PEG10 gene related to CDK4 / 6 inhibitor resistance and anti-cancer agents using an ASO

Abstract

The present specification relates to a PEG10 ASO, and according to one aspect, a PEG10 ASO has an effect of improving CDK4 / 6 inhibitor resistance by inhibiting the PEG10 and EMT mechanisms associated with CDK4 / 6 inhibitor-resistant cancer, and when a PEG10 ASO and a CDK4 / 6 inhibitor are used in combination, the anticancer efficacy is enhanced, so it can be usefully used in treating CDK4 / 6 resistant cancer.

Description

Technology Field

[0001] This relates to an anticancer therapeutic agent using PEG10 and ASO, genes associated with the development of CDK4 / 6 inhibitor resistance. Background Technology

[0002] Breast cancer is the most common cancer among women, accounting for 20.6% of all cancers in women. The number of breast cancer patients in Korea is steadily increasing, and the growth rate of breast cancer in Korea ranks first in the world.

[0003] Breast cancer can be classified into HR-positive breast cancer, HER2-positive breast cancer, or triple-negative breast cancer depending on the expression levels of hormone receptors (HR) and HER2 (human epidermal growth factor receptor 2). Among these, HR-positive breast cancer is known to be the most common type, accounting for about 70% of all breast cancers.

[0004] Hormone therapy and chemotherapy are used to treat the aforementioned breast cancer, and recently, CDK4 / 6 inhibitors, which are therapeutic agents with a new mechanism of action, have been developed and are being used in the treatment of breast cancer. However, since drug resistance eventually develops even with the use of effective drugs, there is a need for treatments capable of overcoming this resistance. The problem to be solved

[0005] One aspect provides an antisense oligonucleotide (ASO) comprising any one polynucleotide selected from the group consisting of SEQ ID NOs 1 to 5 that can inhibit the expression of PEG10 (Paternally Expressed 10).

[0006] Another aspect is to provide a pharmaceutical composition for treating cancer comprising an expression or activity inhibitor of PEG10 (Paternally Expressed 10) as an active ingredient.

[0007] Another aspect provides a method for treating or preventing breast cancer in a subject, comprising administering a composition containing the above-mentioned ASO to a subject in need thereof.

[0008] Another aspect is to provide a composition for diagnosing cancer resistant to CDK4 / 6 inhibitors, comprising a preparation capable of measuring the expression or activity of PEG10. means of solving the problem

[0009] One aspect provides an antisense oligonucleotide (ASO) comprising any one polynucleotide selected from the group consisting of SEQ ID NOs 1 to 5 that can inhibit the expression of PEG10 (Paternally Expressed 10).

[0010] In this specification, the term "PEG10 (Paternally Expressed 10)" refers to a gene known to be involved in placental formation and is known to be derived from a retrotransposon of the Ty3 / Gypsy family. Recent studies have shown that it plays an important role in tumor growth in various cancers, including liver cancer, lung cancer, and prostate cancer.

[0011] In this specification, the term "antisense oligonucleotide" or "Antisense oligonucleotide (ASO)" refers to a single-stranded oligonucleotide that is a drug possessing a mechanism to regulate target expression by binding to target pre-mRNA or mRNA through methods such as target degradation, isoform-switching, and translation inhibition. As a sequence-based drug, it is characterized by having high selectivity for targets with genetic variations. It is similar to siRNA but has the advantage of being more freely chemically modified. Chemically modified ASOs include gapmer ASOs, non-degradable ASOs, and PMOs (Phosphorodiamidatemopholino). The term ASO may be used to include all nucleosides covalently linked through internucleotide linkers, such as naturally occurring nucleosides, or modified forms thereof, such as oligomers or polymers.

[0012] In this specification, the term "siRNA (short interfering RNA, silencing RNA)" refers to a short strand of RNA composed of 21 to 23 nucleotides that interferes with gene expression by inhibiting the production of a specific protein.

[0013] In this specification, the term "Gapmer ASO" or "Gapmer ASO" refers to a single-stranded oligonucleotide consisting of 16 to 24 mers that degrades target mRNA by RNAse H. Structurally, DNA modified with PS (phosphodiester) is located in the middle of the oligomer for several mers, and nucleotides modified with PS / 2'-O-MOE (2'-O-methoxyethyl) or PS / 2'-O-ME (2'-O-Methyl) are located at both ends for several mers each. When an ASO with such modifications binds to a target mRNA having a complementary base sequence, it is recognized as an abnormal binding of DNA and RNA, which calls RNAse H, and eliminates the target mRNA by cleaving the location where only the middle PS is modified.

[0014] Gapmer ASO may be composed of the general formula 5'-ABC-3', where (i) region B is an adjacent sequence of at least 6 DNA units capable of recruiting RNase; (ii) region A is a first wing sequence of 1 to 10 nucleotides, wherein the first wing sequence comprises one or more nucleotide analogs and optionally one or more DNA units, and at least one nucleotide analog is located at the 3' end of A; and (iii) region C is a second wing sequence of 1 to 10 nucleotides, wherein the second wing sequence comprises one or more nucleotide analogs and optionally one or more DNA units, and at least one nucleotide analog may be located at the 5' end of C.

[0015] The above nucleotide analogs or analogs are high-affinity analogs and may be 2'-sugar modified nucleosides independently selected from the group consisting of, for example, locked nucleic acid (LNA); 2'-0-alkyl-RNA; 2'-amino-DNA; 2'-fluoro-DNA; arabino nucleic acid (ANA); 2'-fluoro-ANA, hexitol nucleic acid (HNA), intercalating nucleic acid (INA), bound ethyl nucleoside (cEt), 2'-0-methyl nucleic acid (2'-OMe), 2'-0-methoxyethyl nucleic acid (2'-MOE), and any combination thereof.

[0016] The above nucleotide analogs or analogs may include a bicyclic sugar.

[0017] The above-mentioned dicyclic sugar may be cEt, 2',4'-bound 2'-0-methoxyethyl (cMOE), LNA, α-L-LNA, β 2'-0,4'-C-ethylene-crosslinked nucleic acid (ENA), amino-LNA, oxy-LNA, or thio-LNA, but is not limited thereto.

[0018] Preferably, the ASO may be a gapmer ASO.

[0020] In one embodiment, the ASO may have a length of 8 to 50 nucleotides. Preferably, the ASO may be 8 to 45 nucleotides. More preferably, the ASO may be 8 to 40 nucleotides. More preferably, the ASO may be 9 to 40 nucleotides. More preferably, the ASO may be 9 to 35 nucleotides. More preferably, the ASO may be 9 to 30 nucleotides. More preferably, the ASO may be 10 to 30 nucleotides. More preferably, the ASO may be 11 to 29 nucleotides. More preferably, the ASO may be 12 to 28 nucleotides. More preferably, the ASO may be 13 to 27 nucleotides. More preferably, the ASO may be 14 to 26 nucleotides. More preferably, the ASO may be 15 to 25 nucleotides. More preferably, the ASO may be 16 to 24 nucleotides. More preferably, the ASO may be 17 to 23 nucleotides. More preferably, the ASO may be 18 to 22 nucleotides. More preferably, the ASO may be 19 to 21 nucleotides or 20 nucleotides.

[0021] In one embodiment, the ASO may bind complementarily to PEG10. Preferably, the ASO may bind complementarily to PEG10 with a sequence of 10 nucleotides excluding the 5 nucleotides at both ends.

[0022] The ASO that binds complementarily to the above PEG10 may bind to 4500 to 4600, 12050 to 12100, 12150 to 12200, 12220 to 12300, or 14500 to 14600 nucleotide sequences of the PEG10 genome base sequence.

[0023] In one embodiment, the ASO may be capable of inhibiting a protein associated with the EMT (Epithelial-to-Mesenchymal Transition) mechanism.

[0024] In this specification, the term "EMT (epithelial to mesenchymal transition)" refers to the process in which epithelial cells transform into cells capable of metastasis and invasion. It is involved in fundamental phenomena of morphological development, including the development of tissues and organs during embryonic development, and in adults, it is associated with the wound healing process as well as the formation and progression of cancer.

[0025] In one embodiment, the ASO may induce activation of the genes or proteins of SIAH1 and p21. The activation of the genes or proteins of SIAH1 and p21 may induce inhibition of the EMT-related mechanism.

[0027] Another aspect provides a pharmaceutical composition for treating cancer comprising an expression or activity inhibitor of PEG10 (Paternally Expressed 10) as an active ingredient.

[0028] Another aspect provides a method for treating or preventing breast cancer in a subject, comprising administering a composition containing the above ASO to a subject in need thereof.

[0029] The above inhibitor may be any one selected from the group consisting of antisense oligonucleotides (ASO) that bind complementarily to the gene of PEG10, small hairpin RNA, small interfering RNA (siRNA), and ribozymes, but is not limited thereto.

[0030] The above antisense oligonucleotide may comprise any one polynucleotide selected from the group consisting of SEQ ID NOs 1 to 5.

[0031] In addition, the inhibitor may be any one selected from the group consisting of peptides, peptide mimetics, substrate analogs, aptamers, and antibodies that bind to PEG10 proteins, but is not limited thereto.

[0032] In this specification, the term "antibody" comprises immunoglobulin molecules that are immunologically reactive with a specific antigen, and includes polyclonal antibodies, monoclonal antibodies, and functional fragments thereof. Additionally, the term may include forms produced by genetic engineering, such as chimeric antibodies (e.g., humanized murine antibodies) and heterojunction antibodies (e.g., bispecific antibodies). Among these, monoclonal antibodies are highly specific antibodies directed to a single antigenic region (epitope). Unlike polyclonal antibodies, which include different antibodies directed to different epitopes, monoclonal antibodies are directed only to a single epitope on the antigen, making quality control as a therapeutic agent easier. The above antibody comprises a variable region of the heavy chain and / or light chain constituting the immunoglobulin molecule, and the variable region may include a portion that forms an antigen-binding site of the antibody molecule as its primary structure. The above antibody is not limited thereto if it exhibits the same effect as the anti-PEG10 antibody.

[0033] In this specification, the term “cancer” refers to a physiological condition in animals that is typically characterized by abnormal or uncontrolled cell growth. Cancer and cancer pathology may be associated with, for example, metastasis, interference with normally functioning surrounding cells, release of cytokines or other secretory products at abnormal levels, suppression or amplification of inflammatory or immunological responses, neoplasia, premalignancy, malignancy, surrounding or distant tissues or organs, such as lymph node invasion.

[0034] The above cancer may be a solid tumor or a blood cancer.

[0035] The above solid tumor may be selected from the group consisting of breast cancer, colorectal cancer, head and neck cancer, lung cancer, stomach cancer, skin cancer, colon cancer, prostate cancer, bladder cancer, kidney cancer, rectal cancer, thyroid cancer, liver cancer, cervical cancer, endometrial cancer, choriocarcinoma, skin cancer, rectal cancer, anal cancer, urethral cancer, ovarian cancer, esophageal cancer, gallbladder cancer, testicular cancer, brain cancer, bone metastasis cancer, glioblastoma, malignant melanoma, and pancreatic cancer, but is not limited thereto.

[0036] In one embodiment, the cancer may be a cancer resistant to a CDK (Cyclin Dependent Kinase) 4 / 6 inhibitor.

[0037] In this specification, the term "resistance" refers to the weakening or loss of drug efficacy during the treatment of cancer patients with an anticancer drug. This may be due to a decrease in the therapeutic effect of the anticancer drug on cancer cells that have developed resistance to the drug, resulting in reduced efficacy due to repeated dosing or administration.

[0038] In this specification, the term "susceptibility" refers to the degree to which cancer cells respond to an anticancer agent.

[0039] In one embodiment, the cancer may be CDK4 / 6 inhibitor-resistant breast cancer.

[0040] In this specification, the term "breast cancer" includes, without limitation, all malignant tumors occurring in the breast and may refer to the continued growth of abnormal tissue in the breast or its spread to other organs. Breast cancer is classified according to the site of occurrence into cancers occurring in parenchymal tissues such as the ducts and lobules and cancers occurring in other stromal tissues; cancers of the ducts and lobules may be further classified into invasive and non-invasive types depending on the extent to which cancer cells have spread to surrounding areas. Additionally, based on biological characteristics, it can be classified according to the presence or absence of hormone receptors (HR) and human epidermal growth factor receptor-2 (HER2). Depending on the presence or absence of HR and HER2, it can be classified into HR-positive breast cancer, HER2-overexpressing breast cancer, and triple-negative breast cancer.

[0041] In this specification, the term "prevention" collectively refers to partially or completely delaying or preventing the onset or recurrence of a disease, disorder, or its associated symptoms, preventing the acquisition or reacquisition of a disease or disorder, or reducing the risk of acquiring a disease or disorder. Such prevention refers to any act of suppressing or delaying the occurrence of cancer or cancer-related diseases, disorders, or symptoms through the administration of a composition according to the present invention.

[0042] In this specification, the term "treatment" refers to any act that improves or beneficially alters a disease, disorder, or its associated symptoms.

[0043] In one embodiment, the pharmaceutical composition may be formulated into a preparation selected from the group consisting of tablets, soft or hard capsules, pills, powders, suspensions, syrups, injections, and granules.

[0044] The above pharmaceutical composition may be in an oral or parenteral formulation. The above pharmaceutical composition may be administered parenterally during clinical administration and may be used in the form of a general pharmaceutical preparation. Parenteral administration may refer to administration through routes other than oral administration, such as rectal, intravenous, peritoneal, intramuscular, arterial, transdermal, nasal, inhalation, ocular, and subcutaneous.

[0045] In one embodiment, the pharmaceutical composition may be for oral or parenteral administration.

[0046] The above pharmaceutical composition may include conventional fillers, extenders, binders, disintegrants, anticoagulants, lubricants, wetting agents, pH adjusters, nutrients, vitamins, electrolytes, alginic acid and its salts, pectic acid and its salts, protective colloids, glycerin, flavorings, emulsifiers, or preservatives.

[0047] The above pharmaceutical composition may include a pharmaceutically acceptable carrier, examples of which may be one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, propylene glycol, liquid paraffin and physiological saline.

[0048] The formulation of the above pharmaceutical composition may vary depending on the method of use and may be formulated using methods well known in the art to which the present invention belongs so as to provide rapid, sustained, or delayed release of the active ingredient after administration to mammals.

[0049] Preparations for oral administration include tablets, soft or hard capsules, pills, powders, suspensions, syrups, injections, and granules, and these preparations may be prepared by mixing one or more excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition, lubricants such as magnesium stearate and talc may be used in addition to simple excipients. Preparations for parenteral administration may be creams, lotions, ointments, ointments, liquids, aerosols, fluid extracts, elixirs, infusions, sachets, patches, or injections, etc. Furthermore, they may be preferably formulated according to each disease or component by using methods disclosed in the art.

[0050] According to one embodiment, the composition can inhibit the growth of cancer cells and induce apoptosis.

[0051] The above pharmaceutical composition may vary depending on several factors including the activity of the specific compound used, age, body weight, general health, gender, diet, time of administration, route of administration, elimination rate, drug combination, and the severity of the specific disease to be prevented or treated, and the dosage of the above pharmaceutical composition may be appropriately selected by a person skilled in the art, depending on the patient's condition, body weight, degree of disease, drug form, route of administration, and duration, and may be administered at a dose of 0.0001 to 500 mg / kg or 0.001 to 500 mg / kg per day. Administration may be administered once a day or divided into several doses. The above dosage does not limit the scope of the present invention in any way.

[0053] In one embodiment, the composition may be capable of being administered in combination with an anticancer agent.

[0054] The above anticancer agent may be one or more selected from the group consisting of chemotherapy agents, targeted anticancer agents, immunotherapy agents, CDK4 / 6 inhibitors, and combinations thereof, but is not limited thereto.

[0055] In this specification, the term "chemotherapy agent" is also referred to as an antitumor agent or a cytotoxic agent. It is a collective term for drugs that exhibit anticancer activity by acting directly on DNA to block DNA replication, transcription, and translation processes, or by interfering with the synthesis of nucleic acid precursors in metabolic pathways and inhibiting cell division. Such antitumor agents act on normal cells as well as tumor cells to exhibit cytotoxicity. Chemotherapy agents may be used in maintenance therapy. Furthermore, in this specification, the term "maintenance therapy" refers to treating cancer with drugs after initial anticancer treatment, and signifies a treatment method implemented to prevent or delay the recurrence of cancer.

[0056] In one embodiment, the chemical anticancer agent may be any one selected from the group consisting of mechloretamine, chlorambucil, ifosfamide, melphalan, chlorambucil, thiotepa, altretamine, procarbazine, busulfan, streptozin, camustine, lomustine, dacarbazine, cisplatin, carboplatin, oxaliplatin, docetaxel, velban, oncovin, navelvin, 5-fluorouracil, capecitabine, cytarabine, gemcitabine, fludarabine, methotrexate, pemetrexid, mercaptopurine, hycamtin, camptos, bepecid, paclitaxel, bleocain, adriamycin, and cerubidine, but is not limited thereto.

[0057] In this specification, the term "immuno-anticancer agent" refers to a substance that inhibits the activity of immune checkpoint proteins, which suppress the differentiation, proliferation, and activity of immune cells, and is known to eliminate cancer cells by preventing them from exercising the function of evading the immune system.

[0058] The above immuno-anticancer agent may be any one selected from the group consisting of anti-CTLA-4 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-B7-H4 antibody, anti-HVEM antibody, anti-TIM3 antibody, anti-GAL9 antibody, anti-LAG3 antibody, anti-VISTA antibody, anti-KIR antibody, anti-BTLA antibody and anti-TIGIT antibody, but is not limited thereto.

[0059] In this specification, the term "targeted anticancer drug" refers to a drug that selectively attacks only cancer cells by targeting specific parts of cancer cells that differ from normal cells. It is known to inhibit the proliferation or survival of cancer cells by blocking specific signaling pathways within the cell or specific parts of the cell surface involved in signal transduction or protein regulation processes necessary for cell growth.

[0060] The above targeted anticancer drugs are abciximab, adalimumab, basiliximab, bezlotoxumab, canakinumab, daclizumab, denosumab, epalizumab, golimumab, inflectra, natalizumab, and olaratumab. It may be any one selected from the group consisting of omalizumab, palifizumab, panitumumab, trastuzumab, pertuzumab, cetuximab, rituximab, tocilizumab, secukinumab, ustekinumab, bevacizumab, avelumab, CDNs, SB11285 and DMXAA, but is not limited thereto.

[0061] In this specification, the term "cyclin-dependent protein kinase" or "CDK (cyclin-dependent kinase)" refers to a kinase that has the function of regulating the cell cycle. It can regulate cell division and proliferation, and among the CDK family, CDK4 and CDK6 are responsible for regulating the process of progressing from the G1 phase to the S phase of the cell cycle by phosphorylating cyclin protein D1 to increase its activity.

[0062] The CDK4 / 6 inhibitors that inhibit the above CDK4 / 6 can inhibit both CDK4 and CDK6, and any drug that blocks the formation of the CDK4 / 6-cyclin D1 complex and stops cell division can be used without restriction.

[0063] The above CDK4 / 6 inhibitor may be palbociclib, ribociclib, abemaciclib, and trilaciclib or pharmaceutically acceptable salts thereof, but is not limited thereto.

[0064] Preferably, the anticancer agent may be palbociclib.

[0065] In this specification, the terms “combination therapy,” “combination administration,” or “in combination” refer to any form of simultaneous or concurrent treatment using at least two distinct anticancer agents and anticancer adjuvants. The components of the combination therapy may be administered simultaneously, sequentially, or in any order. The components may be administered in an appropriate manner at different dosages, at different frequencies of administration, or through different routes.

[0066] Specifically, the combination therapy may involve administering the PEG10 ASO and the anticancer agent simultaneously, or administering the anticancer agent sequentially after administering the PEG10 ASO. The combination therapy according to the present invention may be defined as providing a synergistic effect if the efficacy, measured by, for example, the degree of response, the rate of response, the time to disease progression, or the survival period, is therapeutically superior to the efficacy obtainable by administering one or the remainder of the components of the combination therapy at a conventional dose. For example, the efficacy of the combination therapy is synergistic if its efficacy is therapeutically superior to the efficacy obtained by using each of the above components alone. In particular, a synergistic effect is considered to exist if the conventional doses of the PEG10 ASO and the anticancer agent can be reduced without compromising one or more of the degree of response, the rate of response, the time to disease progression, and survival data, particularly without compromising the duration of response, and while reducing / or having fewer problematic side effects than those occurring when each component is used at a conventional dose.

[0067] In this specification, the term "simultaneously administered" is not particularly limited and means that the components of a combination therapy are administered substantially simultaneously, for example, as a mixture or in an immediately following order.

[0068] In this specification, the term “sequentially administered” is not particularly limited and means that the components of the combination therapy are not administered simultaneously, but are administered one by one or in groups with specific time intervals between administrations. The time intervals between each administration of the components of the combination therapy may be the same or different and may be selected, for example, from 2 minutes to 96 hours, 1 day to 7 days, or 1 week, 2 weeks, or 3 weeks. Generally, the time interval between administrations may be in the range of minutes to hours, for example, 2 minutes to 72 hours, 30 minutes to 24 hours, or 1 to 12 hours. Additional examples include time intervals in the range of 24 to 96 hours, 12 to 36 hours, 8 to 24 hours, and 6 to 12 hours.

[0070] In one embodiment, the subject may be an individual suffering from cancer. Additionally, the individual may be a mammal, and preferably a human.

[0071] In one embodiment, the PEG10 ASO may be administered to mammals, including humans, by various routes. The mode of administration may be any commonly used mode, for example, into a tumor, into an artery, into a vein, into a blood vessel, into the pleura, into the abdominal cavity, into the trachea, into the dura mater, into the muscle, endoscopically, into a lesion, percutaneously, subcutaneously, locally, stereotactically, orally, by direct injection or perfusion, specifically by oral administration, intravenous administration, or subcutaneous administration.

[0072] The administration route, dosage, and frequency of the above-mentioned PEG10 ASO may be administered to the subject in various ways and amounts depending on the patient's condition and the presence or absence of side effects, and the optimal administration method, dosage, and frequency may be selected within an appropriate range by a person skilled in the art. In addition, in addition to the above-mentioned active ingredient, it may be administered in combination with other drugs known to have therapeutic effects on cancer (e.g., the anticancer agent described above) or physiologically active substances, or formulated in the form of a combination preparation with other drugs.

[0074] Another aspect provides a composition for diagnosing cancer resistant to CDK4 / 6 inhibitors, comprising a preparation capable of measuring the expression or activity of PEG10.

[0075] As used in this specification, the term "diagnosis" means confirming the existence or characteristics of a pathological condition.

[0076] The term "biomarker" can also be used as a diagnostic marker and refers to a substance capable of diagnosing the development of cancer resistant to CDK4 / 6 inhibitors in biological samples. It may include organic biomolecules such as polypeptides or nucleic acids (e.g., mRNA, etc.), lipids, glycolipids, glycoproteins, or sugars (e.g., monosaccharides, disaccharides, oligosaccharides, etc.) that show an increase in PEG10 in biological samples taken from patients with cancer resistant to CDK4 / 6 inhibitors compared to biological samples obtained from normal individuals. Preferably, the biomarker may be PEG10.

[0077] Measuring the expression or activity level of the above-mentioned PEG10 protein is a process of confirming the presence and expression level of PEG10 in biological samples of an individual to diagnose cancer resistant to CDK4 / 6 inhibitors, and confirming the amount of protein using a molecule that specifically binds to the protein.

[0078] The agent capable of measuring the expression level of the above-mentioned PEG10 protein may be a monoclonal antibody, polyclonal antibody, chimeric antibody, ligand, PNA (Peptide nucleic acid), aptamer, or nanoparticle that specifically binds to the protein, but is not limited thereto.

[0079] Analysis methods for this purpose include, but are not limited to, Western blotting, ELISA (enzyme-linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunoprecipitation assay, immunohistochemical analysis, Complement Fixation Assay, FACS (Fluorescence activated cell sorter), or protein chip.

[0080] Measuring the expression level of the gene encoding the above-mentioned PEG10 protein is a process of confirming the presence and expression level of the PEG10 gene in biological samples of an individual to diagnose cancer resistant to CDK4 / 6 inhibitors, and confirming the amount of the gene using a molecule that specifically binds to the said gene.

[0081] The agent capable of measuring the expression level of the gene encoding the PEG10 protein may be a primer pair, a probe, or an antisense nucleotide that specifically binds to the gene, but is not limited thereto.

[0082] Analysis methods for this include, but are not limited to, reverse transcriptase-polymerase chain reaction (RT-PCR), real-time polymerase chain reaction, RNase protection assay (RPA), Northern blotting, and DNA chips.

[0083] The term "biological sample" means any sample obtained from an individual in which the expression level of the PEG10 protein or the gene encoding it can be measured according to one aspect. The biological sample may be prepared by processing in a manner commonly used in the art of the present invention. Effects of the invention

[0084] According to PEG10 ASOs according to one aspect, there is an effect of improving CDK4 / 6 inhibitor resistance by inhibiting the PEG10 and EMT mechanisms associated with CDK4 / 6 inhibitor-resistant cancer, and when PEG10 ASOs and CDK4 / 6 inhibitors are used in combination, the anticancer efficacy is enhanced, so they can be usefully used in treating CDK4 / 6 resistant cancer. Brief explanation of the drawing

[0085] Figure 1 shows the results of mRNA microarray analysis performed on palbociclib-sensitive breast cancer cell lines and palbociclib-resistant breast cancer cell lines. Figure 2 shows the expression of PEG10 and EMT-related proteins in palbociclib-resistant cell lines by western blot. Figure 3 shows the expression of EMT-related proteins by western blot in palbociclib-sensitive hormone receptor-positive breast cancer cells transfected with a PEG10 overexpression plasmid. Figure 4 shows the resistance to palbociclib in palbociclib-sensitive hormone receptor-positive breast cancer cells transduced with a PEG10 overexpression plasmid, confirmed by an MTT assay. Figure 5 shows the expression of EMT-related proteins by western blot in palbociclib-resistant cells treated with PEG10 siRNA. Figure 6 shows the degree of resistance to palbociclib overcome in palbociclib-resistant cells administered with a combination of PEG10 siRNA and palbociclib using an MTT assay. Figure 7 shows the degree of inhibition of PEG10 by ASO 1 to ASO 5 in MCF7-PR cell lines confirmed by western blot. Figure 8 shows the degree of inhibition of PEG10 by ASO 1, 3, and 4 in the MCF7-PR cell line by western blot. Figure 9 shows the degree of PEG10 inhibition by ASO 2 and ASO 5 in the MCF7-PR cell line confirmed by western blot. Figure 10 shows the degree of inhibition of PEG10 by ASO 1, 3, and 4 in MCF7-PR cell lines at the mRNA expression level. Figure 11 shows the degree of inhibition of PEG10 by ASO3 and ASO4 in MCF7-PR cell lines by western blot. Figure 12 shows the degree of inhibition of PEG10 by ASO 3 and ASO 4 in MCF7-PR cell lines at the mRNA expression level. Figure 13 shows the degree of inhibition of PEG10 by ASO3 and ASO4 in T47D-PR cell lines by western blot. Figure 14 shows the degree of inhibition of PEG10 at low (100 nmol) and high (500 nmol) concentrations of ASO4 in PC3, a prostate cancer cell line, using western blot. Figure 15 shows the expression of EMT-related proteins by western blot in palbociclib-resistant cells treated with PEG10 ASO. Figure 16 shows the results of a CO-IP assay performed on the MCF7-PR cell line. Figure 17 confirms that the EMT signaling pathway is inhibited by western blot in MCF7-PR and T47D-PR cell lines transduced with SIAH1 and p21 overexpression plasmids. Figure 18 shows the effect on cell cycle regulation by western blot in CDK4 / 6 inhibitor-resistant cell lines transduced with a p21 overexpression plasmid. Figure 19 confirms the cancer growth inhibitory effect in a CDK4 / 6 inhibitor-resistant xenograft model. Figure 20 shows the identification of the proliferation marker Ki67 by immunohistochemical staining of tumor tissue from a CDK4 / 6 inhibitor-resistant xenograft model. Specific details for implementing the invention

[0086] Preferred embodiments are presented below to aid in understanding the present invention. However, the following embodiments are provided merely to facilitate a better understanding of the invention and do not limit the scope of the invention. Since the embodiments are subject to various modifications, they are not limited to the embodiments disclosed below but can be implemented in various forms.

[0088] Example 1. Preparation of PEG10 Gapmer ASO

[0089] To prepare a Gapmer ASO targeting PEG10 (Paternally Expressed 10), five ASO sequences containing 10 consecutive nucleotide sequences within the PEG10 coding sequence were randomly selected. To target PEG10, an ASO was designed using the five sequences, and then a phosphorothioate and a 2'-O-methyl group were attached to both ends of each sequence to enhance the intracellular uptake and stability of the ASO. The PEG10 Gapmer ASO prepared by the above method is shown in Table 1.

[0091] No. Sequence (5'→3') Gapmer ASO 1 ACUUC TGTGGGGATG GAGGC (Sequence No. 1) Gapmer ASO 2 GUUGU TGTTGTTGTT GGGGG (Sequence No. 2) Gapmer ASO 3 AGCUC GTCCCTTCTT CGUUC (Sequence No. 3) Gapmer ASO 4 UUUGG TGCTTTAGGA UGUGU (Sequence No. 4) Gapmer ASO 5 AGUUA GAAGGAGGGG UAGGG (Sequence No. 5) Gapmer ASO control CCUUC CCTGAAGGTT CCUCC (Sequence No. 6)

[0093] Reference Example 1. Preparation of Palbociclib-Resistant Breast Cancer Cell Lines

[0094] 1.1. Preparation of Palbociclib-Resistant Breast Cancer Cell Line MCF7-PR

[0095] MCF7, a palbociclib-sensitive breast cancer cell line, was cultured. Subsequently, palbociclib, a CDK4 / 6 inhibitor, was administered for 8 months at stepwise increasing concentrations to produce MCF7-PR, a palbociclib-resistant breast cancer cell line.

[0096] Specifically, to prepare a palbociclib-resistant cell line using MCF7 cells, MCF7 cells were cultured in a cell culture medium containing palbociclib at a concentration of 750 nM, which is the palbociclib IC50 concentration of MCF7. Subsequently, the cell culture medium containing palbociclib and the cell culture medium without palbociclib were alternately replaced every 3 days. During the process of replacing the culture medium, if cell proliferation increased in the cell culture medium containing palbociclib, cell culture was continued while gradually increasing the concentration of palbociclib. Through the above process for 8 months, when the cells began to acquire resistance to palbociclib and the IC50 became 10 times the initial value, it was determined that resistance to palbociclib had been acquired, and the preparation of the palbociclib-resistant MCF7-PR cell line was completed.

[0098] 1.2. Preparation of Palbociclib-resistant breast cancer cell line T47D-PR

[0099] Palbociclib-sensitive resistant cell line T47D-PR was prepared in the same manner as in Reference Example 1.1, except that palbociclib-sensitive breast cancer cell line T47D was used.

[0100] Specifically, to prepare a palbociclib-resistant cell line using T47D cells, T47D cells were cultured in a cell culture medium containing palbociclib at a concentration of 250 nM, which is the palbociclib IC50 concentration of T47D. Subsequently, the cell culture medium containing palbociclib and the cell culture medium without palbociclib were alternately replaced every 3 days. During the process of replacing the culture medium, if cell proliferation increased in the cell culture medium containing palbociclib, cell culture was continued while gradually increasing the concentration of palbociclib. Through the above process for 8 months, when the cells began to acquire resistance to palbociclib and the IC50 became 10 times the initial value, it was determined that resistance to palbociclib had been acquired, and the preparation of the palbociclib-resistant T47D-PR cell line was completed.

[0102] Experimental Example 1. Confirmation of differences in gene expression in palbociclib-sensitive and resistant cell lines

[0103] Genes with differing expression levels were identified through mRNA microassay in palbociclib-sensitive breast cancer cell lines MCF7 and T47D, and palbociclib-resistant breast cancer cell lines MCF7-PR and T47D-PR prepared according to Reference Example 1. The results are shown in Figure 1.

[0104] Figure 1 shows the results of mRNA microarray analysis performed on palbociclib-sensitive breast cancer cell lines and palbociclib-resistant breast cancer cell lines.

[0105] As shown in Figure 1, it was confirmed that the genes with statistically significant differences in expression between palbociclib-sensitive breast cancer cell lines and resistant breast cancer cell lines are PEG10 (Paternally Expressed Gene 10) and EMT (Epithelial-Mesenchymal Transition) related genes.

[0107] Additionally, the expression of EMT-related proteins was confirmed through Western blot, and the results are shown in Figure 2.

[0108] Figure 2 shows the expression of EMT-related proteins in palbociclib-resistant cell lines by western blot.

[0109] As shown in Figure 2, western blot confirmed that the expression of EMT-related proteins was increased in palbociclib-resistant cell lines MCF7-PR and T47D-PR.

[0111] Experimental Example 2. Confirmation of Palbociclib Resistance According to PEG10 Expression

[0112] Changes in anticancer activity against palbociclib were analyzed by regulating the expression of PEG10, which was identified as a resistance-associated gene in Experimental Example 1 above. To increase the expression of PEG10, PEG10 expression was artificially increased by transfecting palbociclib-sensitive breast cancer cell lines MCF7 and T47D with a plasmid overexpressing PEG10. In addition, to decrease the expression of PEG10, PEG10 siRNA was applied to palbociclib-resistant cell lines MCF7-PR and T47D-PR. Subsequently, the expression of EMT-related proteins was confirmed by western blot, and anticancer activity against palbociclib was confirmed by MTT assay. The results are shown in Figures 3 to 6.

[0113] Figure 3 shows the expression of EMT-related proteins by western blot in palbociclib-sensitive hormone receptor-positive breast cancer cells transfected with a PEG10 overexpression plasmid.

[0114] Figure 4 shows the resistance to palbociclib in palbociclib-sensitive hormone receptor-positive breast cancer cells transduced with a PEG10 overexpression plasmid, confirmed by an MTT assay.

[0115] Figure 5 shows the expression of EMT-related proteins by western blot in palbociclib-resistant cells treated with PEG10 siRNA.

[0116] Figure 6 shows the degree of resistance to palbociclib overcome in palbociclib-resistant cells administered with a combination of PEG10 siRNA and palbociclib using an MTT assay.

[0117] As shown in Figures 3 and 4, it was confirmed that when the expression of PEG10 was artificially increased by transfecting MCF7 and T47D with a PEG10 overexpression plasmid, the EMT-related protein increased. In addition, when MCF7 and T47D with increased PEG10 expression were treated with palbociclib at different concentrations and the anticancer activity was confirmed using an MTT assay, it was confirmed that the anticancer activity against palbociclib decreased.

[0118] As shown in Figures 5 and 6, it was confirmed that EMT-related proteins were inhibited when PEG10 was inhibited by treating palbociclib-resistant cell lines MCF7-PR and T47D-PR with PEG10 siRNA. In addition, as a result of confirming anticancer activity using an MTT assay, it was confirmed that when PEG10 siRNA and palbociclib were co-administered to the same cell lines, the anticancer activity increased compared to when palbociclib and PEG10 siRNA were administered alone.

[0119] From the above results, it was confirmed that resistance to palbociclib can be overcome by inhibiting PEG10.

[0121] Experimental Example 3. Confirmation of the effect of PEG10 ASO treatment on CDK4 / 6 inhibitor-resistant breast cancer

[0122] 3.1. Confirmation of PEG10 Inhibitory Efficacy of PEG10 ASOs

[0123] To confirm the efficacy of the PEG10 ASO containing the sequences of SEQ ID NOs. 1 to 5 prepared in Example 1, the inhibitory efficacy of PEG10 in MCF7-PR cell lines was confirmed by western blot and RT-PCR. The results are shown in Figures 7 to 10, and the results of Figure 7 were quantified and shown in Table 2.

[0125] sample RF1 / 2 PEG10 expression in each ASO compared to the control ASO RF1 PEG10 expression in each ASO compared to the control ASO Control ASO 1 1 ASO 1 0.76161 0.49144 ASO 2 0.84857 0.46291 ASO 3 0.79076 0.46643 ASO 4 0.77449 0.53379 ASO 5 1.19255 0.66534

[0127] Figure 7 shows the degree of inhibition of PEG10 by ASO 1 to ASO 5 in MCF7-PR cell lines confirmed by western blot.

[0128] Figure 8 shows the degree of inhibition of PEG10 by ASO 1, 3, and 4 in the MCF7-PR cell line by western blot.

[0129] Figure 9 shows the degree of PEG10 inhibition by ASO 2 and ASO 5 in the MCF7-PR cell line confirmed by western blot.

[0130] Figure 10 shows the degree of inhibition of PEG10 by ASO 1, 3, and 4 in MCF7-PR cell lines at the mRNA expression level.

[0131] As shown in Table 2 and Figures 7 to 10, it was confirmed that PEG10 ASOs 1 to 5 significantly inhibited PEG10 when treated at low concentrations (200 nmol) and high concentrations (500 nmol). In particular, ASOs 1, 3, and 4 showed excellent inhibitory efficacy even at low concentrations.

[0133] Further experiments were conducted using ASO 3 and ASO 4, which were confirmed to have excellent inhibitory efficacy at the mRNA level among the ASOs exhibiting excellent efficacy. The inhibitory efficacy of PEG10 at the protein and mRNA levels of the MCF7-PR cell line was confirmed, and the inhibitory efficacy of PEG10 protein in T47D-PR and the prostate cancer cell line PC3 was confirmed. The results are shown in Figures 11 to 14.

[0134] Figure 11 shows the degree of inhibition of PEG10 by ASO3 and ASO4 in MCF7-PR cell lines by western blot.

[0135] Figure 12 shows the degree of inhibition of PEG10 by ASO 3 and ASO 4 in MCF7-PR cell lines at the mRNA expression level.

[0136] Figure 13 shows the degree of inhibition of PEG10 by ASO3 and ASO4 in T47D-PR cell lines by western blot.

[0137] Figure 14 shows the degree of inhibition of PEG10 at low (100 nmol) and high (500 nmol) concentrations of ASO4 in PC3, a prostate cancer cell line, using western blot.

[0138] As shown in Figures 11 to 14, excellent PEG10 inhibitory efficacy was confirmed at the protein and mRNA levels by ASO 3 and ASO 4, and it was confirmed that they inhibited PEG10 in various cell lines as well. In addition, it was confirmed that the inhibitory efficacy of ASO 4 was superior to that of ASO 3, so experiments were subsequently conducted using ASO 4.

[0140] 3.2. Confirmation of EMT Inhibitory Efficacy of PEG10 ASO

[0141] The expression of EMT-related proteins was confirmed by western blot using ASO 4, which was identified as having the best PEG10 inhibitory efficacy in Experimental Example 3.1, and the results are shown in Fig. 15.

[0142] Figure 15 shows the expression of EMT-related proteins by western blot in palbociclib-resistant cells treated with PEG10 ASO.

[0143] As shown in Figure 15, it was confirmed that the PEG10 and EMT pathways were inhibited when ASO 4 was treated, thereby confirming that resistance to CDK4 / 6 inhibitors can be overcome.

[0145] 3.3 Confirmation of Mechanism for EMT Inhibition and Overcoming CDK4 / 6 Inhibitor Resistance via SIAH1 and p21 Gene Activation by PEG10 ASO

[0146] As confirmed by the results of Experimental Example 3.2 above, an increase in SIAH1 and p21 protein expression was observed upon treatment with PEG10 ASO, so an experiment was conducted to identify the specific mechanism. To confirm the mechanism by which PEG10 regulates SIAH1 and p21, a CO-immunoprecipitation (CO-IP) assay was performed to measure the mutual binding between proteins. The results are shown in Figure 16.

[0147] Figure 16 shows the results of a CO-IP assay performed on the MCF7-PR cell line.

[0148] As shown in Figure 16, it was confirmed that PEG10 binds to SIAH1 and p21 in palbociclib-resistant cell lines and inhibits SIAH1 and p21.

[0150] For further confirmation, SIAH1 overexpression plasmid and p21 overexpression plasmid were transduced into MCF7-PR and T47D-PR cell lines to examine their effects on the EMT signaling pathway and cell cycle. The results are shown in Figures 17 and 18.

[0151] Figure 17 confirms that the EMT signaling pathway is inhibited by western blot in MCF7-PR and T47D-PR cell lines transduced with SIAH1 and p21 overexpression plasmids.

[0152] Figure 18 shows the effect on cell cycle regulation by western blot in CDK4 / 6 inhibitor-resistant cell lines transduced with a p21 overexpression plasmid.

[0153] As shown in Figure 17, it was confirmed that the EMT signaling pathway is inhibited when SIAH1 overexpression plasmid and p21 overexpression plasmid are transduced.

[0154] As shown in Figure 18, it was confirmed that the levels of cell cycle-related proteins, CDK2, Cyclin E, Cyclin D1, and Cyclin A, increased in the MCF7-PR cell line compared to the MCF7 cell line. Additionally, it was confirmed that cell cycle-related proteins decreased in the CDK4 / 6 inhibitor-resistant cell lines MCF7-PR and T47D-PR transfected with the p21 overexpression plasmid, similar to the levels in MCF7-PR treated with PEG10 siRNA.

[0155] From the above results, it was confirmed that PEG10 ASO activates SIAH1 and p21 genes, and that the activation of said SIAH1 and p21 genes is involved in the inhibition of EMT-related mechanisms and the overcoming of CDK4 / 6 inhibitor resistance.

[0157] 3.4. Confirmation of Efficacy of PEG10 ASO in CDK4 / 6 Inhibitor-Resistant Xenograft Model

[0158] The efficacy of PEG10 ASO was confirmed by constructing a xenograft model in which tumors were formed by transplanting the palbociclib-resistant cell line MCF7-PR into mice. Groups treated with Control ASO, palbociclib, and PEG10 ASO 4 alone, as well as groups treated with palbociclib and ASO 4 in combination, were used. The experiment was conducted by administering the samples daily for 4 days, followed by administering the samples three times over a period of 2 weeks. Subsequently, the efficacy was confirmed in tumor tissues, and the results are shown in Figures 19 and 20.

[0159] Figure 19 confirms the cancer growth inhibitory effect in a CDK4 / 6 inhibitor-resistant xenograft model.

[0160] Figure 20 shows the identification of the proliferation marker Ki67 by immunohistochemical staining of tumor tissue from a CDK4 / 6 inhibitor-resistant xenograft model.

[0161] As shown in Figures 19 and 20, tumor growth was not significantly inhibited in the palbociclib-alone treatment group, but was significantly inhibited in the PEG10 ASO4-alone and combination treatment groups. In addition, it was confirmed that the combination treatment of PEG10 ASO4 and palbociclib inhibited tumor growth more than the PEG10 ASO4-alone treatment group.

[0162] In addition, immunohistochemical analysis of Ki67, a cancer cell proliferation marker in tumor tissue, revealed that palbociclib alone did not inhibit cancer cell proliferation, but PEG10 ASO4 alone or in combination was found to significantly reduce cancer cell proliferation.

[0163] From the above results, it was confirmed that PEG10 ASO has the effect of improving resistance to CDK4 / 6 inhibitors by inhibiting PEG10 and EMT mechanisms, and that using PEG10 ASO and CDK4 / 6 inhibitors together exhibits excellent anticancer effects due to synergistic effects.

Claims

Claim 1 An antisense oligonucleotide (ASO) comprising any one polynucleotide selected from the group consisting of SEQ ID NOs 1 to 5, capable of inhibiting the expression of PEG10 (Paternally Expressed 10). Claim 2 The ASO of claim 1, wherein the ASO binds complementarily to PEG10. Claim 3 The ASO of claim 1, wherein the ASO is 8 to 50 nucleotide long. Claim 4 Claim 1, wherein the ASO is capable of inhibiting a protein associated with the EMT (Epithelial-to-Mesenchymal Transition) mechanism. Claim 5 Claim 1, wherein the ASO is capable of increasing the gene activity or protein expression of SIAH1 and p21. Claim 6 A pharmaceutical composition for treating CDK (Cyclin Dependent Kinase) 4 / 6 inhibitor-resistant cancer comprising an expression or activity inhibitor of PEG10 (Paternally Expressed 10) as an active ingredient. Claim 7 A pharmaceutical composition according to claim 6, wherein the inhibitor is any one selected from the group consisting of an antisense oligonucleotide (ASO) that binds complementarily to the gene of PEG10, small hairpin RNA, small interfering RNA (siRNA), and ribozyme. Claim 8 A pharmaceutical composition according to claim 6, wherein the inhibitor is any one selected from the group consisting of a peptide that binds to a PEG10 protein, peptide mimetics, substrate analogs, aptamers, and antibodies. Claim 9 A pharmaceutical composition according to claim 7, wherein the antisense oligonucleotide comprises any one polynucleotide selected from the group consisting of SEQ ID NOs 1 to 5. Claim 10 delete Claim 11 A pharmaceutical composition according to claim 6, wherein the CDK4 / 6 inhibitor-resistant cancer is breast cancer. Claim 12 A pharmaceutical composition according to claim 6, wherein the composition can be administered in combination with an anticancer agent. Claim 13 A pharmaceutical composition according to claim 12, wherein the anticancer agent is one or more selected from the group consisting of chemotherapy agents, targeted anticancer agents, immunotherapy agents, CDK4 / 6 inhibitors, and combinations thereof. Claim 14 A pharmaceutical composition according to claim 12, wherein the combined administration is administered simultaneously or sequentially. Claim 15 A composition for diagnosing cancer resistant to CDK4 / 6 inhibitors comprising a preparation capable of measuring the expression or activity of PEG10.

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