Pharmaceutical composition for prevention or treatment of cancer comprising EZH2 inhibitor and mettl3 inhibitor

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of cancer comprising an EZH2 inhibitor and a METTL3 inhibitor. When co-administered, the EZH2 inhibitor and METTL3 inhibitor based on proteolysis-targeting chimera (PROTAC) technology exhibit a synergistic anticancer effect that is significantly superior to individual treatments, demonstrating that the co-administration can bring about the effects of preventing or treating various cancers such as leukemia, lymphoma, lung cancer, pancreatic cancer, breast cancer, and colorectal cancer.

Description

Pharmaceutical composition for preventing or treating cancer comprising an EZH2 inhibitor and a METTL3 inhibitor

[0001] This patent application claims priority to Republic of Korea Patent Application No. 10-2023-0177312, filed with the Korean Intellectual Property Office on December 8, 2023, the disclosure of which is incorporated herein by reference.

[0002] The present invention was made possible by the support of the Ministry of Science and ICT under the project identification number 1711172425, project number 2022R1F1A1061005 (Industry-Academia Cooperation Group Number: 202203890001). The research management specialized organization of the project is the Industry-Academia Cooperation Group of Inje University, the research project name is "Basic Research", the research project name is "Development of a combination immunotherapy using a PROTAC-based epigenetic regulator EZH2 target drug and an immune checkpoint inhibitor for the treatment of refractory lymphoma", the main organization is the Industry-Academia Cooperation Group of Inje University, and the research period is 2022.06.01 ~ 2025.02.28.

[0003] The present invention relates to a pharmaceutical composition for preventing or treating cancer comprising an EZH2 inhibitor and a METTL3 inhibitor, and more particularly, to a use of a combination therapy of an EZH2 inhibitor and a METTL3 inhibitor for preventing or treating cancer.

[0004] Leukemia is a group of life-threatening malignant diseases of the blood and bone marrow. Genetic errors such as reciprocal chromosomal translocations, chromosomal deletions, point mutations, and epigenetic changes can inhibit stem cell maturation throughout various stages of hematopoiesis, leading to the excessive proliferation of immature white blood cells. Leukemias are classified as myeloid and lymphoid, depending on their cell origin. Leukemia is a cancer that causes a clonal expansion of white blood cells in the bone marrow, increases the number of affected lineage cells in the circulation, and is also a lymphoid malignancy, a type of cancer that causes abnormal cell proliferation in lymphoid tissue, resulting in the suppression of normal white blood cell, red blood cell, and platelet production. A decrease in white blood cell count can lead to immune suppression, leading to sepsis caused by bacterial infection. A decrease in red blood cells can lead to anemia, and a decrease in platelets can lead to a bleeding tendency. Furthermore, hyperproliferating leukemic cells can cause fever, fatigue, pain, decreased consciousness, and a tendency to bleed. Leukemia is classified as acute or chronic depending on the degree of cell differentiation, i.e. the rate of deterioration.

[0005] Lymphoma is a blood cancer that occurs when B or T lymphocytes become cancerous. Lymphoma is a malignant tumor that develops in lymphoid tissue, but it can also develop in non-lymphoid tissue. Lymphoma is broadly classified as Hodgkin lymphoma and non-Hodgkin lymphoma, with Hodgkin lymphoma accounting for approximately 10% of all lymphomas, and non-Hodgkin lymphoma accounting for the remaining 90%. The most well-established risk factor for non-Hodgkin lymphoma is immunosuppression, with patients with HIV at increased risk for high-grade non-Hodgkin lymphoma. Other high-risk groups include organ transplant recipients, patients receiving high-dose chemotherapy in conjunction with stem cell transplantation, and those with inherited immunodeficiency syndromes or autoimmune diseases. Infections also play a significant role in the development of some lymphomas. Epstein-Barr Virus (EBV), a member of the herpes virus family, infects most people and causes an asymptomatic latent infection, but it is known to cause tumors such as Burkitt's lymphoma, nasopharyngeal carcinoma (NPC), and Hodgkin's disease in individuals with weakened immune systems. Helicobacter pylori infection has been identified as a risk factor for gastric mucosa-associated lymphoid tissue lymphoma. Burkitt's lymphoma is a highly aggressive type of lymphoma that targets B lymphocytes and accounts for less than 1% of all non-Hodgkin's lymphomas. Existing treatments for lymphoma include dose reduction of immunosuppressants, use of antiviral agents, chemotherapy, and administration of rituximab antibodies. However, response rates vary, and an effective treatment method has not yet been established.

[0006] For the past several decades, molecular inhibitor-based drugs have been primarily used in cancer treatment. These drugs exhibit efficacy by inhibiting protein function through binding to the active site of the target protein. However, many proteins lack active sites or have sites that are unsuitable for inhibitory action. Consequently, high doses are required to enhance therapeutic efficacy, leading to drug resistance. To overcome these limitations, the ubiquitin-proteasome system (UPS) (PROTAC) strategy can be used. PROTACs are heterobifunctional molecules that bind E3 ligases to target proteins, which then utilize the ubiquitin-based protein degradation system within the cell to degrade the protein. PROTAC-based drugs are expected to play an effective role in the targeted treatment of various diseases, as they reduce side effects and increase efficacy.

[0007] However, there are reports that PROTAC-based tumor protein degradation does not result in an effective level of tumor growth inhibition, or that the target protein degradation effect of PROTAC varies greatly depending on the tissue of administration. Therefore, there is a need to develop a PROTAC-based anticancer therapy that can exhibit an anticancer effect greater than that of conventional chemical drugs.

[0008] EZH2 (Enhancer of zeste homolog 2) is a histone-lysine N-methyltransferase enzyme that catalyzes the methylation of H3K27me3, thereby promoting transcriptional repression. EZH2 has been implicated in the development and progression of various cancers, including hematological malignancies, breast cancer, colon cancer, lymphoma, ovarian cancer, and liver cancer, and its high expression level is known to be correlated with a poor prognosis. Because of this role of EZH2 in tumorigenesis, EZH2 has become a powerful target for anticancer therapy. Because existing EZH2 inhibitors that suppress methyltransferase activity do not effectively suppress the oncogenic activity of EZH2, PROTAC-based EZH2 degraders are being developed.

[0009] METTL3 (N6-adenosine-methyltransferase complex catalytic subunit) regulates mRNA by transferring methyl groups during m6A modification, thereby promoting tumor initiation and progression. METTL3 inhibitors are expected to be effective in tumor-targeted therapy because they inhibit this process and restore chemosensitivity of tumor cells in vitro. The anticancer effects of METTL3 inhibitors have been demonstrated in diffuse large B-cell lymphoma (DLBCL) and acute myeloid leukemia (AML).

[0010] Accordingly, the present inventors have confirmed that when an EZH2 inhibitor and a METTL3 inhibitor are administered in combination based on proteolysis-targeting chimera (PROTAC) technology, there is a synergistic effect that shows significant anticancer activity compared to when they are administered alone, and thus, it can create a preventive or therapeutic effect on various cancers such as leukemia, lymphoma, lung cancer, pancreatic cancer, breast cancer, and colon cancer.

[0011] Accordingly, the purpose of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, which comprises an EZH2 inhibitor and a METTL3 inhibitor as active ingredients.

[0012] Another object of the present invention is to provide a combination kit for preventing or treating cancer comprising (i) an EZH2 inhibitor and (ii) a METTL3 inhibitor.

[0013] Another object of the present invention is to provide a method for preventing or treating cancer using a pharmaceutical composition comprising an EZH2 inhibitor and a METTL3 inhibitor as active ingredients.

[0014] Another object of the present invention is to provide a use of an EZH2 inhibitor and a METTL3 inhibitor for the prevention or treatment of cancer.

[0015] Another object of the present invention is to provide a combination therapy use of an EZH2 inhibitor and a METTL3 inhibitor for the prevention or treatment of cancer.

[0016] The present invention relates to a pharmaceutical composition for preventing or treating cancer comprising an EZH2 inhibitor and a METTL3 inhibitor, and leukemia, lymphoma, lung cancer, pancreatic cancer, breast cancer, or colon cancer can be effectively prevented or treated through the pharmaceutical composition of the present invention.

[0017] EZH2 is a methyltransferase that is an important target for lymphoma treatment but is not widely used in clinical settings. PROteolysis Targeting Chimeras (PROTACs) represent a novel therapeutic strategy aimed at eliminating proteins that are difficult to target with existing small molecules. In a previous study, we compared small molecule-based EZH2 inhibitors used in clinical settings with PROTAC-based EZH2 degraders and found that the PROTAC-based degraders were significantly more effective. Based on this, we investigated the effects of combining a PROTAC-based EZH2 degrader with an established lymphoma drug, a METTL3 inhibitor, on the proliferation and apoptosis of Burkitt's lymphoma cells. Using the CCK-8 assay, we found that treatment with both drugs, either alone or in combination, significantly inhibited the growth of Daudi and Ramos cells in a dose-dependent manner. Combination treatment significantly inhibited cell proliferation and induced apoptosis, as confirmed by Annexin V / PI staining. The results of the present study demonstrated a G2 / M phase arrest with a significant decrease in G0 / G1 phases as determined by flow cytometry. Furthermore, Western blot analysis revealed increased levels of cleaved PARP, cleaved caspase-3, TP53, and PUMA, indicating increased p53-dependent apoptosis. The present study suggests that combination therapy with an EZH2 inhibitor (dEZH2) and a METTL3 inhibitor (iMETTL3) is a promising approach for clinical Burkitt lymphoma.

[0018] Hereinafter, the present invention will be described in more detail.

[0019] One aspect of the present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising an EZH2 inhibitor and a METTL3 inhibitor as active ingredients.

[0020] The term "EZH2" in this specification refers to a histone-lysine N-methyltransferase that catalyzes the methylation of H3K27me3 and promotes transcriptional repression. EZH2 is involved in the development and progression of various cancers, including hematological cancer, breast cancer, colon cancer, lymphoma, ovarian cancer, and liver cancer, and its high expression level is known to be correlated with a poor prognosis. Therefore, drugs such as Tazemetostat, Valemetostat, GSK126, GSK343, CPI-1205, PF-06821497, and SHR2554 are continuously being developed to degrade the EZH2 protein or inhibit gene expression.

[0021] The term “EZH2 inhibitor (EZH2 degrader, dEZH2)” in this specification refers to an agent that targets and degrades the EZH2 protein.

[0022] The term "METTL3" in this specification refers to N 6 -methyladenosine (m 6 A) METTL3 refers to an RNA methyltransferase involved in the biosynthesis, degradation, and translation control of mRNA through modification. METTL3 promotes the translation of important oncogenes such as epidermal growth factor receptor (EGFR) and the Hippo pathway effector TAZ in human cancer cells, and is known to be required for cancer cell growth, survival, and invasion. Inhibiting METTL3 is expected to be effective in tumor-targeted therapy, as it restores chemosensitivity of tumor cells in vitro.

[0023] The term “METTL3 inhibitor (iMETTL3)” in this specification refers to an agent that inhibits the activity of METTL3.

[0024] The term "including as an active ingredient" in this specification means including an amount sufficient to achieve a specific effect of an EZH2 inhibitor or a METTL3 inhibitor, for example, activity for preventing or treating cancer for the purposes of the present invention.

[0025] The term “prevention” in this specification means any action that inhibits or delays the progression of cancer by administering the pharmaceutical composition of the present invention.

[0026] The term “treatment” as used herein means inhibition of cancer development, alleviation of cancer, and elimination of cancer.

[0027] In the present invention, the composition may be for combined administration of an EZH2 inhibitor and a METTL3 inhibitor.

[0028] In the present invention, the EZH2 inhibitor may be based on PROTAC (Proteolysis targeting chimera).

[0029] In the present invention, the PROTAC molecule is a heterobifunctional compound composed of a ubiquitin ligase binding ligand, a target protein binding ligand, and a linker connecting them, which serves to bind an E3 ligase and a target protein, and then degrades the protein using a ubiquitin-based protein degradation system within the cell.

[0030] In one embodiment of the present invention, the EZH2 inhibitor is a PROTAC molecule.

[0031] In one embodiment of the present invention, a PROTAC-based EZH2 inhibitor may refer to a substance aimed at decomposing the EZH2 protein.

[0032] In the present invention, the EZH2 inhibitor may be MS1943 or MS177, but is not necessarily limited thereto.

[0033] In one embodiment of the present invention, the MS1943 is a compound having a structure represented by the following structural formula 1.

[0034] [Structural formula 1]

[0035]

[0036] In one embodiment of the present invention, the MS177 is a compound having a structure of structural formula 2 below.

[0037] [Structural formula 2]

[0038]

[0039] In the present invention, the METTL3 inhibitor may be STM2457, but is not necessarily limited thereto.

[0040] In one embodiment of the present invention, the STM2457 is a compound having a structure of structural formula 3 below.

[0041] [Structural formula 3]

[0042]

[0043] The present inventors confirmed that when the EZH2 inhibitor and METTL3 inhibitor were used alone, the minimum molar concentration at which the effect was observed was 5 μM for both drugs. Accordingly, when the EZH2 inhibitor and METTL3 inhibitor were used in combination, these results were reflected and each drug was included in a pharmaceutical composition at a molar ratio of 1:1, with a minimum molar concentration of 5 μM exhibiting an effect. As a result, compared to the single treatment, the combination treatment showed a superior cancer cell growth inhibition effect, i.e., a superior anticancer activity, confirming that the combination treatment had a synergistic effect.

[0044] In one embodiment of the present invention, the molar ratio of the EZH2 inhibitor and the METTL3 inhibitor is 1:10 to 10:1, for example, 1:10 to 9:1, 1:10 to 8:1, 1:10 to 7:1, 1:10 to 6:1, 1:10 to 5:1, 1:10 to 4:1, 1:10 to 3:1, 1:10 to 2:1, 1:10 to 1:1, 1:9 to 10:1, 1:9 to 9:1, 1:9 to 8:1, 1:9 to 7:1, 1:9 to 6:1, 1:9 to 5:1, 1:9 to 4:1, 1:9 to 3:1, 1:9 to 2:1, 1:9 to 1:1, 1:8 to 10:1, 1:8 to 9:1, 1:8 to 8:1, 1:8 to 7:1, 1:8 to 6:1, 1:8 to 5:1, 1:8 to 4:1, 1:8 to 3:1, 1:8 to 2:1, 1:8 to 1:1, 1:7 to 10:1, 1:7 to 9:1, 1:7 to 8:1, 1:7 to 7:1, 1:7 to 6:1, 1:7 to 5:1, 1:7 to 4:1, 1:7 to 3:1, 1:7 to 2:1, 1:7 to 1:1, 1:6 to 10:1, 1:6 to 9:1, 1:6 to 8:1, 1:6 to 7:1, 1:6 to 6:1, 1:6 to 5:1, 1:6 to 4:1, 1:6 to 3:1, 1:6 to 2:1, 1:6 to 1:1, 1:5 to 10:1, 1:5 to 9:1, 1:5 to 8:1, 1:5 to 7:1, 1:5 to 6:1, 1:5 to 5:1, 1:5 to 4:1, 1:5 to 3:1, 1:5 to 2:1, 1:5 to 1:1, 1:4 to 10:1, 1:4 to 9:1, 1:4 to 8:1, 1:4 to 7:1, 1:4 to 6:1, 1:4 to 5:1, 1:4 to 4:1, 1:4 to 3:1, 1:4 to 2:1, 1:4 to 1:1, 1:3 to 10:1, 1:3 to 9:1, 1:3 to 8:1, 1:3 to 7:1, 1:3 to 6:1, 1:3 to 5:1, 1:3 to 4:1,1:3 to 3:1, 1:3 to 2:1, 1:3 to 1:1, 1:2 to 10:1, 1:2 to 9:1, 1:2 to 8:1, 1:2 to 7:1, 1:2 to 6:1, 1:2 to 5:1, 1:2 to 4:1, 1:2 to 3:1, 1:2 to 2:1, 1:2 to 1:1, 1:1 to 10:1, 1:1 to 9:1, 1:1 to 8:1, 1:1 to 7:1, 1:1 to 6:1, 1:1 to 5:1, 1:1 to 4:1, 1:1 to 3:1, or 1:1 to 2:1, for example, 1:3 to 3:1, but It is not limited to,

[0045] In one embodiment of the present invention, the pharmaceutical composition may comprise an EZH2 inhibitor at a concentration of 1 to 20 μM and a METTL3 inhibitor at a concentration of 1 to 20 μM.

[0046] In one embodiment of the present invention, the pharmaceutical composition may comprise an EZH2 inhibitor at a concentration of 1 to 20 μM, 1 to 15 μM, 1 to 10 μM, 2 to 20 μM, 2 to 15 μM, 2 to 10 μM, 3 to 20 μM, 3 to 15 μM, 3 to 10 μM, 4 to 20 μM, 4 to 15 μM, 4 to 10 μM, 4 to 8 μM, 4 to 6 μM, 5 to 20 μM, 5 to 15 μM, or 5 to 10 μM, for example, at a concentration of 5 μM or 10 μM.

[0047] The composition may comprise a METTL3 inhibitor at a concentration of 1 to 20 μM, 1 to 15 μM, 1 to 10 μM, 2 to 20 μM, 2 to 15 μM, 2 to 10 μM, 3 to 20 μM, 3 to 15 μM, 3 to 10 μM, 4 to 20 μM, 4 to 15 μM, 4 to 10 μM, 4 to 8 μM, 4 to 6 μM, 5 to 20 μM, 5 to 15 μM, or 5 to 10 μM, for example, at a concentration of 5 μM or 10 μM.

[0048] In the present invention, the cancer may be at least one selected from the group consisting of leukemia, lymphoma, lung cancer, pancreatic cancer, breast cancer, and colon cancer.

[0049] In one embodiment of the present invention, the leukemia is acute lymphoblastic leukemia, B cell precursor acute lymphoblastic leukemia, T cell precursor acute lymphoblastic leukemia, acute biphenotypic leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, B cell prolymphocytic leukemia, acute myelogenous leukemia, acute promyelocytic leukemia, acute myeloblastic leukemia, acute megakaryoblastic leukemia, chronic myelogenous leukemia The disease may be at least one selected from the group consisting of, but is not necessarily limited to, chronic myelomonocytic leukemia, hairy cell leukemia, T cell prolymphocytic leukemia, large granular lymphocytic leukemia, adult T cell leukemia, and chronic eosinophilic leukemia.

[0050] In one embodiment of the present invention, the lymphoma may be at least one T-cell lymphoma (TCL) selected from the group consisting of T-cell non-Hodgkin lymphoma, cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL), anaplastic large cell lymphoma (ALCL), adult T-cell leukemia / lymphoma (ATLL), angioimmunoblastic T-cell lymphoma (AITL), extranodal NK / T-cell lymphoma, and hepatosplenic T-cell lymphoma, but is not necessarily limited thereto.

[0051] In one embodiment of the present invention, the lymphoma is Burkitt's lymphoma, B cell non-Hodgkin lymphoma (NHL), B cell chronic lymphocytic leukemia (B-CLL), B cell acute lymphoblastic leukemia (B-ALL), large B cell lymphoma, diffuse large B cell lymphoma (DLBCL), follicular lymphoma, marginal zone B-cell lymphoma, chronic lymphocytic leukemia (CLL), mantle cell lymphoma, primary mediastinal large B-cell lymphoma (PMBCL), intravascular large B cell lymphoma (IVLBCL), It may be one or more types of B-cell lymphoma (BCL) selected from the group consisting of, but not necessarily limited to, primary effusion lymphoma, lymphomatoid granulomatosis, primary central nervous system lymphoma (PCNSL), ALK-positive large B-cell lymphoma, and plasmablastic lymphoma (PBL).

[0052] In one embodiment of the present invention, the lymphoma may be nodular lymphocyte predominant Hodgkin lymphoma, lymphocyte rich classical Hodgkin lymphoma, nodular sclerosis classical Hodgkin lymphoma, mixed cellularity classical Hodgkin lymphoma, or lymphocyte depletion classical Hodgkin lymphoma.

[0053] In another embodiment of the present invention, the lymphoma may be non-Hodgkin's lymphoma that occurs in a human immunodeficiency virus (HIV) patient, an organ transplant recipient, a patient who has received stem cell transplantation and high-dose chemotherapy, a hereditary immunodeficiency syndrome, or an autoimmune disease patient.

[0054] In another embodiment of the present invention, the lymphoma may be Burkitt's lymphoma, nasopharyngeal carcinoma (NPC), or Hodgkin's disease that occurs in an immunocompromised patient infected with Ebstein-Barr Virus (EBV).

[0055] In another embodiment of the present invention, the lymphoma may be gastric mucosa-associated lymphoid tissue lymphoma caused by Helicobacter pylori infection.

[0056] In one embodiment of the present invention, the lung cancer may be, but is not necessarily limited to, non-small cell lung cancer, small cell lung cancer, pancoast tumor, or lung carcinoid tumor.

[0057] In one embodiment of the present invention, the non-small cell lung cancer may be, but is not necessarily limited to, squamous cell carcinoma, adenocarcinoma, or large-cell carcinoma.

[0058] In one embodiment of the present invention, the pancreatic cancer may be at least one pancreatic cystic tumor selected from the group consisting of serous cystadenoma, mucinous cystic neoplasm, intraductal papillary mucinous neoplasm (IPMN), and solid pseudopapillary tumor, or at least one malignant pancreatic tumor selected from the group consisting of pancreatic ductal adenocarcinoma, acinar cell carcinoma, and neuroendocrine tumor, but is not necessarily limited thereto.

[0059] In one embodiment of the present invention, the breast cancer may be at least one selected from the group consisting of triple negative breast cancer (TNBC), metastatic breast cancer, refractory breast cancer, androgen receptor (AR)-positive breast cancer, estrogen receptor (ER)-positive breast cancer, and receptor tyrosine-protein kinase erbB-2 (HER2)-positive breast cancer, or may be breast cancer that has failed treatment with at least one therapeutic agent selected from the group consisting of tamoxifen, toremifene, exemestane, anastrozole, letrozole, trastuzumab, ado-trastuzumab, emtansine, pertuzumab, lapatinib, bevacizumab, and fulvestrant.

[0060] In one embodiment of the present invention, the colon cancer may be, but is not necessarily limited to, adenocarcinoma, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, or lymphoma.

[0061] The pharmaceutical composition of the present invention may additionally comprise a pharmaceutically acceptable carrier.

[0062] The term "pharmaceutically acceptable" as used herein means, as commonly used in the pharmaceutical field, that when administered, it does not stimulate the organism and does not inhibit the biological activity and properties of the administered compound.

[0063] In the present invention, any carrier commonly used in the art may be used as the carrier. Non-limiting examples of the carrier include saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, maltodextrin, glycerol, ethanol, or combinations thereof.

[0064] The pharmaceutical composition of the present invention may be used by adding other pharmaceutically acceptable additives, such as excipients, diluents, antioxidants, buffers, or bacteriostatic agents, if necessary, and may be used by additionally adding fillers, bulking agents, wetting agents, disintegrating agents, dispersing agents, surfactants, binders, or lubricants.

[0065] The pharmaceutical composition of the present invention may be administered in various dosages depending on factors such as the formulation method, administration method, patient age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity. The dosage may be determined or prescribed as an effective dosage for the desired treatment or prevention. For example, the daily dosage of the pharmaceutical composition of the present invention may be 0.0001-1000 mg / kg.

[0066] The pharmaceutical composition of the present invention can be manufactured in a unit dose form or can be manufactured by placing it in a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains, and thereby. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, suppository, powder, granule, tablet or capsule, and may additionally include a dispersant or stabilizer, but is not limited thereto.

[0067] The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's age, weight, sex, dosage form, health condition, and disease severity, and may be administered once or several times a day at regular intervals, depending on the judgment of a doctor or pharmacist. For example, the daily dosage may be 1 to 1000 ug / mL based on the active ingredient content, but this is an example of an average case, and the dosage may be higher or lower depending on individual differences.

[0068] Another aspect of the present invention relates to a combination kit for preventing or treating cancer comprising (i) an EZH2 inhibitor and (ii) a METTL3 inhibitor.

[0069] In one embodiment of the present invention, the (i) EZH2 inhibitor and (ii) METTL3 inhibitor may be contained in the same container or in different containers.

[0070] The above kit may additionally include not only the active ingredients (i) EZH2 inhibitor and (ii) METTL3 inhibitor, but also containers, materials, package inserts, etc. commonly used in the art suitable for use as a combination kit for preventing or treating cancer.

[0071] The above containers may include, but are not necessarily limited to, vials, syringes, bottles, etc.

[0072] The above materials may include, but are not necessarily limited to, diluents such as saline solution, filters, IV bags and lines, needles and syringes, etc.

[0073] The package insert may be instructions for using an EZH2 inhibitor and the METTL3 inhibitor in combination to treat or delay the progression of cancer, or instructions for treating a subject for cancer using an EZH2 inhibitor and the METTL3 inhibitor.

[0074] In the present invention, the EZH2 inhibitor may be MS1943 or MS177, but is not necessarily limited thereto.

[0075] In the present invention, the METTL3 inhibitor may be STM2457, but is not necessarily limited thereto.

[0076] In the present invention, the (i) EZH2 inhibitor and (ii) METTL3 inhibitor may be administered simultaneously or sequentially.

[0077] The term “simultaneously” as used herein means that two preparations or pharmaceutical compositions are administered at the same time.

[0078] The term "sequentially" as used herein means that the active ingredients or pharmaceutical compositions are administered one after the other, rather than simultaneously. For example, "sequentially" administration may mean that one agent / pharmaceutical composition is administered within 1 minute, 5 minutes, 10 minutes, 30 minutes, or several hours, for example, within 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, or 48 hours, of another agent / pharmaceutical composition, provided that the circulating half-life of the first agent administered is sufficiently long to allow both agents to be present simultaneously in therapeutically effective amounts, but is not necessarily limited thereto. The time delay in administration between components may vary depending on the exact nature, interactions, and respective half-lives of the components.

[0079] In one embodiment of the present invention, the (i) EZH2 inhibitor and (ii) METTL3 inhibitor may be administered at the same time or at different time intervals.

[0080] In one embodiment of the present invention, the (i) EZH2 inhibitor and (ii) METTL3 inhibitor may be administered via the same route of administration or different routes of administration.

[0081] In the present invention, the cancer may be at least one selected from the group consisting of leukemia, lymphoma, lung cancer, pancreatic cancer, breast cancer, and colon cancer.

[0082] Since the kit of the present invention comprises the active ingredient of the pharmaceutical composition described above, description of duplicate content is omitted to avoid excessive complexity of this specification.

[0083] Another aspect of the present invention relates to a method for preventing or treating cancer by administering to a subject a pharmaceutical composition comprising an EZH2 inhibitor and a METTL3 inhibitor as active ingredients.

[0084] In one embodiment of the present invention, the EZH2 inhibitor and the METTL3 inhibitor may be administered simultaneously, sequentially, or alternatingly. When the EZH2 inhibitor and the METTL3 inhibitor are administered alternately, each agent may be sequentially administered at a time interval of, but not limited to, about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours, about 96 hours, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 8 weeks, or about 12 weeks.

[0085] In one embodiment of the present invention, the EZH2 inhibitor may be administered at a dose of, but is not limited to, 0.1 mg / kg to 1000 mg / kg, 0.1 mg / kg to 500 mg / kg, 0.1 mg / kg to 100 mg / kg, 0.1 mg / kg to 50 mg / kg, 1 mg / kg to 1000 mg / kg, 1 mg / kg to 500 mg / kg, 1 mg / kg to 100 mg / kg, 1 mg / kg to 50 mg / kg, 5 mg / kg to 1000 mg / kg, 5 mg / kg to 500 mg / kg, 5 mg / kg to 100 mg / kg, or 5 mg / kg to 50 mg / kg.

[0086] In one embodiment of the present invention, the METTL3 inhibitor may be administered at a dose of, but is not limited to, 0.1 mg / kg to 1000 mg / kg, 0.1 mg / kg to 500 mg / kg, 0.1 mg / kg to 100 mg / kg, 0.1 mg / kg to 50 mg / kg, 1 mg / kg to 1000 mg / kg, 1 mg / kg to 500 mg / kg, 1 mg / kg to 100 mg / kg, 1 mg / kg to 50 mg / kg, 5 mg / kg to 1000 mg / kg, 5 mg / kg to 500 mg / kg, 5 mg / kg to 100 mg / kg, or 5 mg / kg to 50 mg / kg.

[0087] In one embodiment of the present invention, the pharmaceutical composition may be administered 1 to 6 times, 1 to 5 times, 1 to 4 times, or 1 to 3 times daily, as needed, but is not limited thereto.

[0088] In one embodiment of the present invention, the pharmaceutical composition may be in the form of, but is not limited to, tablets, capsules, granules, syrups, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, ointments, creams, and injections.

[0089] The term "subject" in this specification may be a mammal including a human, for example, but not limited to, a human, monkey, cow, horse, sheep, pig, cat, dog, mouse, rat, rabbit or guinea pig.

[0090] The term "administration" as used herein means providing a given substance to a subject by any suitable method, and the route of administration of the composition comprising the EZH2 inhibitor and the METTL3 inhibitor of the present invention as active ingredients may be administered orally or parenterally through any common route as long as it can reach the target tissue. In addition, the composition of the present invention may be administered using any device capable of delivering the active ingredient to the target cell. For example, the pharmaceutical composition of the present invention may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, topical administration, intranasal administration, intrapulmonary administration, rectal administration, intrathecal administration, ocular administration, skin administration, and transdermal administration, but is not limited thereto.

[0091] Another aspect of the present invention relates to the use of a pharmaceutical composition comprising an EZH2 inhibitor and a METTL3 inhibitor as active ingredients for the prevention or treatment of cancer.

[0092] Another aspect of the present invention relates to a combination therapy use of an EZH2 inhibitor and a METTL3 inhibitor for the prevention or treatment of cancer.

[0093] Another aspect of the present invention relates to a method for preventing or treating cancer, comprising administering to a subject in need of treatment an effective amount of an EZH2 inhibitor and a METTL3 inhibitor.

[0094] The term “subject” in this specification is meant to include humans (e.g., patients) and mammals (e.g., mice, rats, dogs, cats, rabbits, chickens, monkeys, etc.).

[0095] The term "effective amount" as used herein means an amount (e.g., dose) of a pharmaceutical composition that provides a significant reduction in the clinical symptoms of a disease or condition to be treated without causing excessive toxic side effects.

[0096] The term “dose” as used herein means the weight (e.g., milligrams (mg)) of active substance per kilogram (kg) of body weight of the subject.

[0097] In one embodiment of the present invention, the EZH2 inhibitor and the METTL3 inhibitor may exist as separate formulations, but are not limited thereto.

[0098] In one embodiment of the present invention, the EZH2 inhibitor and the METTL3 inhibitor may be administered simultaneously, sequentially, or alternately. In one embodiment of the present invention, the EZH2 inhibitor and the METTL3 inhibitor may be administered in combination.

[0099] The present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising an EZH2 inhibitor and a METTL3 inhibitor. It has been confirmed that when an EZH2 inhibitor and a METTL3 inhibitor are administered in combination based on proteolysis-targeting chimera (PROTAC) technology, there is a synergistic effect in which they exhibit remarkable anticancer activity compared to when they are administered alone, and thus, it is possible to create a preventive or therapeutic effect on various cancers such as leukemia, lymphoma, lung cancer, pancreatic cancer, breast cancer, and colon cancer.

[0100] Figure 1 is a graph showing the results of measuring the optical density (OD) when an EZH2 inhibitor (MS1943) and a METTL3 inhibitor (STM2457) according to one embodiment of the present invention were treated alone or in combination with a T-cell leukemia cell line (Jurkat).

[0101] Figure 2 is a graph showing the results of measuring absorbance when an EZH2 inhibitor (MS1943) and a METTL3 inhibitor (STM2457) according to one embodiment of the present invention were treated alone or in combination with a B-cell lymphoma cell line (Daudi).

[0102] FIG. 3 is a graph showing the results of measuring cell survival rate (%) when an EZH2 inhibitor (MS1943 or MS177) and a METTL3 inhibitor (STM2457) according to one embodiment of the present invention were treated alone or in combination with a lung cancer cell line (A549).

[0103] Figure 4 is a graph showing the results of measuring cell survival rate when an EZH2 inhibitor (MS1943 or MS177) and a METTL3 inhibitor (STM2457) according to one embodiment of the present invention were treated alone or in combination with a pancreatic cancer cell line (Capan-1).

[0104] FIG. 5 is a graph showing the results of measuring cell survival rate when an EZH2 inhibitor (MS1943 or MS177) and a METTL3 inhibitor (STM2457) according to one embodiment of the present invention were treated alone or in combination with a breast cancer cell line (MDA-MB-231).

[0105] Figure 6 is a graph showing the results of measuring cell survival rate when an EZH2 inhibitor (MS1943 or MS177) and a METTL3 inhibitor (STM2457) according to one embodiment of the present invention were treated alone or in combination with a colon cancer cell line (HCT-116).

[0106] Figure 7 is a graph showing the results of measuring cell survival rate when an EZH2 inhibitor (MS1943 or MS177) and a METTL3 inhibitor (STM2457) were treated alone or in combination with a lymphoma cell line (Daudi) according to one embodiment of the present invention.

[0107] Figure 8a is a graph showing the results of treating Burkitt's lymphoma cell lines (Daudi and Ramos) alone with an EZH2 inhibitor (MS1943) and a METTL3 inhibitor (STM2457) at different concentrations (2.5, 5, and 10 μM) according to one embodiment of the present invention and measuring cell viability 24 hours later.

[0108] Figure 8b is a graph showing the results of treating Burkitt's lymphoma cell lines (Daudi and Ramos) alone with an EZH2 inhibitor (MS1943) and a METTL3 inhibitor (STM2457) at different concentrations (2.5, 5, and 10 μM) according to one embodiment of the present invention and measuring cell viability 48 hours later.

[0109] Figure 8c is a graph showing the results of treating Burkitt's lymphoma cell lines (Daudi and Ramos) alone with an EZH2 inhibitor (MS1943) and a METTL3 inhibitor (STM2457) at different concentrations (2.5, 5, and 10 μM) according to one embodiment of the present invention and measuring cell viability 72 hours later.

[0110] FIG. 8d is a graph showing the results of measuring cell viability by time period (24 hours, 48 ​​hours, or 72 hours) after treating Burkitt's lymphoma cell line (Daudi) with an EZH2 inhibitor (MS1943) and a METTL3 inhibitor (STM2457) at 5 μM alone or in combination according to one embodiment of the present invention.

[0111] Figure 8e is a graph showing the results of measuring cell viability by time period (24 hours, 48 ​​hours, or 72 hours) after treating Burkitt's lymphoma cell line (Ramos) with an EZH2 inhibitor (MS1943) and a METTL3 inhibitor (STM2457) at 5 μM alone or in combination according to one embodiment of the present invention.

[0112] Figure 9a is a graph showing the results of measuring cell viability 24 hours after treating a Burkitt's lymphoma cell line (Ramos) with an EZH2 inhibitor (MS1943) and a METTL3 inhibitor (STM2457) at 5 μM alone or in combination according to one embodiment of the present invention.

[0113] Figure 9b is a graph showing the results of measuring cell viability 48 hours after treating a Burkitt's lymphoma cell line (Ramos) with an EZH2 inhibitor (MS1943) and a METTL3 inhibitor (STM2457) at 5 μM alone or in combination according to one embodiment of the present invention.

[0114] Figure 9c is a graph showing the results of measuring cell viability 72 hours after treating a Burkitt's lymphoma cell line (Ramos) with an EZH2 inhibitor (MS1943) and a METTL3 inhibitor (STM2457) at 5 μM alone or in combination according to one embodiment of the present invention.

[0115] Figures 10a to 10d are graphs showing the results of measuring cell death through annexin V / PI staining analysis 72 hours after treating Burkitt's lymphoma cell line (Daudi) with an EZH2 inhibitor (MS1943) and a METTL3 inhibitor (STM2457) at 5 μM alone or in combination according to one embodiment of the present invention.

[0116] Figures 10e to 10h are graphs showing the results of measuring cell death through annexin V / PI staining analysis 72 hours after treating Burkitt's lymphoma cell line (Ramos) with 5 μM of an EZH2 inhibitor (MS1943) and a METTL3 inhibitor (STM2457) alone or in combination according to one embodiment of the present invention.

[0117] Figures 11a and 11b are graphs showing the results of analyzing the cell cycle using an Annexin V-PI apoptosis kit after treating a Burkitt lymphoma cell line (Daudi) with an EZH2 inhibitor (MS1943) and a METTL3 inhibitor (STM2457) at 5 μM alone or in combination according to one embodiment of the present invention.

[0118] Figures 11c and 11d are graphs showing the results of analyzing the cell cycle using an Annexin V-PI apoptosis kit after treating Burkitt's lymphoma cell line (Ramos) with an EZH2 inhibitor (MS1943) and a METTL3 inhibitor (STM2457) at 5 μM alone or in combination according to one embodiment of the present invention.

[0119] Figures 12a to 12g are graphs showing the results of analyzing the expression of proteins related to the apoptotic p53-dependent pathway by Western blot after treating Burkitt's lymphoma cell line (Daudi) with 5 μM of an EZH2 inhibitor (MS1943) and a METTL3 inhibitor (STM2457) alone or in combination according to one embodiment of the present invention.

[0120] The present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising an EZH2 inhibitor and a METTL3 inhibitor as active ingredients.

[0121] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only intended to illustrate the present invention, and the scope of the present invention is not limited by these examples.

[0122] Unless otherwise stated, all numbers, values ​​and / or expressions expressing ingredients, reaction conditions and quantities of ingredients used in this specification are approximations that inherently reflect, among other things, the various uncertainties of measurement that arise in obtaining such values ​​and therefore should be understood as being modified in all instances by the term "about", and when a numerical range is disclosed herein, such range is continuous and includes every value from the minimum value to the maximum value inclusive, unless otherwise stated.

[0123] Also, the term "or" is intended to mean an inclusive "or" rather than an exclusive "or." That is, where the connection or use between constructions is not otherwise specified or clear from the context, i.e., if X includes A; X includes B; or X includes both A and B, "X includes A or B" can be applied to any of these cases.

[0124] And, throughout the specification, whenever a part is said to "include" a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise specifically stated.

[0125]

[0126] Example 1: Experimental study of combined treatment of METTL3 inhibitor and EZH2 inhibitor on T-cell leukemia cell lines.

[0127] Using Jurkat, a T-cell leukemia cell line, 1.0×10 4After stimulation with PROTAC-based EZH2 inhibitors (MS1943) and METTL3 inhibitors (STM2457) in different conditions, the cells were cultured in a CO2 incubator for 72 h. Then, 10 μl per well of CCK-8 (Cell Counting Kit-8, BIOMAX) reagent was added, and the optical density (OD) was measured at 450 nm after 3 h. Statistical analysis was performed using a t-test, and *p<0.05, **p<0.01, ***p<0.001 indicates significant differences.

[0128] Treatment group Absorbance (OD) Control group 1.474 MS1943 single treatment group (5 μM) 1.0925 MS1943 single treatment group (10 μM) 0.731 STM2457 single treatment group (5 μM) 0.79 STM2457 single treatment group (10 μM) 0.47425 MS1943 (5 μM) + STM2457 (5 μM) combined treatment group 0.4435

[0129]

[0130] As can be seen in Fig. 1 and Table 1 above, in the T-cell leukemia cell line (Jurkat), the PROTAC-based EZH2 inhibitor MS1943 alone treatment group showed an absorbance value of 0.731 at 10 μM, and the METTL3 inhibitor STM2457 alone treatment group showed an absorbance value of 0.47425 at 10 μM, whereas the combination treatment group of MS1943 (5 μM) and STM2457 (5 μM) showed an absorbance value of 0.4435, confirming a statistically significant decrease compared to the alone treatment group.

[0131] In particular, the absorbance of the combined treatment group of EZH2 inhibitor MS1943 (5 μM) and METTL3 inhibitor STM2457 (5 μM) was significantly lower at 0.4435 than the average value of the absorbance of the single treatment group of EZH2 inhibitor MS1943 (10 μM) and the single treatment group of METTL3 inhibitor STM2457 (10 μM) (0.731 + 0.47425) / 2 = 0.602625, confirming that the combined treatment group of EZH2 inhibitor and METTL3 inhibitor had a synergistic effect on the anticancer activity against leukemia compared to the single treatment group.

[0132]

[0133] Example 2: Experimental study of combined treatment with METTL3 inhibitor and EZH2 inhibitor on B-cell lymphoma cell lines.

[0134] Using Daudi, a B-cell lymphoma cell line, 1.0×10 4 After stimulation with PROTAC-based EZH2 inhibitors (MS1943) and METTL3 inhibitors (STM2457) in different conditions, the cells were cultured in a CO2 incubator for 72 h. Then, 10 μl per well of CCK-8 (Cell Counting Kit-8, BIOMAX) reagent was added, and the optical density (OD) was measured at 450 nm after 3 h. Statistical analysis was performed using a t-test, and *p<0.05, **p<0.01, ***p<0.001 indicates significant differences.

[0135] Treatment group Absorbance (OD) Control group 1.019 MS1943 single treatment group (5 μM) 0.682 MS1943 single treatment group (10 μM) 0.316 STM2457 single treatment group (5 μM) 0.515 25 STM2457 single treatment group (10 μM) 0.323 25 MS1943 (5 μM) + STM2457 (5 μM) combined treatment group 0.24 225

[0136]

[0137] As can be seen in Fig. 2 and Table 2 above, in the B-cell lymphoma cell line (Daudi), the PROTAC-based EZH2 inhibitor MS1943 alone treatment group showed an absorbance value of 0.316 at 10 μM, and the METTL3 inhibitor STM2457 alone treatment group showed an absorbance value of 0.32325 at 10 μM, whereas the combined treatment group of MS1943 (5 μM) and STM2457 (5 μM) showed an absorbance value of 0.24225, confirming a statistically significant decrease compared to the single treatment group.

[0138] In particular, the absorbance of the combined treatment group of EZH2 inhibitor MS1943 (5 μM) and METTL3 inhibitor STM2457 (5 μM) was significantly lower at 0.24225 than the average value of the absorbance of the single treatment group of EZH2 inhibitor MS1943 (10 μM) and the single treatment group of METTL3 inhibitor STM2457 (10 μM) (0.316 + 0.32325) / 2 = 0.319625, confirming that the combined treatment group of EZH2 inhibitor and METTL3 inhibitor had a synergistic effect on anticancer activity against lymphoma compared to the single treatment group.

[0139]

[0140] Example 3: Experimental study of combined treatment of METTL3 inhibitor and EZH2 inhibitor in lung cancer cell lines.

[0141] Using lung cancer cell line A549 (A-549), 5.0×10 3 Protease inhibitors MS1943 or MS177, which are PROTAC-based EZH2 inhibitors (dEZH2), and METTL3 inhibitor STM2457, which are iMETTL3 inhibitors, were treated alone or in combination in human cells, and cell viability was determined 48 hours later using a CCK-8 assay (Cell Counting Kit-8, BIOMAX). Statistical analysis was performed using a t-test, and *p<0.05, **p<0.01, ***p<0.001 indicates significant differences.

[0142] Treatment group Cell viability (%) Control group 100 DMSO only treatment group 75.35 dEZH2 (MS1943) only treatment group (5 μM) 87.89 dEZH2 (MS1943) only treatment group (10 μM) 76.51 dEZH2 (MS177) only treatment group (5 μM) 90.14 dEZH2 (MS177) only treatment group (10 μM) 81.24 iMETTL3 only treatment group (5 μM) 78.27 iMETTL3 only treatment group (10 μM) 72.62 dEZH2 (MS1943) (5 μM) + iMETTL3 (5 μM) combination treatment group 53.93 dEZH2 (MS177) (5 μM) + iMETTL3 (5 μM) combination treatment group 59.18

[0143]

[0144] As can be seen in FIG. 3 and Table 3 above, the survival rate inhibition effect of the A549 cell line was not significant in the EZH2 inhibitor (dEZH2) MS1943 or MS177 single treatment group and the METTL3 inhibitor (iMETTL3) STM2457 single treatment group, whereas the combined treatment group of the EZH2 inhibitor (dEZH2) MS1943 and the METTL3 inhibitor (iMETTL3) STM2457 and the combined treatment group of the EZH2 inhibitor (dEZH2) MS177 and the METTL3 inhibitor (iMETTL3) STM2457 statistically significantly inhibited the survival rate of the A549 cell line.

[0145] In particular, the cell viability of the combination treatment group of EZH2 inhibitor MS1943 (5 μM) and METTL3 inhibitor STM2457 (5 μM) was significantly lower at 53.93% than the average cell viability of the EZH2 inhibitor MS1943 (10 μM) monotherapy group and the METTL3 inhibitor STM2457 (10 μM) monotherapy group (76.51% + 72.62%) / 2 = 74.565%. Therefore, it was confirmed that the combination treatment group of EZH2 inhibitor MS1943 and METTL3 inhibitor STM2457 had a synergistic effect on anticancer activity against lung cancer compared to the monotherapy group.

[0146] In addition, the average cell viability of the EZH2 inhibitor MS177 (10 μM) single treatment group and the METTL3 inhibitor STM2457 (10 μM) single treatment group was (81.24% + 72.62%) / 2 = 76.93%, which is significantly lower than the cell viability of the combination treatment group of EZH2 inhibitor MS177 (5 μM) and METTL3 inhibitor STM2457 (5 μM), which confirmed that the combination treatment group of EZH2 inhibitor MS177 and METTL3 inhibitor STM2457 had a synergistic effect on anticancer activity against lung cancer compared to the single treatment group.

[0147]

[0148] Example 4: Experimental study of combined treatment of METTL3 inhibitor and EZH2 inhibitor in pancreatic cancer cell lines.

[0149] Using the pancreatic cancer cell line Capan-1, 5.0×10 3 Protease inhibitors MS1943 or MS177, which are PROTAC-based EZH2 inhibitors (dEZH2), and METTL3 inhibitor STM2457, which are iMETTL3 inhibitors, were treated alone or in combination in human cells, and cell viability was determined 48 hours later using a CCK-8 assay (Cell Counting Kit-8, BIOMAX). Statistical analysis was performed using a t-test, and *p<0.05, **p<0.01, ***p<0.001 indicates significant differences.

[0150] Treatment group Cell viability (%) Control group 100.02 DMSO only treatment group 73.77 dEZH2 (MS1943) only treatment group (5 μM) 87.74 dEZH2 (MS1943) only treatment group (10 μM) 68.55 dEZH2 (MS177) only treatment group (5 μM) 80.38 dEZH2 (MS177) only treatment group (10 μM) 76.2 iMETTL3 only treatment group (5 μM) 81.38 iMETTL3 only treatment group (10 μM) 74.26 dEZH2 (MS1943) (5 μM) + iMETTL3 (5 μM) combination treatment group 50.93 dEZH2 (MS177) (5 μM) + iMETTL3 (5 μM) combination treatment group 59.88

[0151]

[0152] As can be seen in FIG. 4 and Table 4 above, the survival rate inhibition effect of the Capan-1 cell line was not significant in the EZH2 inhibitor (dEZH2) MS1943 or MS177 single treatment group and the METTL3 inhibitor (iMETTL3) STM2457 single treatment group, whereas the combined treatment group of the EZH2 inhibitor (dEZH2) MS1943 and METTL3 inhibitor (iMETTL3) STM2457 and the combined treatment group of the EZH2 inhibitor (dEZH2) MS177 and METTL3 inhibitor (iMETTL3) STM2457 statistically significantly inhibited the survival rate of the Capan-1 cell line.

[0153] In particular, the cell viability of the combination treatment group of EZH2 inhibitor MS1943 (5 μM) and METTL3 inhibitor STM2457 (5 μM) was significantly lower at 50.93% than the average cell viability of the EZH2 inhibitor MS1943 (10 μM) monotherapy group and the METTL3 inhibitor STM2457 (10 μM) monotherapy group (68.55% + 74.26%) / 2 = 71.405%. Therefore, it was confirmed that the combination treatment group of EZH2 inhibitor MS1943 and METTL3 inhibitor STM2457 had a synergistic effect on anticancer activity against pancreatic cancer compared to the monotherapy group.

[0154] In addition, the average cell viability of the EZH2 inhibitor MS177 (10 μM) single treatment group and the METTL3 inhibitor STM2457 (10 μM) single treatment group was (76.2% + 74.26%) / 2 = 75.23%, which is significantly lower than the cell viability of the combination treatment group of EZH2 inhibitor MS177 (5 μM) and METTL3 inhibitor STM2457 (5 μM), which confirmed that the combination treatment group of EZH2 inhibitor MS177 and METTL3 inhibitor STM2457 had a synergistic effect on anticancer activity against pancreatic cancer compared to the single treatment group.

[0155]

[0156] Example 5: Experimental combination treatment of METTL3 inhibitor and EZH2 inhibitor on breast cancer cell lines.

[0157] Using the breast cancer cell line MDA-MB-231, 5.0×10 3Protease inhibitors MS1943 or MS177, which are PROTAC-based EZH2 inhibitors (dEZH2), and METTL3 inhibitor STM2457, which are iMETTL3 inhibitors, were treated alone or in combination in human cells, and cell viability was determined 48 hours later using a CCK-8 assay (Cell Counting Kit-8, BIOMAX). Statistical analysis was performed using a t-test, and *p<0.05, **p<0.01, ***p<0.001 indicates significant differences.

[0158] Treatment group Cell viability (%) Control group 100 DMSO only treatment group 70.57 dEZH2 (MS1943) only treatment group (5 μM) 80.28 dEZH2 (MS1943) only treatment group (10 μM) 77.42 dEZH2 (MS177) only treatment group (5 μM) 39.69 dEZH2 (MS177) only treatment group (10 μM) 36.17 iMETTL3 only treatment group (5 μM) 69.66 iMETTL3 only treatment group (10 μM) 55.38 dEZH2 (MS1943) (5 μM) + iMETTL3 (5 μM) combination treatment group 43.76 dEZH2 (MS177) (5 μM) + iMETTL3 (5 μM) combination treatment group 26.96

[0159]

[0160] As can be seen in FIG. 5 and Table 5 above, the survival rate inhibition effect of the MDA-MB-231 cell line was not significant in the EZH2 inhibitor (dEZH2) MS1943 or MS177 single treatment group and the METTL3 inhibitor (iMETTL3) STM2457 single treatment group, whereas the combined treatment group of the EZH2 inhibitor (dEZH2) MS1943 and METTL3 inhibitor (iMETTL3) STM2457 and the combined treatment group of the EZH2 inhibitor (dEZH2) MS177 and METTL3 inhibitor (iMETTL3) STM2457 statistically significantly inhibited the survival rate of the MDA-MB-231 cell line.

[0161] In particular, the average cell viability of the EZH2 inhibitor MS1943 (10 μM) single treatment group and the METTL3 inhibitor STM2457 (10 μM) single treatment group was (77.42% + 55.38%) / 2 = 66.4%, which is significantly lower than the cell viability of the combination treatment group of EZH2 inhibitor MS1943 (5 μM) and METTL3 inhibitor STM2457 (5 μM), which confirmed that the combination treatment group of EZH2 inhibitor MS1943 and METTL3 inhibitor STM2457 had a synergistic effect on anticancer activity against lung cancer compared to the single treatment group.

[0162] In addition, the average cell viability of the EZH2 inhibitor MS177 (10 μM) single treatment group and the METTL3 inhibitor STM2457 (10 μM) single treatment group was (36.17% + 55.38%) / 2 = 45.775%, which is significantly lower than the cell viability of the combination treatment group of EZH2 inhibitor MS177 (5 μM) and METTL3 inhibitor STM2457 (5 μM), which was 26.96%. Therefore, it was confirmed that the combination treatment group of EZH2 inhibitor MS177 and METTL3 inhibitor STM2457 had a synergistic effect on anticancer activity against lung cancer compared to the single treatment group.

[0163]

[0164] Example 6: Experimental study of combined treatment of METTL3 inhibitor and EZH2 inhibitor on colon cancer cell lines.

[0165] Using the colon cancer cell line HCT-116, 5.0×10 3Protease inhibitors MS1943 or MS177, which are PROTAC-based EZH2 inhibitors (dEZH2), and METTL3 inhibitor STM2457, which are iMETTL3 inhibitors, were treated alone or in combination in human cells, and cell viability was determined 48 hours later using a CCK-8 assay (Cell Counting Kit-8, BIOMAX). Statistical analysis was performed using a t-test, and *p<0.05, **p<0.01, ***p<0.001 indicates significant differences.

[0166] Treatment group Cell viability (%) Control group 99.99 DMSO only treatment group 97.94 dEZH2 (MS1943) only treatment group (5 μM) 79.08 dEZH2 (MS1943) only treatment group (10 μM) 83.88 dEZH2 (MS177) only treatment group (5 μM) 88.64 dEZH2 (MS177) only treatment group (10 μM) 74.71 iMETTL3 only treatment group (5 μM) 103.97 iMETTL3 only treatment group (10 μM) 92.36 dEZH2 (MS1943) (5 μM) + iMETTL3 (5 μM) combination treatment group 86.38 dEZH2 (MS177) (5 μM) + iMETTL3 (5 μM) combination treatment group 81.38

[0167]

[0168] As can be seen in FIG. 6 and Table 6 above, the survival rate inhibition effect of the HCT-116 cell line was not significant in the EZH2 inhibitor (dEZH2) MS1943 or MS177 single treatment group and the METTL3 inhibitor (iMETTL3) STM2457 single treatment group, whereas the combined treatment group of the EZH2 inhibitor (dEZH2) MS1943 and METTL3 inhibitor (iMETTL3) STM2457 and the combined treatment group of the EZH2 inhibitor (dEZH2) MS177 and METTL3 inhibitor (iMETTL3) STM2457 statistically significantly inhibited the survival rate of the HCT-116 cell line.

[0169] In particular, the cell viability of the combination treatment group of EZH2 inhibitor MS1943 (5 μM) and METTL3 inhibitor STM2457 (5 μM) was significantly lower at 86.38% than the average value of the cell viability of the EZH2 inhibitor MS1943 (10 μM) single treatment group and the METTL3 inhibitor STM2457 (10 μM) single treatment group (83.88% + 92.36%) / 2 = 88.12%. Therefore, it was confirmed that the combination treatment group of EZH2 inhibitor MS1943 and METTL3 inhibitor STM2457 had a synergistic effect on anticancer activity against colon cancer compared to the single treatment group.

[0170] In addition, the average cell viability of the EZH2 inhibitor MS177 (10 μM) single treatment group and the METTL3 inhibitor STM2457 (10 μM) single treatment group was (74.71% + 92.36%) / 2 = 83.535%, which is significantly lower than the cell viability of the combination treatment group of EZH2 inhibitor MS177 (5 μM) and METTL3 inhibitor STM2457 (5 μM), which confirmed that the combination treatment group of EZH2 inhibitor MS177 and METTL3 inhibitor STM2457 had a synergistic effect on anticancer activity against colon cancer compared to the single treatment group.

[0171]

[0172] Example 7: Experimental combination treatment of METTL3 inhibitor and EZH2 inhibitor on B-cell lymphoma cell lines.

[0173] Using Daudi, a B-cell lymphoma cell line, 5.0×10 3Protease inhibitors MS1943 or MS177, which are PROTAC-based EZH2 inhibitors (dEZH2), and METTL3 inhibitor STM2457, which are iMETTL3 inhibitors, were treated alone or in combination in human cells, and cell viability was determined 48 hours later using a CCK-8 assay (Cell Counting Kit-8, BIOMAX). Statistical analysis was performed using a t-test, and *p<0.05, **p<0.01, ***p<0.001 indicates significant differences.

[0174] Treatment group Cell viability (%) Control group 99.95 DMSO only treatment group 51.97 dEZH2 (MS1943) only treatment group (5 μM) 77.22 dEZH2 (MS1943) only treatment group (10 μM) 42.31 dEZH2 (MS177) only treatment group (5 μM) 43.63 dEZH2 (MS177) only treatment group (10 μM) 34.79 iMETTL3 only treatment group (5 μM) 66.03 iMETTL3 only treatment group (10 μM) 52.01 dEZH2 (MS1943) + iMETTL3 (5 μM) combination treatment group 32.83 dEZH2 (MS177) + iMETTL3 (5 μM) combination treatment group 34.22

[0175]

[0176] As can be seen in FIG. 7 and Table 7 above, the survival rate inhibition effect of the Daudi cell line was not significant in the EZH2 inhibitor (dEZH2) MS1943 or MS177 single treatment group and the METTL3 inhibitor (iMETTL3) STM2457 single treatment group, whereas the combined treatment group of the EZH2 inhibitor (dEZH2) MS1943 and METTL3 inhibitor (iMETTL3) STM2457 and the combined treatment group of the EZH2 inhibitor (dEZH2) MS177 and METTL3 inhibitor (iMETTL3) STM2457 statistically significantly inhibited the survival rate of the Daudi cell line.

[0177] In particular, the average cell viability of the EZH2 inhibitor MS1943 (10 μM) monotherapy group and the METTL3 inhibitor STM2457 (10 μM) monotherapy group was (42.31% + 52.01%) / 2 = 47.16%, which is significantly lower than the cell viability of the combination treatment group of EZH2 inhibitor MS1943 (5 μM) and METTL3 inhibitor STM2457 (5 μM), which was 32.83%. Therefore, it was confirmed that the combination treatment group of EZH2 inhibitor MS1943 and METTL3 inhibitor STM2457 had a synergistic effect on anticancer activity against B-cell lymphoma compared to the monotherapy group.

[0178] In addition, the average cell viability of the EZH2 inhibitor MS177 (10 μM) single treatment group and the METTL3 inhibitor STM2457 (10 μM) single treatment group was (34.79% + 52.01%) / 2 = 43.4%, which is significantly lower than the cell viability of the combination treatment group of EZH2 inhibitor MS177 (5 μM) and METTL3 inhibitor STM2457 (5 μM), confirming that the combination treatment group of EZH2 inhibitor MS177 and METTL3 inhibitor STM2457 had a synergistic effect on anticancer activity against B-cell lymphoma compared to the single treatment group.

[0179]

[0180] Example 8: Dose-dependent cell growth inhibition effects of METTL3 inhibitors and EZH2 inhibitors in Burkitt's lymphoma cell lines.

[0181] 8-1. Cell culture method

[0182] Burkitt lymphoma cell lines, Daudi and Ramos (Korea Cell Line Bank, Republic of Korea), were used. The cell lines were cultured in RPMI 1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco), 2 mM L-glutamine (Gibco), and 1% antibiotics (10 U / mL penicillin and 10 g / mL streptomycin; Gibco) at 37°C and 5% CO2.

[0183] 8-2. How to prepare the drug

[0184] EZH2 inhibitor (MS1943) and METTL3 inhibitor (STM2457) were purchased from MedChemExpress (NJ, USA). Both drugs were dissolved in dimethyl sulfoxide (DMSO) as recommended by the manufacturer and stored at -80°C. Before use in cell line treatment, both drugs were diluted in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, and 1% antibiotics.

[0185] 8-3. Cell proliferation analysis method

[0186] Cell growth was assessed using the CCK-8 assay (Dojindo, Rockville, MD, USA) according to the manufacturer's protocol. Daudi and Ramos cell lines were cultured in 96-well plates at a density of 2 × 10 4Cells were seeded at an initial cell density of 100 μL / well. Cells were treated with various doses of EZH2 inhibitors and METTL3 inhibitors (2.5 μM, 5 μM, and 10 μM). Synergistic effects were evaluated using 5 μM for both drugs. Cell cultures were maintained under 5% CO2 and 37°C. 10 μL of CCK-8 solution was added to each well after 24, 48, and 72 h. Plates were incubated in a CO2 incubator for 1–4 h, and the optical density (OD) was measured at 450 nm using a microplate reader.

[0187] 8-4. Statistical Analysis Methods

[0188] All data were statistically evaluated using the Student's t-test using GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA). All experiments were performed at least three times. Values ​​are expressed using the mean and SEM. Differences between group means were considered statistically significant if *P < 0.05, **P < 0.01, and ***P < 0.001.

[0189] 8-5. Experimental Results

[0190] We investigated the effects of MS1943, an EZH2 inhibitor (dEZH2), and STM2457, a METTL3 inhibitor (iMETTL3), on the proliferation of Burkitt's lymphoma cell lines Daudi and Ramos cells using the CCK-8 assay.

[0191] As can be seen in Figures 8a to 8c, when cell viability was examined at different time points, i.e., 24 hours (Figure 8a), 48 hours (Figure 8b), and 72 hours (Figure 8c) after drug treatment, with various concentrations of EZH2 inhibitor and METTL3 inhibitor (ranging from 2.5 to 10 μM) in both cell lines, a dose-dependent decrease in cell viability was observed at all time points in both cell lines.

[0192] In addition, as can be seen in FIGS. 8d and 8e, the cell viability (%) was checked when the same concentration of dEZH2 was treated alone (5 μM), iMETTL3 was treated alone (5 μM), or dEZH2 (5 μM) and iMETTL3 (5 μM) were combined (Comb) at different time points in the Burkitt lymphoma cell lines Daudi (FIG. 8d) and Ramos (FIG. 8e), and both cell lines showed a statistically significant decrease in cell viability in the combined treatment compared to the single treatment.

[0193] From the above results, the inventors of the present invention were able to clearly confirm that not only dEZH2 treatment alone and iMETTL3 treatment alone inhibit the proliferation of Burkitt's lymphoma cells, but also that combined treatment with dEZH2 and iMETTL3 significantly inhibits the proliferation of Burkitt's lymphoma cells compared to treatment alone.

[0194]

[0195] Example 9: Significant cell proliferation inhibition effect in Burkitt's lymphoma cells by combined treatment with a METTL3 inhibitor and an EZH2 inhibitor.

[0196] To evaluate the inhibitory effects of METTL3 inhibitor (iMETTL3) and EZH2 inhibitor (dEZH2) on the proliferation of Burkitt's lymphoma cells in vitro, we performed cell proliferation assays on Daudi and Ramos cells cultured with or without these drugs. Cell viability of Daudi and Ramos cell lines was analyzed using CCK-8 (Cell Counting Kit-8). 1 × 10 4 / Wells were plated and treated with 5 μM dEZH2 alone, 5 μM iMETTL3 alone, or a combination of 5 μM dEZH2 and 5 μM iMETTL3 for 24, 48, or 72 hours, and dimethyl sulfoxide (DMSO) was used as a control.

[0197] As shown in Figures 9a to 9c, combined treatment with dEZH2 and iMETTL3 significantly reduced Burkitt's lymphoma cell growth compared to single treatment at 24 hours (Figure 9a), 48 hours (Figure 9b), and 72 hours (Figure 9c). We also performed a toxicity assay in HEK293T cells and confirmed that there was no significant difference between dEZH2 and iMETTL3.

[0198] From the above results, the inventors were able to confirm that the combination therapy effectively suppressed the survival and proliferation of Burkitt lymphoma cells, thereby exhibiting a synergistic effect.

[0199]

[0200] Example 10: Significantly enhanced apoptotic effect in Burkitt's lymphoma cells by combined treatment with a METTL3 inhibitor and an EZH2 inhibitor.

[0201] To determine whether METTL3 inhibitors and EZH2 inhibitors induce apoptosis, we performed an annexin V / PI (propidium iodide) staining assay. Controls were treated with dimethyl sulfoxide (DMSO), and cells were treated with 5 μM of the METTL3 inhibitor alone, 5 μM of the EZH2 inhibitor alone, or a combination of 5 μM of the METTL3 inhibitor and 5 μM of the EZH2 inhibitor for 72 hours. Surviving cells were annexin V-negative and PI-negative, early mitotic cells were annexin V-positive and PI-negative, and late mitotic cells were annexin V-positive and PI-positive, as measured by flow cytometry.

[0202] As shown in Figures 10a and 10e, both cell lines, Daudi and Ramos, exhibited a marked combined effect of the two drugs in the Q2 (late apoptosis) and Q3 (early apoptosis) quadrants. As shown in Figures 10a to 10h, this combined treatment promoted both early and late apoptosis in Daudi (Figures 10a to 10d) and Ramos cells (Figures 10e to 10h).

[0203]

[0204] Example 11: Induction of G2 / M phase arrest in Burkitt lymphoma cells by combined treatment with a METTL3 inhibitor and an EZH2 inhibitor.

[0205] To investigate the relationship between cell proliferation inhibition and cell cycle regulation, we performed flow cytometry to assess cell distribution across various cell cycle phases.

[0206] 11-1. Cell cycle analysis method

[0207] 5×10 of Daudi or Ramos, Burkitt's lymphoma cell lines 5Cells were treated with DMSO, METTL3 inhibitor, EZH2 inhibitor, or METTL3 inhibitor and EZH2 inhibitor. After culturing for 72 h in a CO2 incubator, cells were harvested and centrifuged at 2,000 rpm for 5 min with DPBS (Gibco). Cells were washed twice and the supernatant was discarded. For cell cycle analysis, the Annexin V-PI Apoptosis Kit (BioVision, London, UK) was used according to the manufacturer's protocol. Cell cycle was detected using Novocyte Advanteon (Agilent, CA, USA).

[0208] 11-2. Cell cycle analysis results

[0209] As shown in Figures 11a and 11c, the major peaks in each cell cycle phase of Ramos and Daudi cell lines exposed to 5 μM dEZH2, 5 μM iMETTL3, or 5 μM dEZH2 and 5 μM iMETTL3 for 72 h are indicated from left to right: G0 / G1 phase, S phase, and G2 / M phase. Cell cycle phases were analyzed as percentage ratios, excluding some cells outside the measurement range.

[0210] As shown in Figures 11a to 11d, combined treatment with a METTL3 inhibitor and an EZH2 inhibitor resulted in a significant increase in Burkitt lymphoma cells in the S phase and G2 / M phase after 72 hours. Simultaneously, the number of cells in the G0 / G1 phase decreased in both Daudi cells (Figures 11a and 11b) and Ramos cells (Figures 11c and 11d).

[0211]

[0212] Example 12: Induction of cell death through the p53-dependent pathway by combined treatment with a METTL3 inhibitor and an EZH2 inhibitor.

[0213] After confirming the induction of apoptosis by dual inhibition of EZH2 and METTL3, we investigated the involvement of specific apoptosis-related pathways using western blot analysis.

[0214] 12-1. Western blot method

[0215] One million Ramos or Daudi cells treated with 5 μM dEZH2, 5 μM iMETTL3, or 5 μM dEZH2 and 5 μM iMETTL3 were lysed in 2× Laemmli sample buffer (Bio-Rad) containing β-mercaptoethanol and boiled at 95°C for 10 min. After removal of the insoluble fraction by centrifugation at 10,000× g for 10 min, protein samples were separated by SDS gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were stained overnight at 4°C with cleaved PARP, PARP, cleaved caspase-3, TP53, and PUMA antibodies (Cell Signaling Technology, Danvers, MA, USA) diluted 1:1000 or GAPDH antibody (Cell Signaling Technology) diluted 1:5000. After overnight incubation at 4°C, HRP-conjugated secondary antibodies were added. After washing with Tris-buffered saline and Tween 20, hybridized bands were detected using an enhanced chemiluminescence (ECL) detection kit (Amersham Pharmacia Biotech, Buckinghamshire, UK).

[0216] The protein expression levels of cleaved caspase-3 (c-caspase-3), poly (ADP-ribose) polymerase (PARP), and cleaved PARP (c-PARP) in Daudi cell lines were analyzed in comparison with GAPDH (glyceraldehyde 3-phosphate dehydrogenase). The protein expression levels of TP53 (tumor protein P53) and TP53-related PUMA (p53 upregulated modulator of apoptosis) were also analyzed in comparison with GAPDH. Statistical analysis was performed using a two-tailed, unpaired t-test, and error bars represent ±SD.

[0217] 12-2. Western blot results

[0218] PARP, a key protein in DNA repair, is known to be cleaved by caspase-3 upon activation, leading to DNA damage and cell death. As shown in Figures 12a to 12d, combined treatment with dEZH2 and iMETTL3 significantly increased the levels of cleaved PARP and cleaved caspase-3 compared to treatment with dEZH2 or iMETTL3 alone.

[0219] We further investigated the role of TP53, an apoptotic gene that promotes apoptosis in cancer cells by inducing cell cycle arrest and activating PUMA. As shown in Figures 12e to 12g, combined therapy with dEZH2 and iMETTL3 increased the expression of TP53 and PUMA in Daudi cells.

[0220] Through the above results, the inventors demonstrated that combination therapy increases p53-dependent apoptosis through upregulation of cleaved PARP, cleaved caspase-3, TP53, and PUMA.

[0221]

[0222] Sintering

[0223] From the above results, the inventors of the present invention confirmed that when PROTAC-based EZH2 inhibitors (MS1943, MS177) and METTL3 inhibitors (STM2457) were combined, compared to when treated alone, there was a synergistic effect on anticancer activity that significantly inhibited the growth and cell viability of leukemia (Jurkat), lymphoma (Daudi), lung cancer (A549), pancreatic cancer (Capan-1), breast cancer (MDA-MB-231), and colon cancer (HCT-116) cell lines.

[0224] PROTAC-based drugs offer several distinct advantages over existing small-molecule inhibitors. First, PROTAC-based drugs completely eliminate disease-causing proteins by inducing target protein degradation, ensuring sustained therapeutic efficacy as the target protein must be resynthesized to restore function. Second, this approach can overcome drug resistance, as PROTAC-based drugs can degrade mutant proteins that are not effectively inhibited by existing small molecules. Third, PROTAC-based drugs can target a wider range of protein conformations and mutations, increasing therapeutic versatility. Furthermore, PROTACs often offer lower doses than existing inhibitors, reducing potential side effects. These advantages collectively highlight the transformative potential of PROTACs in developing more effective and sustainable cancer therapies.

[0225] Previous literature has demonstrated the superior efficacy of the PROTAC-based EZH2 inhibitor MS1943 compared to the clinically active EZH2 inhibitor tazemetostat in B-cell lymphoma cells. In the present invention, we extend these findings by exploring the synergistic effects of combining an EZH2 inhibitor with a METTL3 inhibitor in Burkitt's lymphoma cells. The combination therapy significantly reduced Ramos and Daudi cell proliferation and significantly increased levels of apoptosis-related proteins, highlighting the therapeutic potential of the PROTAC-based strategy.

[0226] The present invention provides compelling evidence that the combined use of a METTL3 inhibitor and an EZH2 inhibitor can effectively inhibit the growth of Burkitt's lymphoma cells and induce apoptosis via a p53-dependent pathway. Our results are significant given the challenges associated with targeting EZH2 in clinical settings. Furthermore, we demonstrated that combined treatment with a METTL3 inhibitor and an EZH2 inhibitor significantly increased G2 / M phase. Furthermore, we observed an increase in S phase of the cell cycle following combined treatment. These results suggest that METTL3 inhibition increases PLK1 expression, leading to accumulation in S phase and potentially G2 / M phase arrest.

[0227] The clinical implications of these findings are significant for the treatment of Burkitt lymphoma. The enhanced efficacy of the combination of a METTL3 inhibitor and an EZH2 inhibitor offers a promising, novel approach to treating Burkitt lymphoma. The combination therapy according to the present invention, which targets and degrades disease-related proteins more effectively than existing inhibitors, highlights the potential of targeted protein degradation (TPD) technology in oncology.

[0228] Furthermore, successful use of the combination therapy according to the present invention requires conducting preclinical models for potential clinical trials. Broad application of PROTAC-based strategies is needed in various cancer types, such as breast, lung, and prostate cancers, where EZH2 is overexpressed and has been particularly difficult to treat with existing small molecule inhibitors.

[0229] In conclusion, the present invention demonstrates that the combination of an EZH2 inhibitor and a METTL3 inhibitor significantly inhibits Burkitt lymphoma cell growth and induces a p53-dependent apoptosis pathway. These results provide a strong basis for the continued development and clinical evaluation of PROTAC-based therapies, offering new hope for improving treatment outcomes in patients with Burkitt lymphoma and potentially other hematological malignancies.

[0230]

[0231] The present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising an EZH2 inhibitor and a METTL3 inhibitor. It has been confirmed that when an EZH2 inhibitor and a METTL3 inhibitor are administered in combination based on proteolysis-targeting chimera (PROTAC) technology, a synergistic effect is achieved in which a remarkable anticancer activity is exhibited compared to when they are administered alone, and thus, it is possible to create a preventive or therapeutic effect on various cancers such as leukemia, lymphoma, lung cancer, pancreatic cancer, breast cancer, and colon cancer.

Claims

1. A pharmaceutical composition for preventing or treating cancer, comprising an EZH2 inhibitor and a METTL3 inhibitor as active ingredients.

2. A pharmaceutical composition according to claim 1, wherein the composition is for combined administration of an EZH2 inhibitor and a METTL3 inhibitor.

3. A pharmaceutical composition according to claim 1, characterized in that the EZH2 inhibitor is PROTAC (Proteolysis targeting chimera)-based.

4. A pharmaceutical composition in claim 1, wherein the EZH2 inhibitor is MS1943 or MS177.

5. A pharmaceutical composition according to claim 1, wherein the METTL3 inhibitor is STM2457.

6. A pharmaceutical composition according to claim 1, wherein the molar ratio of the EZH2 inhibitor and the METTL3 inhibitor is 1:10 to 10:

1.

7. A pharmaceutical composition according to claim 1, wherein the pharmaceutical composition comprises an EZH2 inhibitor at a concentration of 1 to 20 μM and a METTL3 inhibitor at a concentration of 1 to 20 μM.

8. A pharmaceutical composition according to claim 1, wherein the cancer is at least one selected from the group consisting of leukemia, lymphoma, lung cancer, pancreatic cancer, breast cancer, and colon cancer.

9. (i) A combination kit for preventing or treating cancer comprising an EZH2 inhibitor and (ii) a METTL3 inhibitor.

10. A combination kit in claim 9, wherein the EZH2 inhibitor is MS1943 or MS177.

11. A combination kit according to claim 9, wherein the METTL3 inhibitor is STM2457.

12. A combination kit according to claim 9, wherein (i) the EZH2 inhibitor and (ii) the METTL3 inhibitor are administered simultaneously or sequentially.

13. A combination kit according to claim 9, wherein the cancer is at least one selected from the group consisting of leukemia, lymphoma, lung cancer, pancreatic cancer, breast cancer, and colon cancer.

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