition of the present invention may be variously prepared by mixing with the above-described pharmaceutically acceptable carrier. For example, for oral administration, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, it can be prepared in unit dosage ampoules or multiple dosage forms. there is. In addition, it may be formulated into solutions, suspensions, tablets, capsules, sustained-release preparations, and the like.

On the other hand, examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil and the like can be used. In addition, fillers, anti-coagulants, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, and the like may be further included.

The route of administration of the pharmaceutical composition according to the present invention is, but is not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical , sublingual or rectal. Oral or parenteral administration is preferred.

In the present invention, "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intracapsular, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration.

The pharmaceutical composition of the present invention depends on various factors including the activity of the specific compound used, age, body weight, general health, sex, diet, administration time, route of administration, excretion rate, drug combination and severity of the specific disease to be prevented or treated. The dosage of the pharmaceutical composition may be variously varied, and the dosage of the pharmaceutical composition varies depending on the patient's condition, weight, disease severity, drug form, administration route and period, but may be appropriately selected by those skilled in the art, and is 0.0001 to 50 mg per day. /kg or 0.001 to 50 mg/kg. Administration may be administered once a day, or may be administered in several divided doses. The dosage is not intended to limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated into a pill, dragee, capsule, liquid, gel, syrup, slurry, or suspension.

In addition, the composition for preventing, improving or treating cancer in the present invention may be administered in combination with other anticancer agents. In this way, when used in combination with other anticancer agents, it is possible to exert a remarkable effect in preventing or treating cancer by more effectively suppressing cancer growth or metastasis.

The anticancer agent of the present invention is nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, neratinib, lapatinib, gefitinib, vandetanib, nirotinib, semasanib, bosutinib, axitinib, cedi ranib, lestaurtinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, sorafenib, bevacizumab, cisplatin, cetuximab, viscum album, asparaginase, tretinoin, hydroxyl Carbamide, dasatinib, estramustine, gemtuzumab ozogamicin, ibritumomabtucetan, heptaplatin, methylaminolevulinic acid, amsacrine, alemtuzumab, procarbazine, alprostadil, nitric acid Holmium chitosan, gemcitabine, doxifluridine, pemetrexed, tegafur, capecitabine, gimeracin, oteracil, azacitidine, methotrexate, uracil, cytarabine, fluorouracil, fludabine, eno Citabine, flutamide, capecitabine, decitabine, mercaptopurine, thioguanine, cladribine, carmophor, raltitrexed, docetaxel, paclitaxel, irinotecan, belotecan, topotecan, vinorelbine, etoposide , vinblastine, idarubicin, mitomycin, bleromycin, dactinomycin, pirarubicin, aclarubicin, pepromycin, temsirolimus, temozolomide, busulfan, ifosfamide, cyclophos Pamid, melpharan, altretmin, dacarbazine, thiotepa, nimustine, chlorambucil, mitolactol, leucovorin, tretonin, exmestane, aminoglutecimid, anagrelide, olaparib , navelbine, fadrazole, tamoxifen, toremifene, testolactone, anastrozole, letrozole, vorozole, bicalutamide, lomustine, 5FU, vorinostat, entinostat and carmustine It may be at least one selected, but is not limited thereto.

According to another embodiment of the present invention, it relates to a method of accompanying diagnosis for a cancer treatment application.

In the present invention, the method may include measuring the expression level of a PUM1-TRAF3 (pumilio RNA binding family member 1-TNF receptor associated factor 3) fusion protein or a gene encoding the same in a biological sample isolated from a subject of interest. there is.

In the present invention, the subject may be used interchangeably with a subject of interest, and the subject of interest may be a mammal including a human, for example, a human, rat, mouse, guinea pig, hamster, rabbit, monkey, dog, cat , may be selected from the group consisting of cows, horses, pigs, sheep and goats, preferably human, but is not limited thereto.

In the present invention, the "human" may refer to a person who has developed cancer or is suspected of having a high possibility of developing cancer, and may mean a patient who needs or is expected to receive appropriate treatment for cancer disease, preferably requires appropriate treatment for biliary tract cancer or It may be an expected patient, but is not limited thereto.

The "biological sample" of the present invention refers to any material, biological fluid, tissue, or cell obtained from or derived from a patient with a cancer disease or a subject suspected of having a cancer disease, for example, whole blood Whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, blood including plasma and serum, sputum, tears ), mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, pelvis Pelvic fluids, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid ), nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, organ secretions, cell, cell extract Or cerebrospinal fluid (cerebrospinal fluid) may be included, but is not limited thereto.

The cancer therapeutic agent of the present invention is a nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) inhibitor or an NF-κB-inducing kinase (NIK) inhibitor. It may include, but is not limited to, any drug that is expected to have a preventive or therapeutic effect on cancer-related diseases.

In the present invention, the NF-κB inhibitor is QNZ or a pharmaceutically acceptable salt thereof; MG132 or a pharmaceutically acceptable salt thereof; At least one selected from the group consisting of pyrrolidinedithiocarbamate ammonium or a pharmaceutically acceptable salt thereof and caffeic acid phenethyl ester or a pharmaceutically acceptable salt thereof; It can be done, but is not limited thereto.

In the present invention, the NIK inhibitor is B022 or a pharmaceutically acceptable salt thereof; XT2 or a pharmaceutically acceptable salt thereof; And NIK SMI1 or a pharmaceutically acceptable salt thereof; may include at least one selected from the group consisting of, but is not limited thereto.

In the present invention, Companion Diagnostics using the biomarker of PUM1-TRAF3 can pre-select a subject predicted to be effective in preventing or treating cancer-related diseases due to high drug reactivity when the cancer treatment is applied. and measuring the expression level of the subject's PUM1-TRAF3 fusion protein or the gene encoding it can provide essential information for the effective use of the therapeutic agent.

The accompanying diagnosis method of the present invention may further include selecting an individual whose expression level of the PUM1-TRAF3 fusion protein measured above or a gene encoding the same is higher than that of a normal control group.

In the present invention, an individual whose expression level of the PUM1-TRAF3 fusion protein or the gene encoding it is higher than that of the normal control group activates the non-canonical NF-κB signaling induced by the expression of the PUM1-TRAF3 fusion gene (non-canonical NF-κB signaling ) Since the efficacy of therapeutic agents having an inhibitory effect is predicted to be high, cancer, preferably cholangiocarcinoma, most preferably by overexpression of the PUM1-TRAF3 fusion gene through administration of the composition for preventing or treating cancer of the present invention The resulting bile duct cancer can be more effectively prevented or treated.

In the method of the present invention, the description of the PUM1-TRAF3 fusion protein or the gene encoding it, the cancer, the type of cancer drug, accompanying diagnosis, etc. is the same as that described in the composition for determining the suitability of a cancer drug, thereby avoiding the excessive complexity of the present specification. omitted to avoid

The companion diagnosis method of the present invention has the meaning of precisely presenting a patient's treatment method according to the patient's characteristics, and thus increases the efficiency of cancer treatment or reduces the side effects of the treatment by selecting the patient's drug reactivity before prescribing a cancer treatment. It can reduce, and furthermore, the economic effect of preventing unnecessary medical expenses is expected.

The composition according to the present invention maximizes the therapeutic effect of cholangiocarcinoma by applying an NF-κB inhibitor or a NIK inhibitor to selected patients with high expression of the PUM1-TRAF3 fusion gene using a companion diagnostic biomarker called the PUM1-TRAF3 fusion gene. can

1 is a diagram showing a flow chart for fusion gene analysis from a biliary tract cancer (BTC) patient according to an embodiment of the present invention.
2A is a diagram showing a PUM1-TRAF3 fusion gene including PUM1 (PUF domain, chromosome 1) and TRAF3 (MATH domain, chromosome 14) according to an embodiment of the present invention.
Figure 2b shows fusion signals (indicated by arrows) through fluorescence in situ hybridization (FISH) analysis using two probes for PUM1 (red) and TRAF3 (green) in P1 tissue slides according to an embodiment of the present invention. It is also
Figure 2c is a P1 patient tissue slide (left) in which the PUM1-TRAF3 fusion gene was detected using the PUM1 antibody and the TRAF3 antibody according to an embodiment of the present invention and a patient tissue slide (right) in which the PUM1-TRAF3 fusion gene was not detected. It is a diagram showing the results (DAPI: blue, PT (PUM1-TRAF3 fusion gene): red) of performing in situ proximal ligation assay (PLA) to visualize PUM1-TRAF3 protein expression in .
Figure 2d is a diagram showing the results of PCR analysis (NL: normal, CA: cancer) of P1 to P26 tissue samples according to an embodiment of the present invention.
Figure 3a shows the results of confirming the cell proliferation effect upon expression of PUM1-TRAF3 in SNU1196 cells or SNU308 cells according to an embodiment of the present invention (C: control vector transduced cells, PT: PUM1-TRAF3 fusion gene transduced cells) is the diagram shown.
Figure 3b is a result of confirming cell metastasis and cell invasiveness upon expression of PUM1-TRAF3 in SNU1196 cells or SNU308 cells according to an embodiment of the present invention (C: control vector transduced cells, PT: PUM1-TRAF3 fusion gene transduced cells) ) is shown.
Figures 4a and 4b show the results of measuring the tumor size at 7-day intervals from mice of the PUM1-TRAF3 transduced group and the control vector transduced group according to an embodiment of the present invention (C: control vector transduced cells, PT: It is a diagram showing PUM1-TRAF3 fusion gene-transduced cells).
Figure 5a shows the results of analyzing proteins differentially expressed in PUM1-TRAF3 transduced cells according to an embodiment of the present invention (C: control vector transduced cells, PT: PUM1-TRAF3 fusion gene transduced cells). It is also
Figure 5b shows the immunohistochemical staining results of NF-κB2, RelB and green fluorescent protein (GFP) from mouse tumor tissue according to an embodiment of the present invention (C: control vector transduced cells, PT: PUM1-TRAF3 fusion gene trait It is a diagram showing introduced cells).
5C is a diagram showing immunohistochemical staining results of NF-κB and RelB in the nucleus from BTC patient tissues (P1 to P11) expressing the PUM1-TRAF3 fusion protein according to an embodiment of the present invention.
6a to 6c are diagrams confirming target sites for gRNA design for generation of the TRAF3 knockout cell line of the present invention and protein expression of cells derived from cells transfected with CRISPR through Western blotting.
7a to 7c are diagrams confirming target sites for gRNA design for generation of NF-κB2 knockout cell lines according to an embodiment of the present invention and protein expression of cells derived from cells transfected with CRISPR through Western blotting; am.
Figure 8a is a control vector transduced SNU1196 cells and PUM1-TRAF3 fusion gene transduced cells or a TRAF3 knockout control vector transduced SNU1196 cells and PUM1-TRAF3 transduced SNU1196 cells according to an embodiment of the present invention in NF-κB2 and RelB It is a diagram showing the result of immunofluorescence staining for.
8B is a diagram showing the results of immunofluorescence staining for NF-κB2 and RelB when NF-κB2 was knocked out in control vector-transduced SNU1196 cells or PUM1-TRAF3-transduced SNU1196 cells according to an embodiment of the present invention.
Figure 9a is a diagram showing the results of confirming cell proliferation changes after treatment with NF-κB inhibitor QNZ to SNU1196 and SNU308 cells transduced with PUM1-TRAF3 or a control vector according to an embodiment of the present invention.
Figure 9b shows changes in NF-κB2 and RelB compared to administration of DMSO, a control drug, after treatment with QNZ, an NF-κB inhibitor, in SNU1196 and SNU308 cells transduced with PUM1-TRAF3 or a control vector according to an embodiment of the present invention. It is a diagram showing the result of immunofluorescence staining for.
10a and 10b show p52 and RelB expression levels in the nucleus or cytoplasm through Western blotting after treatment of SNU1196 cells transduced with PUM1-TRAF3 or a control vector according to an embodiment of the present invention with the NIK inhibitor B022. It is a diagram showing the result of confirming the change in .
11 is a diagram showing the results of immunofluorescence staining for NIK, NF-κB2 and RelB after treating SNU1196 cells transduced with PUM1-TRAF3 or a control vector according to an embodiment of the present invention with the NIK inhibitor B022 .
12a and 12b are images obtained by immunohistochemistry analysis by dividing NIK-weak and NIK-strong groups in cholangiocarcinoma patients according to an embodiment of the present invention, and NIK according to PUM1-TRAF3 positive/negative groups. It is a diagram showing the result of confirming the relative frequency of NIK expression in the -weak and NIK-strong groups.
13a and 13b show disease-free survival (DFS) and overall survival according to PUM1-TRAF3 positive/negative group or NIK-strong/weak in BTC patients (n=55) according to an embodiment of the present invention. This diagram confirms the Kaplan-Meier analysis results for overall survival (OS).
14a and 14b are diagrams illustrating the results of confirming DFS and OS through multivariate analysis using a Cox proportional hazards model according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Preparation Example 1: Sample collection of biliary tract cancer patients

In order to confirm the genetic characteristics of patients with biliary tract cancer in the examples according to the present invention, the present inventors obtained written consent among patients who underwent surgery for the purpose of treatment for biliary tract cancer (BTC) at Severance Hospital, Yonsei University College of Medicine. Patient samples and clinical information were obtained from the recipient patients. The study protocol complied with the ethical standards of the institutional research committee and the 1964 Declaration of Helsinki and its subsequent amendments or similar ethical standards, and was approved by the ethics committee and institutional review committee of Yonsei University College of Medicine (IRB approval code: 4-2011-0625). ) was received and the experiment was performed. Tumors were staged according to the American Joint Committee on Cancer (AJCC) stage classification, and information on collected patient samples is shown in Table 1 below.

Fusion gene (-)
n=27 (49.1%) Fusion gene (+)
N=28 (50.9%) P-value* Mean age at diagnosis (standard deviation) 67.0 (±9.5) 68.7 (±7.2) 0.259 gender(%) 0.019 female 7 (25.9%) 16 (57.1%) male 20 (74.1%) 12 (42.9%) Origin of tumor (%) 0.533 Intrahepatic cholangiocarcinoma (CC) 2 (7.4%) 7 (25.0%) Extrahepatic cholangiocarcinoma (CC) 24 (88.9%) 20 (71.4%) GB cancer 1 (3.7%) 1 (3.6%) ordnance(%) 0.285 I 6 (22.2%) 9 (32.1%) Ⅱ 20 (74.1%) 12 (42.9%) III 1 (3.7%) 4 (14.3%) IVa 0 (0.0%) 3 (10.7%) [Ⅰ + Ⅱ] 26 (96.3%) 21 (75.0%) [III + IVa] 1 (3.7%) 7 (25.0%) differentiation(%) 0.389 Well 4 (14.8%) 3 (10.7%) Moderate 15 (55.6%) 19 (67.9%) Poor 7 (25.9%) 5 (17.9%) Undifferentiated 1 (3.7%) 0 (0.0%) CA19-9, IU/mL mean (standard deviation) 997.1 (±2910.2) 311.4 (±865.2) 0.242 Resection margin (%) 0.469 R0 20 (74.1%) 23 (82.1%) R1 7 (25.9%) 5 (17.9%) Adjuvant chemotherapy (%) 0.166 you 13 (48.1%) 18 (64.3%) radish 14 (51.9%) 20 (35.7%) Recurrence after surgery (%) 0.080 you 11 (40.7%) 18 (64.3%) radish 16 (59.3%) 20 (35.7%) Distant metastasis at recurrence (%) 9 (81.8%) 17 (94.4%) 0.324 liver 7 (63.6%) 8 (42.1%) 0.369 lung 1 (9.1%) 4 (21.1%) 0.364 brain 1 (9.1%) 3 (15.8%) 0.562 peritoneum 1 (9.1%) 6 (31.6%) 0.143 Transient chemotherapy after relapse (%) 0.199 you 7/11 (63.6%) 15/18 (83.3%) radish 4/11 (36.4%) 3/18 (16.7%) Survival, months, median (range) Disease free survival (DFS) 29.8 (0.2-122.5) 12.1 (0.8-65.6) 0.088 Overall survival (OS) 36.6 (1.8-122.7) 26.1 (1.2-67.0) 0.190

Preparation Example 2: Cultivation of cell lines

Human BTC cell lines SNU245, SNU308, SNU478, SNU869, SNU1079 and SNU1196 were obtained from the Korean Cell Line Bank (KCLB). Each of the above cell lines was cultured in RPMI1640 (Invitrogen) medium containing 10% fetal bovine serum (FBS; Hyclone, Logan), 2mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin according to the guidelines of the Korean Cell Line Bank. cultured.

Hucct-1, OZ and KKU 100 cell lines were obtained from the Japan Collection of Research Bioresources Cell Bank (JCRB) and cultured in Dulbecco's Modified Eagle Medium (DMEM; Invitrogen) supplemented with 10% FBS (Hyclone). did 293FT cells were purchased from Invitrogen and cultured in DMEM (Invitrogen) medium supplemented with 10% FBS (Hyclone). All cell lines were cultured in an incubator at 37 °C and 5% CO ₂ , and were used for experiments after contamination testing.

statistical analysis

Data were analyzed using χ2 and Fisher's exact test for categorical data and Student's t-test and Mann 190 Whitney U test for continuous variables. Multivariate analysis was performed to evaluate potentially significant factors considering the influence of confounding clinical variables, and hazard ratios (HRs), 95% confidence intervals (95% CI), and P-values of multivariate analysis were used for DFS and OS. It was calculated using the Cox proportional hazards model. All statistical analyzes were performed using IBM SPSS Statistics version 25.0 for Windows (IBM Corp., Armonk, NY, US), and a value of P < 0.05 was considered statistically significant.

Example 1: Discovery and Verification of Fusion Genes

RNA-seq of 5 tissue samples from 201 Korean BTC patients was performed, including 5 tumors and 5 matched normal bile duct tissues for initial detection (see Table 2).

patient age gender Primary site Operation Pathology Differentiation stage One 66 female intrahepatic Lobectomy of the liver cholangiocarcinoma
(Cholangiocarcinoma) commonly
(moderate) T2N1M0 2 72 female intrahepatic Lobectomy of the liver cholangiocarcinoma
(Cholangiocarcinoma) commonly
(moderate) T3N1M0 3 77 female extrahepatic PPPD cholangiocarcinoma
(Cholangiocarcinoma) commonly
(moderate) T2N0M0 4 80 male intrahepatic Lobectomy of the liver cholangiocarcinoma
(Cholangiocarcinoma) bad
(poor) T2N1M0 5 67 male extrahepatic PPPD cholangiocarcinoma
(Cholangiocarcinoma) commonly
(moderate) T3N0M0

Fusion genes were discovered through step-by-step filtering through a custom bioinformatics pipeline using ChimeraScan (version 0.4.5), FusionCatcher (version 0.99.4d), and JAFFA (version 1.06) (see Figure 1). The two fusion genes finally selected after filtering were identified with high confidence, and the PUM1-TRAF3 and ASH1L 204 DOCK7 fusion genes were identified in patients with intrahepatic cholangiocarcinoma (P1 or B01). Since ASH1L-DOCK7 was out of frame, it was excluded from further verification, and then additional verification experiments of PUM1-TRAF3 were performed.

To validate the PUM1-TRAF3 fusion gene, primers of forward 5'-TGT ATG GCT GCC GTG TTA TC -3' and reverse 5'- ATG TCG TGC ACA CTC AGC AT-3' covering putative breakpoints were used. PCR was performed by design, and the fusion gene in patient P1 could be successfully identified from subsequent Sanger sequencing.

PUM-TRAF3, an in-frame transcript, consists of 2,988 bp of PUM1 (located at chr1:p35.2 of 3558 bp) and 744 bp of TRAF3 (located at chr14:q32.32 of 1704 bp), as shown in FIG. TRAF3 transcripts conserve the MATH domain of TRAF3 required for physical binding to NF-κB-inducing kinase (NIK), and the other two domains of TRAF3 required for ubiquitination and degradation of NIK (e.g. For example, it was confirmed that RING finger and zinc finger domains) were lost.

FISH analysis using PUM1 and TRAF3 probes at the gene or protein level was performed to confirm genomic translocation (see Fig. 2b), and in situ PLA was performed to confirm the presence of the endogenous PUM1-TRAF3 fusion protein in patient tissue to dissociate the protein complex. Detected and visualized, it is shown in Figure 2c. By applying the PUM1 mouse antibody and the TRAF3 rabbit antibody, the fusion protein could be visually observed in a speckle-like pattern of PLA signals on patient tissue slides under a fluorescence microscope. Taken together, it was confirmed that the PUM1-TRAF3 fusion event occurred by genomic translocation and was actively expressed to produce a fusion protein in BTC.

To determine the frequency of PUM1-TRAF3 expression, PCR analysis was performed on 26 pairs of frozen tissues (normal and cancer tissues) to be applied to additional BTC cases and validated in an expanded cohort, and the results are shown in Figure 2d. Reanalysis of 26 sample pairs by ddPCR confirmed the presence of the fusion gene in tissue and plasma samples of 7 patients expressing the fusion transcript, and it was confirmed that the PUM1-TRAF3 fusion transcript was not detected in the healthy control group.

Example 2: Effect of expression of PUM1-TRAF3 on tumorigenicity

In order to confirm the expression level of PUM1-TRAF3 in cholangiocarcinoma (BTC) cell lines, mRNA levels and protein expression were analyzed by PCR and Western blotting. Among the nine BTC cell lines including SNU245, SNU308, SNU478, SNU869, SNU1079, SNU1196, Hucct-1, OZ, and KKU-100, PUM1-TRAF3 mRNA and PUM1-TRAF3 protein expression were detected only in SNU1096 and SNU11.

In addition, to evaluate the functional effects of the PUM1-TRAF3 fusion protein on tumorigenicity, including cell proliferation, metastasis, invasion and motility, PUM1-TRAF3 was stably transduced into BTC cell lines to generate a PUM1-TRAF3 expressing cell line (PT cell line). established. More specifically, the Lentiviral vector and Packaging vector (OriGene) were co-transfected into 293FT cells using Lipofectamine 2000 (Invitrogen), and the supernatant was collected and added to the BTC cell line to transduce the PT protein. Green fluorescent protein cells were selected using flow cytometry and used in the experiments.

2.1 Proliferation assay

Cells were dissociated and plated in triplicate at a density of 2×10 ³ cells/well in 100 μl of complete medium in 96-well plates. After 24 hours incubation, WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium) (EZ-Cytox, Daeil Lab , Korea) was added and incubated for 2 hours. Absorbance at 450 nm was measured using a microplate reader and is shown in FIG. 3A. Compared to cells treated with control vectors (308C and 1196C cell lines), significantly increased cell proliferation of 69.16 ± 1.90% and 26.88 ± 0.77%, respectively, was confirmed in cells expressing the PUM1-TRAF3 fusion protein (308PT and 1196PT cell lines). .

2.2 Cell migration and invasion assay

For the metastasis assay, cells were dissociated and resuspended at 1.5 x 10 ⁵ cells/mL in 1% FBS and seeded in 24-well transwell plates (Costar, Bethesda, MD) at a density of 3 x 10 ⁴ cells/well. For the invasion assay, the upper chamber was pre-coated with Matrigel and cells were seeded at 3 x 10 ⁴ cells/well. The bottom chamber was filled with culture medium containing 20% FBS, and after incubation at 37° C., the cells were fixed with 5% glutaraldehyde for 30 minutes and stained with 0.1% crystal violet. Cells were completely removed from the upper surface of the membrane with a wet cotton swab, and migrating and infiltrating cells were counted and the photograph was analyzed under a microscope, as shown in FIG. 3B. As a result of the experiment, it was confirmed that cell transition (69.95 ± 1.91% and 75.79 ± 22.55%, P < 0.001) and invasion (150.00 ± 42.31% and 75.00 ± 41.11%, P < 0.031) significantly increased in PT cells, and soft agar Through soft agar colony formation assay, it was confirmed that the number of colonies was similar in the PT cell line, but the colony size significantly increased (see FIG. 3B ).

2.3 Evaluation of tumorigenicity

For in vivo tumorigenicity analysis, mice were injected with SNU1196 cells expressing PT and control vectors or TRAF3 knockout SNU1196 cells (5 x 10 ⁶ cells/site). All animal studies were performed using protocols approved by the Institutional Animal Care and Use Committee. As a result of evaluating the tumorigenicity of two groups of mice (N = 5/group) injected subcutaneously with tumor cells of the PT cell line or injected with CV cells, mice injected with PT cells injected subcutaneously with tumor cells of the PT cell line (2528.38 ± 371.97 mm ³ ) was confirmed to form a tumor about 1.55 times larger than that of mice injected with CV cells (1632.16 ± 310.57 mm ³ ) (see FIGS. 4a and 4b ).

B 신호 전달과의 관계 Example 3: Expression of PUM1-TRAF3 and NF-

Relationship with B signaling

To determine the oncogenic role of related genes, we investigated the expression and activity of PUM1 and TRAF3-related signaling pathways by western blotting. We found that the expression of p27, a cell cycle inhibitor directly inhibited by PUM1, was similar between PT and CV cell lines, suggesting that PUM1 signaling does not exert an oncogenic effect. On the other hand, the noncanonical nuclear factor kappa-light chain enhancer of the activated B cell (NF-κB) pathway, including NF-κB-induced kinase (NIK), phospho-IKKα (activated IKKα), p52 and RelB (the non -canonical nuclear factor kappa-light-chain-enhancer) gene expression was significantly increased in PT cells (see FIG. 5a). Accordingly, the expression levels of p52 and RelB in tumors were analyzed using immunohistochemical analysis, and compared to control vector-transduced cells (1196C), p52 and RelB were analyzed in the nucleus of mouse tumor tissue (1196PT) expressing the PUM1-TRAF3 fusion protein. It was confirmed that the expression of RelB was elevated (see FIG. 5b). Upon immunohistochemical analysis, it was confirmed that the expression of p52 and RelB was increased in the nucleus in BTC patient tissues expressing the PUM1-TRAF3 fusion protein (see FIG. 5c ).

From this, it can be seen that PUM1-TRAF3 induces activation of non-canonical NF-κB signaling through NF-κB2 accumulation.

Example 4: Effect on PUM1-TRAF3-induced tumorigenicity upon inhibition of NF-κB

4.1 Acquisition of TRAF3/NF-κB2 knockout cell line

The SNU1196 cell line was used to obtain TRAF3 and NF-κB2 knockout cells using Macrogen (Macrogen Korea, Seoul Korea) CRISPR/CAS technology, and specific target locations are shown in FIGS. 6a and 7a. Cells transfected with CRISPR were selected with puromycin, and protein expression of colonies derived from single cells was further analyzed by Western blotting (see Figs. 6b, 6c, 7b and 7c).

As a result of the experiment, it was confirmed that TRAF3 knockout enhanced NF-κB2 and RelB translocation into the nucleus in SNU1196 PUM1-TRAF3 transduced cells, but not in control vector-transduced cells (see FIG. 8a ). As a result of analyzing the expression of NF-κB2 and RelB through immunofluorescence staining in CV and PT cell lines in which NF-κB2 was knocked out, as shown in FIG. It was confirmed that translocation was not observed in both CV and PT cell lines. Through these results, it was found that the PUM1-TRAF3 fusion protein affects cell proliferation through a mechanism that interferes with the NF-κB signaling pathway or inhibits NF-κB2 gene expression. This suggests that the tumor formation process can be efficiently suppressed through inhibition of the NF-κB signaling pathway in BTC patients carrying a fusion gene by PUM1-TRAF3.

4.2 NF-κB inhibitor treatment

PUM1-TRAF3 transduced PT and CV cell lines and control vector-transduced cell lines (SNU1196 and SNU308) were treated with diluted QNZ (CAS number; 545380-34-5) among NF-κB inhibitors within the concentration range of 0 to 500 nM to obtain PUM1 - The effect of TRAF3 on induced cell proliferation was compared and confirmed. The above concentration range was applied by approximating the IC70 value for the cell line. As a result of the experiment, referring to Figure 9a, the difference in proliferation between the PT and CV cell lines was reduced by QNZ treatment, and the difference in proliferation between the two cell lines was statistically significant from concentrations exceeding about IC20 concentrations (38.9 nM for SNU1196 and 29.5 nM for SNU308). It was confirmed that it was not significant.

As above, further analysis through immunofluorescence staining was performed and is shown in FIG. 9B. It was confirmed that NF-κB2 and RelB showed reduced expression in the nuclei of 1196PT cells treated with QNZ at 30 nM (approximately IC20) compared to cells treated with DMSO.

Taken together, the above results used RNA sequencing to identify BTC-specific molecular markers and RNA sequencing of tissue samples from BTC patients revealed a novel pumilio RNA binding family member 1 (PUM1) arising from a 1:14 chromosomal translocation. A fusion gene of TNF receptor-related factor 3 (TRAF3) was discovered, and tissue samples from cholangiocarcinoma patients were analyzed to verify that the fusion gene was specific to cholangiocarcinoma. Furthermore, after establishing fusion protein-expressing cells, proliferative ability and motility and degree of invasion in xenograft tumors were analyzed, and PUM1 for non-canonical NF-κB signaling affecting cancer cell survival -The effect of the TRAF3 fusion gene was confirmed. In BTC cells expressing the fusion gene, proliferation, metastasis and tumorigenicity of BTC cells were increased by the PUM1-TRAF3 fusion gene, and the fusion gene activates non-canonical NF-κB signaling It was confirmed that BTC tumor formation was induced by induction. Through this, in applying a drug with a mechanism of inhibiting NF-κB, by using the PUM1-TRAF3 fusion gene as an index that can preemptively determine the suitability of treatment, among patients with biliary tract cancer, the PUM1-TRAF3 fusion gene was highly expressed. It is expected that the treatment effect of bile duct cancer patients can be maximized.

Example 5: Relationship between PUM1-TRAF3 induced NF-κB signaling and NIK

In order to determine the oncogenic role of the PUM1-TRAF3 fusion gene in the above example, the expression and activity of markers related to PUM1 and TRAF3-related signal transduction pathways were measured by western blotting, NF-κB-induced kinase (NIK) , expression of phospho-IKKα (activated IKKα), p52 and RelB, the non-canonical nuclear factor kappa-light-chain-enhancer gene of the activated B cell (NF-κB) pathway Since it was confirmed that the NF-κB activity induced by the PUM1-TRAF3 fusion gene was significantly increased in these cholangiocarcinoma (PT) cells, the relationship between the NF-κB activity and NIK carcinogenicity was further confirmed. Specifically, in order to determine whether the NF-κB signaling pathway induced by the fusion protein can be reversed by NIK inhibition (reversal of PUM1-TRAF3-induced NF-κB activation), B022 (CAS) belonging to the NIK inhibitor number; 1202764-53-1) was used.

As a result of treating PUM1-TRAF3 transduced SNU1196 (PT and C cell lines) with B022 among the NIK inhibitors in a 2-fold serial dilution in the concentration range of 0 to 100 μM, NIK expression was sufficiently inhibited in all cells treated with 50 μM and 100 μM doses. Confirmed. As a result of the experiment, it was observed that the expression patterns of p52 and RelB in the nucleus were reduced in cells treated with 100 μM of B022 (see FIGS. 10A and 10B ). Further analysis by immunofluorescence staining confirmed that treatment with B022 reduced the expression of p52 and RelB in the nuclei of TRAF3 knockout C and PT cell lines as well as 1196PT when compared to cells treated with DMSO (dimethyl sulfoxide). (see FIG. 11). These results indicate that NF-κB signaling induced and activated by PUM1-TRAF3 can be effectively reversed by inhibiting NIK expression, and from this, patients with high PUM1-TRAF3 marker expression can be treated with NF-κB inhibitors as well as This means that the activation of NF-κB can be effectively inhibited by treatment with a NIK inhibitor. The therapeutic potential of NIK inhibitors as drugs that inhibit the activation of non-canonical NF-κB signaling that induces BTC tumorigenesis was confirmed.

Example 6: Confirmation of clinical significance of expression of NIK

In order to evaluate the clinical significance of the PUM1-TRAF3 fusion gene in BTC patients from the relationship between NF-κB activity induced by the PUM1-TRAF3 fusion gene identified above and NIK carcinogenicity, the present inventors performed IHC on samples from BTC patients. (immunohistochemistry) analysis was performed, and NIK staining was scored as the intensity of NIK-positive (+) tumor cells (no expression; 0, low expression; 1+, moderate expression; 2+ and strong expression; 3+). , Patients with a NIK IHC staining score of 0 or 1+ were classified as NIK-weak, and patients with a score of 2+ or 3+ were classified as NIK-strong, and the results are shown in FIGS. 12a and 12b. In addition, disease-free survival (DFS) and overall survival; OS) to determine whether NIK expression is associated with the expression of the PUM1-TRAF3 fusion and poor prognosis in BTC patients.

As a result, all patients with PUM1-TRAF3 belonged to the NIK-strong group, and it was confirmed that the fusion gene expression rate was significantly higher than that of the NIK-weak group (p=0.004). When comparing DFS and OS between the NIK-strong and NIK-weak groups, the NIK-weak group was found to have a significantly higher 2-year survival rate in DFS (DFS; 62.9% vs. 18.0%, Long-Rank p=0.001) and OS ;46.3% vs. 18.0%, Long-Rank p=0.001), it was confirmed that the DFS and OS of the PUM1-TRAF3 positive group and the PUM1-TRAF3 negative group showed the same pattern (see FIGS. 13a and 13b). These results indicate that NIK expression is related to the expression of the PUM1-TRAF3 fusion marker in BTC patients and also to a worse prognosis. Through this, in applying a drug with a mechanism of inhibiting NIK, by using the PUM1-TRAF3 fusion gene as an index that can preemptively determine the suitability of treatment, cholangiocarcinoma in which the PUM1-TRAF3 fusion gene was highly expressed among cholangiocarcinoma patients. It is expected to maximize the treatment effect for patients.

Having described specific parts of the present invention in detail above, it is clear that these specific techniques are only preferred embodiments for those skilled in the art, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (25)

A composition for determining the suitability of a cancer therapeutic, comprising an agent for measuring the expression level of a pumilio RNA binding family member 1-TNF receptor associated factor 3 (PUM1-TRAF3) fusion protein or a fusion gene encoding the same. According to claim 1,
Wherein the PUM1-TRAF3 fusion protein comprises an amino acid sequence represented by at least one of SEQ ID NO: 1 or SEQ ID NO: 2. According to claim 1,
Wherein the PUM1-TRAF3 fusion gene comprises a nucleic acid sequence represented by at least one of SEQ ID NO: 3 or SEQ ID NO: 4. According to claim 1,
The agent for measuring the expression level of the protein comprises at least one selected from the group consisting of antibodies, oligopeptides, ligands, PNAs (peptide nucleic acids) and aptamers that bind specifically to the protein, composition . According to claim 1,
An agent for measuring the expression level of the gene comprises at least one selected from the group consisting of primers, probes and antisense nucleotides that specifically bind to the gene, composition. According to claim 1,
The cancer includes biliary tract cancer, stomach cancer, breast cancer, colon cancer, lung cancer, liver cancer, esophageal cancer, pancreatic cancer, gallbladder cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, colon cancer, cervical cancer, endometrial cancer, choriocarcinoma, skin cancer, ovarian cancer, At least one composition selected from the group consisting of thyroid cancer, brain cancer, blood cancer, head and neck cancer, malignant melanoma, and lymphoma. According to claim 1,
The composition, wherein the therapeutic agent is effective in suppressing non-canonical NF-κB signaling. According to claim 7,
The therapeutic agent is a nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) inhibitor or an NF-κB-inducing kinase (NIK) inhibitor, composition. According to claim 8,
The NF-κB inhibitor is QNZ or a pharmaceutically acceptable salt thereof; MG132 or a pharmaceutically acceptable salt thereof; At least one selected from the group consisting of pyrrolidinedithiocarbamate ammonium or a pharmaceutically acceptable salt thereof and caffeic acid phenethyl ester or a pharmaceutically acceptable salt thereof, composition. According to claim 8,
The NIK inhibitor is B022 or a pharmaceutically acceptable salt thereof; XT2 or a pharmaceutically acceptable salt thereof; And NIK SMI1 or a pharmaceutically acceptable salt thereof; at least one selected from the group consisting of, a composition. A kit for determining the suitability of a cancer therapeutic agent comprising the composition of any one of claims 1 to 10. According to claim 11,
The kit is an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or a multiple reaction monitoring (MRM) kit. Information on suitability for cancer treatment, including measuring the expression level of a PUM1-TRAF3 (pumilio RNA binding family member 1-TNF receptor associated factor 3) fusion protein or a gene encoding it in a biological sample isolated from a subject of interest How to provide. According to claim 13,
When the expression level of the PUM1-TRAF3 fusion protein or the gene encoding it measured in the biological sample is higher than that of the normal control group, it is predicted that the effect as a therapeutic agent for treating cancer in the target subject will be high. . According to claim 13,
The cancer includes biliary tract cancer, stomach cancer, breast cancer, colon cancer, lung cancer, liver cancer, esophageal cancer, pancreatic cancer, gallbladder cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, colon cancer, cervical cancer, endometrial cancer, choriocarcinoma, skin cancer, ovarian cancer, At least one method selected from the group consisting of thyroid cancer, brain cancer, blood cancer, head and neck cancer, malignant melanoma, and lymphoma. According to claim 13,
Wherein the therapeutic agent is a nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) inhibitor or an NF-κB-inducing kinase (NIK) inhibitor. (a) a measurement unit for measuring the expression level of a PUM1-TRAF3 (pumilio RNA binding family member 1-TNF receptor associated factor 3) fusion protein or a gene encoding the same with respect to a biological sample obtained from a subject of interest; and
(b) a detection unit that outputs the suitability of the target subject for cancer treatment from the expression level of the fusion protein measured by the measurement unit or the gene encoding the same; According to claim 17,
The cancer includes biliary tract cancer, stomach cancer, breast cancer, colon cancer, lung cancer, liver cancer, esophageal cancer, pancreatic cancer, gallbladder cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, colon cancer, cervical cancer, endometrial cancer, choriocarcinoma, skin cancer, ovarian cancer, A diagnostic device that is at least one selected from the group consisting of thyroid cancer, brain cancer, blood cancer, head and neck cancer, malignant melanoma, and lymphoma. According to claim 17,
The therapeutic agent is a nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) inhibitor or an NF-κB-inducing kinase (NIK) inhibitor, diagnostic device. Cancer, including a nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) inhibitor or an NF-κB-inducing kinase (NIK) inhibitor as an active ingredient A pharmaceutical composition for the prevention or treatment of 21. The method of claim 20,
The cancer includes biliary tract cancer, stomach cancer, breast cancer, colon cancer, lung cancer, liver cancer, esophageal cancer, pancreatic cancer, gallbladder cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, colon cancer, cervical cancer, endometrial cancer, choriocarcinoma, skin cancer, ovarian cancer, Thyroid cancer, brain cancer, blood cancer, head and neck cancer, at least one selected from the group consisting of malignant melanoma and lymphoma, the composition. 21. The method of claim 20,
The NF-κB inhibitor is QNZ or a pharmaceutically acceptable salt thereof; MG132 or a pharmaceutically acceptable salt thereof; At least one selected from the group consisting of pyrrolidinedithiocarbamate ammonium or a pharmaceutically acceptable salt thereof and caffeic acid phenethyl ester or a pharmaceutically acceptable salt thereof, composition. 21. The method of claim 20,
The NIK inhibitor is B022 or a pharmaceutically acceptable salt thereof; XT2 or a pharmaceutically acceptable salt thereof; And NIK SMI1 or a pharmaceutically acceptable salt thereof; at least one selected from the group consisting of, a composition. 21. The method of claim 20,
The composition is for treating a subject having a high expression level of a PUM1-TRAF3 (pumilio RNA binding family member 1-TNF receptor associated factor 3) fusion protein or a fusion gene encoding the same. 21. The method of claim 20,
The composition includes nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, neratinib, lapatinib, gefitinib, vandetanib, nirotinib, semasanib, bosutinib, axitinib, cediranib, lesta urtinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, sorafenib, bevacizumab, cisplatin, cetuximab, viscum album, asparaginase, tretinoin, hydroxycarbamide, Dasatinib, estramustine, gemtuzumab ozogamicin, ibritumomabtucetan, heptaplatin, methylaminolevulinic acid, amsacrine, alemtuzumab, procarbazine, alprostadil, chitosan holmium nitrate, gemcitabine, doxifluridine, pemetrexed, tegafur, capecitabine, gimeracin, oteracil, azacytidine, methotrexate, uracil, cytarabine, fluorouracil, fludabine, enocitabine, flutamide, capecitabine, decitabine, mercaptopurine, thioguanine, cladribine, carmophor, raltitrexed, docetaxel, paclitaxel, irinotecan, belotecan, topotecan, vinorelbine, etoposide, vinbla Steen, idarubicin, mitomycin, bleromycin, dactinomycin, pirarubicin, aclarubicin, pepromycin, temsirolimus, temozolomide, busulfan, ifosfamide, cyclophosphamide, Melpharan, altretmin, dacarbazine, thiotepa, nimustine, chlorambucil, mitolactol, leucovorin, tretonin, exmestane, aminoglutecimide, anagrelide, olaparib, navelbine at least selected from the group consisting of fadrazole, tamoxifen, toremifene, testolactone, anastrozole, letrozole, vorozole, bicalutamide, lomustine, 5FU, vorinostat, entinostat and carmustine A composition further comprising one.

Images