Pharmaceutical composition for preventing or treating cancer comprising a FOXM1 inhibitor and an immune checkpoint inhibitor

Abstract

The present invention relates to a pharmaceutical composition for preventing, ameliorating, or treating cancer, comprising a FOXM1 inhibitor and an immune checkpoint inhibitor as active ingredients. The FOXM1 inhibitor of the present invention suppresses PD-L1 expression on the cell membrane, inhibits FOXM1 translocation to the nucleus, reduces cancer cell proliferation and survival, and induces increased cell death, thereby preventing, ameliorating, or treating cancer. Furthermore, when administered in combination with an immune checkpoint inhibitor, the FOXM1 inhibitor effectively suppresses tumor growth compared to the FOXM1 inhibitor or immune checkpoint inhibitor administered alone, making it useful as a cancer preventive or therapeutic agent.

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical composition for the prevention or treatment of cancer, which comprises a FOXM1 inhibitor and an immune checkpoint inhibitor as active ingredients. [Background technology]

[0002] Lung cancer is the second most common cancer in the world after breast cancer, and despite significant advances in cancer treatment, it still has the highest mortality rate among all cancers. Non-small cell lung cancer (NSCLC) accounts for 80-85% of all lung cancer patients, and while immune cells infiltrate many NSCLC tissues, cytotoxic T lymphocytes in tumor tissue are known to be functionally suppressed by various mechanisms, including the aberrant overexpression of immune checkpoint proteins utilized by tumor cells.

[0003] Immune checkpoint inhibitors (ICIs), a form of cancer immunotherapy, are drugs that block immune checkpoint proteins expressed by immune cells such as T cells and cancer cells. Immunotherapy using immune checkpoint inhibitors has recently significantly improved the therapeutic efficacy of various cancer types, including lung cancer. PD-L1 (Programmed Cell Death-Ligand-1), encoded by the CD274 gene, is a key immune checkpoint molecule that mediates interactions between cancer cells and tumor-infiltrating T cells. PD-1 (Programmed Cell Death-Ligand-1) is expressed on the surface of T cells, while PD-L1 is expressed on normal tissues, suppressing the immune system's self-attack. However, overexpression of PD-L1 on the surface of cancer cells allows them to evade immune cell attack. Therefore, ICIs, such as anti-PD-1 or anti-PD-L1 antibodies, that inhibit the interaction between PD-1 and PD-L1 may enhance the therapeutic efficacy of cancers such as lung cancer by reactivating immune cell attack on cancer cells. However, expensive ICI treatment not only places a significant economic burden on cancer patients, but also has been reported to cause various immune-related side effects in non-target organs, including cardiac complications. Furthermore, it has been reported that ICIs are less effective in treating tumors with large tumors or rapid tumor growth. Therefore, the development of new strategies and therapeutic agents to address these issues with ICIs is necessary.

[0004] FOXM1 (Forkhead box protein M1) is a key protein in regulating various processes related to lung cancer tumor development, including cell cycle progression, anticancer therapy resistance, and metastasis. Its function is partially attributed to its ability to translocate to the nucleus via various forkhead or DNA-binding domains and bind to regulatory regions of multiple target genes, which are important for cancer cell survival. FOXM1 inhibitors, such as thiostrepton (TST), siomycin A, Robert Costa Memorial drug-1 (RCM-1), and forkhead domain inhibitor-6 (FDI-6), have been identified. Among these, TST, a natural thiazole antibiotic isolated from Streptomyces azureus, inhibits the interaction of the forkhead domain of FOXM1 with target DNA. Furthermore, TST has been used to treat mastitis caused by Gram-negative bacteria and has been reported to be active against breast cancer.

[0005] Against this background, the inventors conducted research using a FOXM1 inhibitor and found that the inhibitor not only significantly reduced PD-L1 expression but also suppressed tumor cell proliferation, demonstrating its effectiveness in preventing or treating cancer, leading to the completion of the present invention. Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer, which comprises a FOXM1 inhibitor and an immune checkpoint inhibitor as active ingredients.

[0007] Another object of the present invention is to provide an anti-cancer adjuvant comprising a FOXM1 inhibitor and an immune checkpoint inhibitor as active ingredients.

[0008] Another object of the present invention is to provide a use of a composition comprising a FOXM1 inhibitor and an immune checkpoint inhibitor for the prevention or treatment of cancer.

[0009] Another object of the present invention is to provide a use of a composition comprising a FOXM1 inhibitor and an immune checkpoint inhibitor for the manufacture of a medicament for the prevention or treatment of cancer.

[0010] Another object of the present invention is to provide a method for preventing or treating cancer, comprising administering to an individual a composition comprising a FOXM1 inhibitor and an immune checkpoint inhibitor. [Means for solving the problem]

[0011] This will be explained in more detail as follows. Meanwhile, each description and embodiment disclosed in this application can also be applied to each of the other descriptions and embodiments. In other words, all combinations of various elements disclosed in this application belong to the category of this application. Furthermore, the specific descriptions described below are not considered to limit the category of this application.

[0012] In one aspect to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising a FOXM1 inhibitor and an immune checkpoint inhibitor as active ingredients.

[0013] In the present invention, "FOXM1 (Forkhead box protein M1)" is a protein belonging to the FOX family of transcription factors, is encoded by the FOXM1 gene, and is known to play an important role in the progression of the cell cycle.

[0014] In the present invention, a "FOXM1 inhibitor" is a substance that suppresses FOXM1. Any substance that suppresses or reduces FOXM1 expression can be used without limitation. Specifically, the inhibitor may be a thiazolidinedione, a diarylheptanoid, RCM-1, thiostrepton, honokiol, FDI-6, siomycin A, monensin, a FOXM1 aptamer, the peptide 9R-P201, or a pharmaceutically acceptable salt thereof.

[0015] The thiazolidinedione compound is a type of heterocyclic compound consisting of a five-membered C3NS ring that has been reported as a pharmaceutical agent that can be used to lower blood glucose levels and treat type 2 diabetes, and may be, for example, TFI-1 to TFI-10, preferably TFI-10, but is not limited to these.

[0016] The TFI-10 compound is a thiazolidinedione forkhead domain inhibitor, and is (5Z)-5-[(3-methoxy-4-{[4-(trifluoromethyl)phenoxy]methyl}phenyl)methylidene]-1,3-thiazolidine-2,4-dione, which means a substance having a skeleton having the structure of Chemical Formula 1.

[0017] [ka]

[0018] Diarylheptanoids are a general term for secondary metabolites of plants that consist of two aromatic rings (aryl groups) connected by a seven-carbon chain (heptane) and have various substituents, and are known to have antioxidant activity.

[0019] RCM-1 is a FOXM1 inhibitor known to block nuclear localization and increase proteasomal degradation of FOXM1, and refers to a substance having a structure of Chemical Formula 2.

[0020] [ka]

[0021] Thiostrepton is a natural cyclic oligopeptide antibiotic of the thiopeptide family derived from various Streptomyces strains such as Streptomyces azureus and Streptomyces laurentii, and refers to a substance having a structure of Chemical Formula 3.

[0022] [ka]

[0023] Honokiol is a lignan isolated from the bark, seeds, and leaves of trees belonging to the Magnolia genus. It is known to be highly bioavailable as it can easily pass through the blood-brain barrier and the blood-cerebrospinal fluid barrier. It refers to a substance having the structure of chemical formula 4.

[0024] [ka]

[0025] FDI-6 is a FOXM1 inhibitor that directly binds to FOXM1 protein to induce transcriptional downregulation, and refers to a substance having a structure of Chemical Formula 5.

[0026] [ka]

[0027] Siomycin A is a thiazole antibiotic that has been reported to downregulate not only the transcriptional activity of FOXM1 but also mRNA and protein expression, and refers to a substance having a structure of Chemical Formula 6.

[0028] [ka]

[0029] Monensin, also known as monene acid, monensin A, monensina, monensinum, or rumensin, is a polyether antibiotic isolated from Streptomyces cinnamonensis and has the structure of Chemical Formula 7.

[0030] [ka]

[0031] The FOXM1 aptamer (FOXM1 apt) is a general term for FOXM1-specific aptamers that target the FOXM1 DNA-binding domain, and may consist of, for example, the sequence of SEQ ID NO: 9, but is not limited to this.

[0032] The peptide 9R-P201 is a peptide that has been reported to downregulate FOXM1 expression, suppress the survival, proliferation, and migration of cancer cells, and induce cell death, and refers to a peptide substance in which nine arginines are conjugated to P201 (peptide) (Jian Cui et al. Int J Pept Res Ther20, 447-456(2014)).

[0033] The FOXM1 inhibitor of the present invention may, for example, reduce the gene or protein level of FOXM1 or reduce the translocation of FOXM1 to the nucleus.

[0034] In the present invention, FOXM1 inhibitors can be obtained from biological sources known in the art, or can be chemically synthesized or commercially available.

[0035] In the present invention, the FOXM1 inhibitor includes pharmaceutically or food-acceptable salts having similar efficacy, such as basic salts or acid salts, and the basic salts can be used in the form of either organic or inorganic base salts, and can be selected from the group consisting of sodium salts, potassium salts, calcium salts, lithium salts, magnesium salts, cesium salts, aminium salts, ammonium salts, triethylaminium salts, and pyridinium salts.

[0036] Examples of such salts include acid salts, and acid addition salts formed with free acids are useful. The free acids can be inorganic or organic acids, such as hydrochloric acid, bromic acid, sulfuric acid, sulfurous acid, phosphoric acid, diphosphoric acid, and nitric acid. The organic acids can be citric acid, acetic acid, maleic acid, malic acid, fumaric acid, gluconic acid, methanesulfonic acid, benzenesulfonic acid, camphorsulfonic acid, oxalic acid, malonic acid, glutaric acid, acetic acid, glyconic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, citric acid, aspartic acid, and stearic acid, but are not limited to these. The free acids can include all salts formed with various inorganic and organic acids commonly used in the art.

[0037] In the present invention, the term "immune checkpoint inhibitor" refers to a form of cancer immunotherapy that blocks proteins known as immune checkpoints expressed in some immune cells, such as T cells, and cancer cells. Specific examples of such drugs include, but are not limited to, PD-L1 inhibitors, anti-PD-L1 antibodies, PD-1 inhibitors, anti-PD-1 antibodies, CTLA inhibitors, anti-CTLA4 antibodies, anti-PD-L2 antibodies, LTF2 regulatory antibodies, anti-LAG3 antibodies, anti-A2aR antibodies, anti-TIGIT antibodies, anti-TIM-3 antibodies, anti-B7-H3 antibodies, anti-B7-H4 antibodies, anti-VISTA antibodies, anti-CD47 antibodies, anti-BTLA antibodies, anti-KIR antibodies, anti-IDO antibodies, and anti-4-1BB antibodies.

[0038] In the present invention, the term "active ingredient" means an ingredient that exhibits a desired activity alone or that can exhibit an activity together with a carrier that is inactive by itself.

[0039] In the present invention, "cancer (or tumor)" does not distinguish between primary cancer and metastatic cancer, and may be solid cancer or blood cancer. Specifically, the cancer may be, but is not limited to, lung cancer, pancreatic cancer, pharyngeal cancer, laryngeal cancer, esophageal cancer, gastric cancer, duodenal cancer, small intestine cancer, colon cancer, rectal cancer, breast cancer, ovarian cancer, prostate cancer, liver cancer, thyroid cancer, kidney cancer, uterine cancer, brain tumor, skin cancer, melanoma, malignant bone tumor, bladder cancer, or blood cancer. More specifically, the cancer may be lung cancer or colon cancer.

[0040] The term "prevention" as used herein means any action of suppressing or delaying the onset of cancer by administering the composition of the present invention, "improvement" means showing the effect of applying the composition of the present invention to delay the progression of cancer or alleviate the symptoms of cancer, and "treatment" means any action of administering the composition of the present invention to result in a favorable change, such as completely suppressing the progression of cancer or ameliorating the symptoms.

[0041] As an example, the composition of the present invention reduces the proliferation and survival of cancer cells and induces increased cell death by reducing or suppressing the expression of PD-L1, thereby having the effect of preventing, ameliorating, or treating cancer.

[0042] The decrease in PD-L1 expression may mean that the expression level of PD-L1 protein on the cell membrane is reduced.

[0043] In one embodiment of the present invention, when the FOXM1 inhibitor of the present invention is a hydrophobic compound with low water solubility, the hydrophobic compound may be provided in a form carried by a liposome as a system for effectively delivering the hydrophobic compound to tumors, specifically, but not limited to, on the surface, inside, or phospholipid bilayer of the liposome.

[0044] The liposome refers to a bilayer membrane structure composed of phospholipids, and preferably refers to a micelle structure.

[0045] The phospholipids constituting the liposomes were 1,2-dioleoyl-sn-glycero-2-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000), cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methyl(polyethylene glycol) The oleoyl phosphate may be at least one selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methyl(polyethylene glycol)-5000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methyl(polyethylene glycol)-5000]ammonium salt, dioleoylphosphatidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, dipalmitoylphosphatidylcholine, and cholesteryl hemisuccinate, but is not necessarily limited thereto.

[0046] There is no particular limitation on the content ratio of the phospholipids constituting the liposomes, and this ratio can be freely set taking into consideration the size of the liposomes to be produced.

[0047] In one embodiment to achieve the above object, the liposome of the present invention is composed of DOPC, cholesterol, and DSPE-PEG2000, and specifically, the molar ratio of each component may be, but is not limited to, 5 to 15, 3 to 13, and 1 to 12.

[0048] The pharmaceutical composition of the present invention may be for combined administration of a FOXM1 inhibitor and an immune checkpoint inhibitor. Specifically, the pharmaceutical composition may be in the form of a mixture of the FOXM1 inhibitor and the immune checkpoint inhibitor, or the FOXM1 inhibitor and the immune checkpoint inhibitor may be formulated separately and administered simultaneously or sequentially, but is not limited thereto.

[0049] When a FOXM1 inhibitor and an immune checkpoint inhibitor are co-administered, the administration concentration ratio of the FOXM1 inhibitor to the immune checkpoint inhibitor may be 1:0.01 to 1:1000, specifically 1:0.01 to 1:500, 1:0.05 to 1:300, 1:0.1 to 1:200, or 1:1 to 1:100, and more specifically 1:0.01, 1:0.02, 1:0.03, 1:0.05, 1:0.08, 1:0.1, 1:0.3, 1:0.5, 1:0.7, 1:0.8, 1:1, 1:1.5, 1:2, 1:3, 1:5, 1:7, 1:8, 1:10, 1:25, 1:50, 1:100, 1:200, or 1:500, but is not limited thereto.

[0050] When a liposomal FOXM1 inhibitor and an immune checkpoint inhibitor are co-administered, the administration concentration ratio of the liposomal FOXM1 inhibitor and the immune checkpoint inhibitor may be 1:0.01 to 1:1000, specifically 1:0.01 to 1:500, 1:0.05 to 1:300, 1:0.1 to 1:200, or 1:1 to 1:1 The ratio may be 1:0.00, and more specifically may be, but is not limited to, 1:0.01, 1:0.02, 1:0.03, 1:0.05, 1:0.08, 1:0.1, 1:0.3, 1:0.5, 1:0.7, 1:0.8, 1:1, 1:1.5, 1:2, 1:3, 1:5, 1:7, 1:8, 1:10, 1:25, 1:50, 1:100, 1:200, or 1:500.

[0051] The pharmaceutical compositions of the present invention may further contain pharmaceutically acceptable carriers, excipients, or diluents according to conventional methods. Pharmaceutically acceptable carriers are well known in the art depending on the route of administration or formulation, and specific examples can be found in the pharmacopoeias of various countries, including the Korean Pharmacopoeia. Carriers, excipients, and diluents that can be included in the compositions of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. Furthermore, carriers, excipients, and diluents that can be included in the compositions of the present invention may be, but are not limited to, non-natural carriers.

[0052] The pharmaceutical compositions of the present invention can be formulated and used in the form of oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, topical preparations, suppositories, or sterile injectable solutions according to conventional methods. Specifically, they can be prepared using diluents or excipients commonly used in formulations, such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like. These solid preparations can be prepared by mixing the compound with at least one or more excipients, such as starch, calcium carbonate, sucrose, lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Furthermore, in addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid formulations for oral administration include suspensions, oral solutions, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients, such as wetting agents, sweeteners, flavorings, and preservatives, are also used. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, lauric butter, and glycerogelatin. Specific formulations of pharmaceutical compositions are known in the art and can be found in, for example, Remington's Pharmaceutical Sciences (19th ed. 1995), which is incorporated herein by reference.

[0053] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment and not to cause side effects. The effective dose level can be determined based on factors including the patient's health condition, the type and severity of the disease, the activity of the drug, sensitivity to the drug, the method of administration, the administration time, the administration route and excretion rate, the duration of treatment, coadministered or co-administered drugs, and other factors well known in the medical field. The amount and frequency of administration do not in any way limit the scope of the present invention.

[0054] The pharmaceutical compositions of the present invention can be administered to mammals such as rats, dogs, cats, cows, horses, pigs, and humans via a variety of routes.

[0055] The pharmaceutical compositions of the present invention can be applied to any formulation containing a FOXM1 inhibitor and an immune checkpoint inhibitor as active ingredients, and can be prepared as oral or parenteral formulations. Specifically, the mode of administration is not limited, and includes, but is not limited to, formulations suitable for oral, rectal, nasal, topical (including buccal and sublingual), subcutaneous, vaginal, or parenteral (including intramuscular, subcutaneous, and intravenous) administration, or formulations suitable for inhalation or injection.

[0056] In one aspect to achieve the above object, the present invention provides an anticancer adjuvant comprising a FOXM1 inhibitor and an immune checkpoint inhibitor as active ingredients.

[0057] The "FOXM1 inhibitor," "immune checkpoint inhibitor," "active ingredient," and "cancer" are as described above.

[0058] The anticancer adjuvant of the present invention refers to any form for enhancing the anticancer effect of an anticancer drug or suppressing or ameliorating the side effects of an anticancer drug. The anticancer adjuvant of the present invention can be administered in combination with various types of anticancer drugs or anticancer adjuvant drugs, and can exhibit the same level of anticancer therapeutic effect even when the anticancer drug is administered at a lower dose than the usual anticancer drug during combined administration, thereby enabling safer anticancer treatment.

[0059] The anticancer adjuvant agent can be administered via any common route as long as it can reach the target tissue. Depending on the intended purpose, the anticancer adjuvant agent of the present invention may be in a form suitable for oral, rectal, nasal, topical (including buccal and sublingual), subcutaneous, vaginal, or parenteral (including intramuscular, subcutaneous, and intravenous) administration, or by inhalation or injection, but is not limited thereto. Furthermore, the anticancer adjuvant agent can be administered by any device that can deliver the active substance to the target cells.

[0060] The anti-cancer adjuvant of the present invention can be preferably formulated as an anti-cancer adjuvant by further comprising one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration.The carrier, excipient or diluent that can be contained in the anti-cancer adjuvant of the present invention includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0061] In one aspect to achieve the above object, the present invention provides use of a composition comprising a FOXM1 inhibitor and an immune checkpoint inhibitor for the prevention or treatment of cancer.

[0062] The "cancer", "prevention", "treatment", "FOXM1 inhibitor", "immune checkpoint inhibitor", and "composition" are as described above.

[0063] In one aspect to achieve the above object, the present invention provides use of a composition comprising a FOXM1 inhibitor and an immune checkpoint inhibitor for the manufacture of a medicament for the prevention or treatment of cancer.

[0064] The "cancer", "prevention", "treatment", "FOXM1 inhibitor", "immune checkpoint inhibitor", and "composition" are as described above.

[0065] In one embodiment to achieve the above object, the present invention provides a method for preventing or treating cancer, comprising administering to an individual a composition comprising a FOXM1 inhibitor and an immune checkpoint inhibitor.

[0066] The "cancer", "prevention", "treatment", "FOXM1 inhibitor", "immune checkpoint inhibitor", and "composition" are as described above. [Effects of the Invention]

[0067] The pharmaceutical composition of the present invention comprising a FOXM1 inhibitor and an immune checkpoint inhibitor more effectively suppresses tumor growth, induces cell death, and exhibits cancer prevention, amelioration, or treatment effects than the FOXM1 inhibitor or immune checkpoint inhibitor alone, and can therefore be useful as an agent for the prevention or treatment of such cancer. [Brief explanation of the drawings]

[0068] [Figure 1]Figure 1 shows the downregulation of PD-L1 in FOXM1-knockdown lung adenocarcinoma (LUAD) cell lines. Figure 1a shows the overall survival curves for lung adenocarcinoma patients with (red line) or without (blue line) concomitant alteration in FOXM1 and PD-L1 expression. Figure 1b shows the immunohistochemical analysis of FOXM1 and PD-L1 expression levels in human lung cancer tissues and normal lung tissues. Figures 1c and 1d show the results of RNA-seq and gene ontology functional activation analysis of siFOXM1-transfected H1299 and PC9 cells. Figures 1e, 1f, and 1g show the semiquantitative PCR results of FOXM1 and CD274 (PD-L1) expression levels in H1299 and PC9 cells following FOXM1 knockdown, overexpression, or thiostrepton (TST) treatment. [Figure 2] Figure 2 shows the correlation between thiostrepton (TST)-mediated downregulation of FOXM1 and decreased cell proliferation, survival, and cell death. The cell death effect of FOXM1 reduction by siFOXM1 transfection or thiostrepton (5 μM) treatment was confirmed (Figures 2a and 2b). FACS analysis of DNA content by PI staining (Figures 2c and 2d) and FACS analysis of dead cells after staining with Annexin V and PI (Figures 2e and 2f) were performed. Figures 2g and 2h show the protein expression levels of FOXM1, c-MYC, cyclin B1, cyclin E1, and cyclin D1 in cells treated with siFOXM1 knockdown or thiostrepton. [Figure 3] Figure 3 shows the results of confirming that downregulation of FOXM1 by thiostrepton (TST) is associated with reduced PD-L1 expression at the cell membrane. Fluorescence imaging results are shown for H1299 cells (Figures 3a-c) and PC9 cells (Figures 3b-d) treated with siFOXM1 knockdown or thiostrepton. Figures 3e and 3f show quantitative analysis of PD-L1 fluorescence signals from the fluorescence images. [Figure 4]Figure 4 shows that thiostrepton (TST) treatment suppresses the nuclear migration of FOXM1. Changes in FOXM1 protein levels were assessed by Western blotting (Fig. 4a and 4b) and fluorescence imaging (Fig. 4c to 4f) in the cytoplasmic and nuclear fractions of H1299 and PC9 cells treated with FOXM1 knockdown, overexpression, or TST. [Figure 5] Figure 5 shows the results of immunoblot analysis (Figures 5a and 5b) and fluorescent imaging (Figures 5c to 5f) confirming that reduced PD-L1 expression was restored by overexpression of FOXM1. [Figure 6] Figure 6 shows the results confirming that FOXM1 binds to the promoter of the gene encoding PD-L1 and mediates PD-L1 expression. [Figure 7] Figure 7 shows the results of confirming that thiostrepton (TST) inhibits tumor growth and PD-L1 expression in vivo. Figures 7a to 7c show the timeline of tumor formation and thiostrepton administration in BALB / c mice, along with the associated tumor volume, body weight, and expression levels. Figure 7d shows the results of blood biochemistry analysis of control and thiostrepton-treated mice. Figure 7e shows the results of hematoxylin and eosin (H&E) and TUNEL staining of tissue sections from the liver, kidney, and spleen of control and thiostrepton-treated mice to analyze the rate of cell death in these normal tissues. [Figure 8]Figure 8 shows the anti-tumor effect of a combination of thiostrepton (TST) and anti-4-1BB antibody in a syngeneic Lewis lung carcinoma (LLC-1) animal model. Figures 8a to 8c show the timeline of tumor formation and drug administration in C57BL / 6 mice, along with the corresponding tumor volume and body weight. Figures 8d and 8e show the results of TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay to analyze cell death and CD3+ T cell levels by immunohistochemistry in the mice shown in Figure 8a. [Figure 9] Figure 9 shows the synergistic anti-tumor effect of combined treatment with thiostrepton (TST) and immune checkpoint inhibitors in a syngeneic Lewis lung carcinoma (LLC-1) animal model using C57BL / 6 mice. Figure 9a shows the timeline of tumor formation and drug administration in the animal model. Figure 9b shows the synergistic anti-tumor effect of combined treatment compared to treatment with thiostrepton (TST) and anti-4-1BB antibody alone. Figure 9c shows the synergistic anti-tumor effect of combined treatment compared to treatment with thiostrepton (TST) and anti-PD-1 antibody alone. [Figure 10] Figure 10 shows the results of immunoblotting analysis of FOXM1 and PD-L1 expression in human colon cancer cell lines treated with thiostrepton (TST). [Figure 11]Figure 11 shows the effects of thiostrepton (TST) treatment on the suppression of PD-L1 expression and the increase in cancer cell death in MC38 colon cancer cells. Figure 11a shows immunoblotting analysis confirming that thiostrepton (TST) treatment resulted in a concentration-dependent decrease in FOM1 and PD-L1 expression. Figure 11b shows fluorescence imaging analysis confirming that thiostrepton (TST) treatment resulted in a concentration-dependent decrease in PD-L1 expression on the cancer cell surface. Figure 11c shows data on cell viability in MC38 cancer cells as a function of thiostrepton (TST) treatment concentration. Figure 11d shows data on cell viability in normal human primary coronary artery smooth muscle cells (SMCs) as a function of thiostrepton (TST) treatment concentration. Figure 11e shows flow cytometric analysis of cell death in MC38 cells in the control and thiostrepton (TST)-treated groups. [Figure 12] Figure 12 shows the results of characterizing thiostrepton-loaded liposomes (ThioLipos). Figure 12a shows the chemical structure of thiostrepton (TST). Figures 12b and 12c show the size distribution measurement results and transmission electron microscope (TEM) images of ThioLipos in the aqueous solution phase. Figure 12d shows the change in size distribution of ThioLipos over time, evaluating the stability of ThioLipos. [Figure 13] FIG. 13 shows the results of analyzing the change in blood concentration over time after the same volume of free thiostrepton (TST) and ThioLipo were intravenously administered. [Figure 14]Figure 14 shows the synergistic antitumor effect of combined administration of ThioLipo and immune checkpoint inhibitors in a syngeneic colon cancer model. Figure 14a shows the timeline for administration of immune checkpoint inhibitors and ThioLipo to mice. Figure 14b shows tumor growth curves for mice treated with ThioLipo and anti-4-1BB antibody, alone or in combination, or ThioLipo and anti-PD-1 antibody, alone or in combination. Figure 14c shows tumor growth curves for individual mice for each treatment group in Figure 14b. [Figure 15] Figure 15 shows the results of quantitative analysis of the expression of TUNEL, CD3, and PD-L1 after immunohistochemical staining of tumor sections from mice administered ThioLipo and immune checkpoint inhibitors alone or in combination. [Figure 16] Figure 16 confirms that no side effects occur when ThioLipo and immune checkpoint inhibitors are administered alone or in combination. Figure 16a shows the results of TUNEL staining of sections of major organs (heart, kidney, liver, lung, and spleen), and no histopathological changes or increased normal cell death were observed. Figure 16b shows the results of analyzing the biochemical data of the mouse blood, and no side effects were observed. [Figure 17] Figure 17 shows the results of confirming that FDI-6 down-regulates FOXM1 and PD-L1 in lung adenocarcinoma (LUAD) and colon cancer cell lines. Figure 17a shows the results of immunoblotting, which demonstrates that the expression of FOXM1 and PD-L1 is simultaneously decreased in proportion to the concentration of FDI-6 administered to lung cancer cell lines. Figure 17b shows the results of immunoblotting, which demonstrates that the expression of FOXM1 and PD-L1 is simultaneously decreased in proportion to the concentration of FDI-6 administered to colon cancer cell lines. [Figure 18]Figure 18 shows the results confirming that RCM1 treatment down-regulated FOXM1 and PD-L1 expression in lung cancer cells, colon cancer cells, and brain cancer cells. Figure 18a shows the results confirmed by immunoblotting that FOXM1 and PD-L1 expression was all suppressed in lung cancer, colon cancer, and brain cancer cell lines in proportion to the RCM1 treatment concentration. Figure 18b shows the results confirmed by fluorescence imaging that PD-L1 expression on the cancer cell surface was reduced when human lung cancer cell lines were treated with RCM1. DETAILED DESCRIPTION OF THE INVENTION

[0069] The present invention will be described in more detail with reference to the following examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0070] Example 1: Confirmation of the association between FOXM1 and PD-L1 expression 1.1. Information analysis of FOXM1 and PD-L1 in lung adenocarcinoma (LUAD) (1) Dataset for informatics of FOXM1 and PD-L1 in lung adenocarcinoma (LUAD) To determine the role of FOXM1 and PD-L1 in the prognosis of LUAD, the Cancer Genome Atlas (TCGA) LUAD Firehose Legacy (n=586), available on cBioPortal, for cancer genome open access resources, was used.

[0071] (2) Geneset enrichment analysis (GEA) In response to siFOXM1 knockdown, DEGs were listed, and significantly downregulated genes (less than 0.5-fold between NC siRNA and siFOXM1 knockdown H1299 and PC9 cells) were applied to GSEA using the Enrichr database.

[0072] 1.2. Cell culture and transfection H1299 and PC9 cells, purchased from ATCC (Manassas, VA, USA), were cultured at 37°C and 5% CO in RPMI-1640 medium (#10-040-CV; Corning, New York, NY, USA) supplemented with 10% human serum albumin (#35-010-CV; Corning) and penicillin-streptomycin antibiotic cocktail (#15240062; Thermo Fisher Scientific, Waltham, MA, USA).

[0073] H1299 and PC9 LUAD cells were transfected with 5 nM negative control (NC) or FOXM1 siRNA (siFOXM1) (QIAGEN, Hilden, Germany) using Lipofectamine RNAiMAX (#13778150; Thermo Fisher Scientific). Human FOXM1 isoform b (pFLAG-FOXM1)-expressing plasmid or empty vector (pFLAG-CMV; mock) were transfected using Lipofectamine 2000 reagent (#11668019; Thermo Fisher Scientific) according to the manufacturer's instructions.

[0074] 1.3. Reverse transcription and semi-quantitative PCR Total RNA was extracted and purified using the RNeasy kit (#74004; QIAGEN) according to the manufacturer's protocol. mRNA reverse transcription was performed using the PrimeScript 1st Strand cDNA Synthesis Kit (#6110A; TaKaRa Bio, Otsu, Japan). Quantitative PCR was performed using primers specific to the CD274 and FOXM1 genes designed using Primer 3 software. The β-actin gene was used as an internal control to normalize target gene expression. The sequences of the primer pairs used in this study are listed in Table 1.

[0075] [Table 1]

[0076] 1.4. Confirmation of the relationship between FOXM1 and PD-L1 expression in lung cancer cells To investigate the clinical relevance of FOXM1 and PD-L1 in patients with adenocarcinoma, we analyzed survival curves of lung adenocarcinoma (LUAD) using the TCGA (The Cancer Genome Atlas) dataset on cBioPortal. Compared with 443 patients with no changes in FOXM1 or PD-L1 expression, 70 patients with higher FOXM1 and PD-L1 expression had a lower median overall survival (31.21 months vs. 50.20 months, p=8.716×10 -4 , Figure 1a).

[0077] To confirm whether FOXM1, a putative transcription factor (TF) that regulates PD-L1 expression, is associated with a poor prognosis in LUAD patients, we immunostained lung cancer tissues and corresponding normal tissues with antibodies against FOXM1 and PD-L1 to compare their expression levels. The results showed that FOXM1 and PD-L1 were upregulated in LUAD tissues compared with adjacent normal tissues (Figure 1b).

[0078] RNA-seq analysis of H1299 and PC9 cells with siRNA-mediated FOXM1 knockdown revealed that CD274, encoding PD-L1, was a putative target of FOXM1 among 67 differentially expressed genes (DEGs) expressed at 0.5-fold or less in the two cell lines (Figure 1c). To confirm the biological functions of the downregulated genes after FOXM1 knockdown in H1299 and PC9 cells, we performed gene set enrichment analysis (GSEA) on the downregulated genes (67 in total) using the Enrichr database. Positive transcriptional regulation by RNA polymerase II and cell cycle regulation were significantly activated in the target genes (Figure 1d), suggesting that FOXM1 may also contribute to CD274 regulation. The encoded PD-L1 protein plays an important role in immune evasion in lung cancer cells. After knocking down FOXM1 with siRNA, we confirmed PD-L1 expression and found that PD-L1 transcription levels were significantly reduced in both cell lines (Fig. 1e).

[0079] Additionally, we assessed PD-L1 transcription levels after FOXM1 overexpression using pFLAG-FOXM1 isoform b, the major isoform expressed under physiological conditions. We found that FOXM1 overexpression significantly increased PD-L1 mRNA expression in H1299 and PC9 cells (Fig. 1f).

[0080] We evaluated the potential effects of FOXM1 on PD-L1 expression in NSCLC cells using FOXM1 inhibition strategies other than siRNA knockdown. Several FOXM1 inhibitors, including the FOXM1-inhibiting natural product TST, were tested in H1299 and PC9 cells, and TST was the most effective inhibitor, significantly reducing FOXM1 levels.

[0081] To induce increased PD-L1 expression, we analyzed PD-L1 transcription levels in cells treated with TST in the presence of IFN-γ. The results showed that TST-induced reduction of FOXM1 resulted in a concentration-dependent decrease in PD-L1 mRNA expression in both cell lines (Figure 1g).

[0082] Example 2: Confirmation of the effects of FOXM1 inhibition on cells 2.1. Cell analysis method (1) Cell proliferation analysis H1299 and PC9 cells were grown in 96-well plates and treated with 5 μM TST or transfected with siFOXM1 at a density of 3,000 cells / well in 6-well plates. Cell growth was monitored for 2 hours at 37°C by adding 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzenesulfonate (WST-1, #MK400, TaKaRa Bio) at the indicated times, and absorbance was measured at 450 nm. All experiments were performed in quadruplicate.

[0083] (2) Cell cycle distribution analysis Both H1299 and PC9 cells were enzymatically dissociated using TrypLE (#12605-010; Thermo Fisher Scientific), collected by centrifugation at 2,000 × g for 2 minutes, resuspended in PBS, fixed in 70% ethanol, and stored at 4°C overnight. Fixed cells were pelleted, washed twice with PBS, resuspended in FxCycle PI (propidium iodide) / RNase staining solution (#F10797; Thermo Fisher Scientific), and incubated in the dark at 37°C for 30 minutes. Flow cytometry was performed using a FACS-LSR Fortessa flow cytometer (BD Biosciences, Santa Clara, CA, USA).

[0084] (3) Apoptotic cell death analysis (Annexin V / PI staining) H1299 and PC9 cells (1 × 10 5) were cultured in 6-well plates and transfected with siFOXM1 or NC siRNA or treated with 5 μM TST or dimethyl sulfoxide (DMSO). Cells were then stained using the fluorescein isothiocyanate (FITC) Annexin V Apoptosis Detection Kit I (#556547; BD Biosciences). Cells were analyzed within 1 h to reduce side effects from staining. Flow cytometry was performed using a FACSVerse cell analyzer (BD Biosciences).

[0085] 2.2. Cell growth suppression and cell death induction by FOXM1 inhibition To confirm the effect of FOXM1 on cell proliferation and survival in NSCLC, we performed 24-, 48-, and 72-hour time-course experiments. We found that cell proliferation was reduced in two cell lines in response to siRNA-mediated knockdown and 5 μM TST treatment in a time-dependent manner (Figures 2a and 2b). Furthermore, flow cytometry analysis of protein content in cells treated with siFOXM1 or TST revealed that FOXM1 knockdown with siFOXM1 or inhibition with TST increased the sub-G0 / G1 population and time-dependently reduced the S-phase population compared to negative control (NC) siRNA or dimethyl sulfoxide (DMSO) treatment (Figures 2c and 2d). TST-treated two cell lines showed an increase in the G2 population, accompanied by a decrease in the S-phase population compared to DMSO treatment. This confirms that FOXM1 reduction alters cell cycle progression in NSCLC cells and plays an important role in the G1-S and G2-M transitions. Furthermore, we confirmed that the apoptotic cell population (Annexin V / PI-positive cells) significantly increased in a time-dependent manner in cells whose FOXM1 expression was reduced by siRNA or TST treatment (Fig. 2e and 2f).

[0086] Western blot analysis further confirmed the effect of reducing FOXM1 expression on c-MYC, cyclin B1, cyclin E1, and cyclin D1 in the two cell lines. The protein levels of c-MYC, cyclin B1, and cyclin D1 were significantly reduced after reducing FOXM1 expression by siRNA or TST treatment (Figure 2g and Figure 2h).

[0087] 2.3. Downregulation of PD-L1 on lung cancer cell membranes with decreased FOXM1 expression PD-L1 is a transmembrane protein, and we performed immunocytochemistry (ICC) to assess whether the effect of FOXM1 knockdown by siRNA or TST treatment on reducing total PD-L1 protein levels was phenotypically observed at the cell membrane. We also performed ICC to assess membrane PD-L1 enrichment in H1299 and PC9 cells transfected with siFOXM1 or NC siRNA for 72 hours. The membrane expression of PD-L1 in control H1299 cells was significantly higher than that in siFOXM1-transfected cells (Figure 3a). Downregulation of FOXM1 reduced PD-L1 expression by 67.4% (Figure 3e), and siFOXM1-transfected PC9 cells downregulated FOXM1 and reduced PD-L1 expression by 81.68% (Figures 3b and 3f).

[0088] Considering that TST treatment in the presence of IFNγ reduces total PD-L1 protein levels in lung cancer cells, we investigated the effect of TST on PD-L1 enrichment on the cell membrane. IFNγ-boosted (IFNγ(+)) membrane PD-L1 levels were observed in both cell lines (Figures 3c and 3d). TST-treated H1299 and PC9 cells also showed reduced PD-L1 on the membrane (Figures 3c and 3d), and FOXM1 downregulation reduced PD-L1 levels in H1299 and PC9 cells by 87.23% and 85.82%, respectively, compared to the DMSO control (Figures 3e and 3f).

[0089] Example 3: Confirmation of the inhibitory effect of TST on FOXM1 3.1. Protein Analysis (1) Nucleocytoplasmic fractionation To confirm the subcellular localization of proteins, H1299 and PC9 cell pellets were lysed in Buffer A (10 mM HEPES, 10 mM KCl, 1.5 mM MgCl, 0.5 mM dithiothreitol (DTT), and 0.05% IGEPAL CA-630 (pH 7.9)) for 10 min. The Buffer A lysate was centrifuged at 1000 × g for 10 min at 4 °C to collect the nuclear fraction. After removing the cytoplasmic fraction, Buffer B containing 5 mM HEPES, 0.2 mM EDTA, 1.5 mM MgCl, 0.5 mM DTT, and 26% glycerol (v / v) (pH 7.9) was added to the nuclear fraction and lysed on ice for 30 min.

[0090] (2) Immunoblotting analysis of protein expression Cells were lysed in RIPA buffer supplemented with Complete Mini Protease Inhibitor Cocktail (#04693124001; Roche, Basel, Switzerland) and stored on ice for 30 min. Equal amounts of protein were uniformly loaded and analyzed by SDS-PAGE (sulfate-polyacrylamide gel electrophoresis) and transferred to a PVDF (polyvinylidene difluoride) membrane (#IPVH 00010; Millipore, Billerica, MA, USA) at 100 V for 1 h. Proteins of interest were probed using specific primary antibodies against FOXM1 (#sc-271746), cyclin E1 (#sc-481), α-tubulin (#sc-8035), and β-actin (#sc-47778) (Santa Cruz Biotechnology, TX, USA); PD-L1 (#13684S), cyclin D1 (#2978S), cyclin B1 (#4138S), c-MYC (#5605S), and poly(ADP-ribose) polymerase (PARP, #9542S; Cell Signaling Technology, Beverly, MA, USA). Horseradish peroxidase-conjugated secondary antibodies specific for the primary antibodies were used for protein detection using Pico Enhanced Chemiluminescence Substrate (#34577; Thermo Fisher Scientific).

[0091] (3) Immunocytochemistry (ICC) Cells were fixed with 4% paraformaldehyde (#30525-89-4; FUJIFILM Wako Chemical, Wako, Japan) for 15 minutes and blocked with 1% bovine serum albumin (BSA) in PBS-0.01% Tween-20 (PBS-T) to reduce nonspecific antibody binding. Cells were then incubated overnight with anti-PD-L1 antibody (#14598382; eBioscience, San Diego, CA, USA) diluted 1:200 and anti-FOXM1 antibody (#sc-271746; Santa Cruz Biotechnology) diluted 1:50, followed by 1 hour of incubation with Alexa Fluor 488-conjugated secondary antibody (#A11029; Thermo Fisher Scientific). Nuclei were stained with Hoechst 33342. After incubation, cells were washed with PBS and mounted on glass slides using Prolong Glass Antifade mounting medium (#P36982; Thermo Fisher Scientific). Confocal microscopy was performed using a Zeiss 730 Meta microscope (Carl Zeiss, Oberkochen, Germany) and analyzed using Axiovision software (Carl Zeiss).

[0092] 3.2. Confirmation of the inhibitory effect of TST on FOXM1 nuclear translocation Because the nuclear potential of FOXM1 has been highlighted as a prominent feature in various cancers, we examined the enrichment of FOXM1 in the nuclei of pFLAG-FOXM1-transfected H1299 and PC9 cells, followed by treatment with IFN-γ (20 ng mL -1siFOXM1 knockdown or TST (5 μM) treatment was performed in the presence of 5 μM FOXM1. FOXM1 is present in both the nucleus and cytoplasm, and overexpression of FOXM1 increased the nuclear potential of cytoplasmic FOXM1. siFOXM1-mediated FOXM1 inhibition or TST treatment reduced FOXM1 protein levels and nuclear potential in H1299 and PC9 cells (Figures 4a and 4b). Compared to mock transfection, overexpression of FOXM1 restored FOXM1 levels by 48.85% and 84.33% with siFOXM1 and TST treatment in H1299 cells, respectively. In PC9 cells, the restoration values were 30.95% and 57.56% with siFOXM1 and TST treatment, respectively. ICC analysis showed that FOXM1 was primarily localized in the nucleus of pFLAG-FOXM1-transfected H1299 cells and NC siRNA-transfected PC9 cells compared with siFOXM1-transfected cells (Figures 4c and 4d). Similarly, in pFLAG-FOXM1-transfected H1299 and PC9 cells, FOXM1 expression was primarily localized in the nucleus compared with TST-treated cells, in which FOXM1 expression and nuclear potential were suppressed. pFLAG-FOXM1-mediated FOXM1 overexpression was significantly reduced by 54.22% (p<0.01) in H1299 cells and 35% (p<0.001) in PC9 cells by siRNA-mediated knockdown. In TST-treated cells, FOXM1 overexpression was significantly reduced by 63.7% (p<0.05) in H1299 cells and 44% (p<0.01) in PC9 cells.

[0093] Example 4: Elucidation of the regulatory mechanism of PD-L1 expression by FOXM1 4.1. Confirmation of restoration of PD-L1 expression by FOXM1 overexpression We confirmed the restoration of PD-L1 expression by inducing FOXM1 overexpression in FOXM1-knockdown cell lines. Specifically, H1299 and PC9 cells were transfected with NC siRNA or siFOXM1, followed by pFLAG-FOXM1 or empty vector for 72 hours. Western blot analysis and confocal cell imaging confirmed that siFOXM1-induced PD-L1 downregulation was not restored in mock-transfected cells, but was restored in pFLAG-FOXM1-transfected cells. FOXM1 restored PD-L1 expression on the H1299 and PC9 cell membranes by 55.9% and 51.8%, respectively (Figures 5a, 5c, and 5d). Similarly, we investigated whether FOXM1 could restore PD-L1 expression, which was reduced in H1299 and PC9 cells treated with TST (5 μM) for 48 hours in the presence of IFNγ. FOXM1 overexpression restored PD-L1 expression, which was reduced in FOXM1-knockdown cells, and TST treatment also restored the reduced expression in H1299 and PC9 cells. As shown by Western blotting (Figure 5b) and confocal microscopy (Figures 5e and 5f), FOXM1 restored PD-L1 expression at the plasma membrane of H1299 and PC9 cells by 38.7% and 46%, respectively. Furthermore, elevated PD-L1 levels induced by pFLAG-FOXM1 overexpression (mimicking the malignant tumor mass) were significantly reduced by 52.6% (p<0.05) in H1299 cells and 59.2% (p<0.01) in PC9 cells with siRNA, and by 41.9% (p<0.01) in H1299 cells and 68.0% (p<0.05) in PC9 cells with TST treatment. Collectively, these results further support the hypothesis that PD-L1 is a downstream target of and positively regulated by FOXM1, implying that PD-L1 levels can be precisely regulated by inhibiting FOXM1.

[0094] 4.2. Confirmation of the mechanism by which FOXM1 regulates PD-L1 expression (1) Chromatin immunoprecipitation (ChIP) To cross-link proteins to DNA, H1299 and PC9 cells were treated with 0.7% formaldehyde for 10 minutes at 37°C. Cross-linking was quenched by adding glycine to the culture medium at a final concentration of 0.125 M for 5 minutes. Cells were sonicated for 5 minutes using a Bioruptor Next Gen sonicator (Diagenode, Denville, NJ, USA) with a 30-second on / 30-second off cycle to obtain ~500-bp DNA fragments. The sonicated cell lysate, containing genomic DNA fragments, was precleared to remove unwanted nonspecific components. ChIP was performed overnight at 4°C with rotation using an antibody against FOXM1 (#sc-271746, Santa Cruz Biotechnology), followed by various washing steps, reverse cross-linking, and proteolysis using protease K (#P2308; Sigma-Aldrich, St. Louis, Missouri, USA). The immunoprecipitated DNA fragments were purified using the phenol-chloroform method, and the purified DNA pellet was resuspended in TE buffer (pH 8.0) and used for PCR. Primers specifically detecting the FOXM1-binding region were designed using the Primer3 tool for ChIP-PCR (Table 1).

[0095] (2) Dual-luciferase assay The pGL3 plasmid (#107003; Addgene, Watertown, MA, USA) containing the 2 kb promoter of CD274 was provided by Julian Downward's laboratory (Coelho MA, de Carne Trecesson S, Rana S, Zecchin D, Moore C, Molina-Arcas M, East P, Spencer-Dene B, Nye E, Barnouin K, Snijders AP, Lai WS, Blackshear PJ, Downward J, Immunity 2017, 47, 1083.). H1299 and PC9 cells were co-transfected with the pGL3 2 kb promoter. Cells containing CD274 or control pGL3-basic plasmid and pGL4.70 hRluc Renilla luciferase vector (Promega, Madison, WI, USA) were transfected with siFOXM1 or treated with TST in the presence of interferon-gamma (IFNγ). Cells were harvested 48 hours post-transfection and subjected to dual luciferase assays according to the manufacturer's protocol (#E1910; Promega). At least four independent biological replicates were performed to ensure reliable results.

[0096] (3) FOXM1 directly binds to the promoter and regulates PD-L1 expression To elucidate the mechanism by which FOXM1 induces PD-L1 expression, we analyzed ChIP-seq data from the ENCODE 3 database accessed through the UCSC Genome Browser (Human assembly Dec 2013, GRCh38 / hg38). In K562 (a human immortalized myeloid leukemia cell line), FOXM1 enrichment was confirmed in the promoter region with a peak signal approximately 167 bp upstream of the CD274 transcription start site. The TAAAC FOXM1 consensus DNA-binding domain (DBD) was detected in two distant regions (-1,988 bp and -2,310 bp) as well as in a nearby region (approximately 142 bp) from the CD274 transcription start site (Figure 6a).

[0097] Based on these in silico data, we hypothesized that the CD274 promoter region in lung cancer cells is a potential target of the FOXM1 transcription factor and performed ChIP-PCR analysis to isolate FOXM1-CD274 DNA complexes using an anti-FOXM1 antibody. The CD274 promoter region flanking the putative FOXM1 binding site (~167 nt) was detected by semiquantitative PCR in H1299 and PC9 cells, compared with the dramatically reduced FOXM1 binding to the CD274 promoter observed in siFOXM1-transfected and TST-treated cells, along with their respective controls. pFLAG-FOXM1-transfected cells showed increased FOXM1 binding to the CD274 promoter compared to mock-transfected cells, indicating that FOXM1 directly binds to the CD274 promoter (Figure 6b). Thus, FOXM1 directly regulates PD-L1 transcription by binding to the promoter, and targeting FOXM1 disrupts FOXM1-mediated regulation of PD-L1 expression.

[0098] To further confirm whether reduction of FOXM1 binding by siRNA or TST treatment significantly reduced CD274 transcriptional activity, we performed dual-luciferase reporter analysis using a CD274 promoter-luciferase construct. Consistent with the ChIP-PCR results, reduction of FOXM1 by siRNA significantly reduced relative luciferase activity by 40% in H1299 cells (p<0.001) and 42% in PC9 cells (p<0.01) (Figure 6c). Similarly, inhibition of FOXM1 by TST in the presence of IFNγ also significantly reduced CD274 promoter activity, as indicated by a 53% reduction in relative luciferase activity in H1299 cells (p<0.001) and a 66% reduction in relative luciferase activity in PC9 cells (p<0.001) (Figure 6d).

[0099] These results support the conclusion that nuclear FOXM1 translocation positively regulates PD-L1 expression by directly binding to the CD274 promoter in both H1299 and PC9 cell lines. A new model for FOXM1-mediated regulation of PD-L1 expression in lung cancer has been proposed, in which FOXM1 regulates PD-L1 expression by directly binding to the CD274 promoter and inducing PD-L1 transcriptional activation (Figure 6g). Because FOXM1-induced PD-L1 upregulation causes immune evasion in lung tumor cells, targeting FOXM1 with TST, a natural FOXM1 inhibitor, may be a novel therapeutic strategy for preventing immune evasion and reducing tumor growth in lung cancer cells.

[0100] Example 5: Confirmation of FOXM1 inhibitory effect in vivo Animal models (1) Xenograft tumor model All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of the National Cancer Center Research Institute (NCC-21-619). Mice (n=6) were intraperitoneally injected with TST dissolved in DMSO at a dose of 17 mg / kg every 2 days before euthanasia and sacrifice. Tumor volume and body weight of the mice were measured every other day. Tumor volume (V) was calculated as AB 2 The mean diameter was calculated as ≈ 1 / 2 (A and B are the major and minor diameters (mm) respectively). Two days after the final TST injection, all mice were euthanized, and tumor tissues were collected for immunohistochemical (IHC) staining of FOXM1 and PD-L1. The tissues were fixed in 10% neutralized formaldehyde and embedded in paraffin blocks. Tissue microarray (TMA) slides were prepared with a diameter of 4 mm per sample and cut at a thickness of 4 μm for IHC with anti-FOXM1 (#20459S) or anti-PD-L1 (#13684S) (Cell Signaling Technology) antibodies. An Autostainer Link48 (Dako, Agilent Technologies, Santa Clara, CA, USA) was used for analysis.

[0101] IHC staining was performed using an anti-rabbit-HRP antibody (K4003, EnVision+ HRP Labeled Polymer Anti-Rabbit, Dako, Agilent Technologies, Carpinteria, CA, USA) and 3,3'-diaminobenzidine (DAB) (K3468, DAB+ Chromogen, Dako, Agilent Technologies, Santa Clara, CA, USA) as the substrate. Sections were counterstained with hematoxylin (K8008, EnVision FLEX Hematoxylin, Dako, Agilent Technologies Singapore, Singapore). To analyze the in vivo toxicity of the test drugs, four xenografted mice from each group were used for serum biochemistry analysis (Table 2). On day 9, serum samples were collected from the control and TST-treated mice. Furthermore, vital organs (liver, spleen, and kidney) of the mice were collected and sectioned, and H&E staining was performed to monitor histopathological changes. To analyze cell death in response to the test drugs, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay (ApopTag Plus Proxidase In Situ kit, EMD Millipore, Temecula, CA, USA) was also performed on the fragmented tissues.

[0102] (2) A syngeneic tumor model using mouse lung cancer cell line LLC-1 The animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of the National Cancer Center Research Institute (NCC-22-675B). Lewis Lung Carcinoma LLC-1, murine lung carcinoma cells (5 × 10 5 ) were subcutaneously implanted (OrientBio, Seoul, Korea) into 5-week-old C57BL / 6N mice (n = 6 per group) and left for 10 days to grow to approximately 150 mm 3Tumors of 1000 μg / mL were formed. Mice were then injected with 17 mg / kg TST and 0.2 mg / mouse anti-4-1BB antibody at the time points indicated by the black arrows (days 1, 3, 5, and 7). Tumor volume and body weight were measured every other day. On day 9, all mice were euthanized, and tumor tissues were collected, fixed in formalin, embedded in paraffin, and sectioned for IHC. Furthermore, TUNEL analysis was performed on fragmented tissues to analyze cell death in response to TST and anti-4-1BB antibody treatment.

[0103] CD3 in tumor sections + T-cell assessment was performed as previously described (Huang Y, Lin C, Kao HK, Hung SY, Ko HJ, Huang YC, Chang YL, Chang KP, Cancer Manag Res 2020, 12, 8275). CD3 + T-cells were detected with anti-CD3 antibody (ab16669, Abcam, Cambridge, UK) according to the manufacturer's instructions. + The positive signal intensity of T-cells was analyzed using computer image analysis with ImageJ software (LOCI, University of Wisconsin) and CD3 + Signal intensity was calculated as the relative ratio of total positive area / total surface area. Pathological images were acquired using an Aperio VERSA slide scanning system (Leica Biosystems Imaging, Vista, CA, USA). Three independent, intact computerized microscope fields of each tissue sample were captured by Aperio Imagescope software (Leica Biosystems Imaging, Vista, CA, USA) at 100x magnification. CD3 was detected in the entire microscope field. + The cells were used for signal intensity analysis.

[0104] The in vivo synergistic effect between TST and anti-4-1BB antibody treatment groups was determined by calculating the CDI (drug interaction coefficient), with CDI values <1, <0.7, =1, and >1 indicating synergy, significant synergy, additivity, and antagonism, respectively.

[0105] In all statistical analyses, the differences between groups were χ 2 The results were evaluated using the σ test and Student's t test. Statistical significance was set at p<0.05, and deviations were expressed as standard deviations.

[0106] 5.2. FOXM1 inhibition inhibits tumor growth and modulates PD-L1 expression To investigate the effects of the FOXM1 inhibitor TST on tumor growth and PD-L1 expression in vivo, human H1299 or PC9 cells (5 × 10 6 ) were subcutaneously transplanted into BALB / c nude mice (n = 6 per group). The average size of the xenografted tumor mass was approximately 170 mm 3 When the tumor volume reached 1000 mg / kg, mice were intraperitoneally injected with TST at a dose of 17 mg / kg four times every two days (Figure 7a). Periodic monitoring of tumor volume during inhibitor treatment showed a significant decrease in tumor volume in the TST-treated group compared with the control group. After 9 days, no difference in body weight was observed between the two groups (Figure 7b). Two days after the final TST treatment, mice were euthanized, and tumor tissues were collected, sectioned, and stained to analyze changes in FOXM1 and PD-L1 expression levels. Immunohistochemical analysis demonstrated that FOXM1 and PD-L1 were overexpressed in lung tumors from control mice but significantly downregulated in TST-treated tumors (Figure 7c). FOXM1 was primarily localized in the nuclei of tumor cells in the control group, whereas the nuclei of TST-treated tumor cells showed slight immunopositivity. These results indicated that, in accordance with FOXM1 expression, PD-L1 was positively expressed in control tumors of H1299 and PC9 xenografts. In contrast, PD-L1 expression was significantly reduced by TST treatment (Fig. 7c).

[0107] Furthermore, serum biochemistry analysis was performed to evaluate the potential toxicity of TST in vivo. Blood biomarker levels showed no significant changes in the TST-treated group compared with the DMSO control group (Figure 7d and Table 2). Furthermore, hematoxylin and eosin (H&E) staining results showed no significant histological changes in vital organs in the TST-treated group (Figure 7e). TUNEL analysis of tissue sections demonstrated that TST treatment did not affect cell death in normal tissues compared with the DMSO control group (Figure 7e). Collectively, these results suggest that TST-mediated FOXM1 inhibition may inhibit immune evasion and effectively reduce tumor growth.

[0108] [Table 2]

[0109] Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; Glu, glucose; BUN, blood urea nitrogen; Crea, creatinine; T-Bill, total bilirubin; T-chol, total cholesterol; TG, triglycerides; TP, total protein; Alb, serum albumin; Glo, serum globulin; A / G ratio, serum albumin / serum globulin ratio

[0110] Example 6: Confirmation of antitumor synergistic effect by combined administration of FOXM1 inhibitor and immune checkpoint inhibitor To evaluate the therapeutic potential of TST in cancer immunotherapy, we used the LLC-1 syngeneic tumor model, which was reported to be resistant to ICI treatment (Figure 8a). Lewis lung carcinoma (LLC-1) mouse lung cancer cells (5 × 10 5 ) was subcutaneously injected into C57BL / 6N mice (n = 6 per group) and administered at 150 mm 3Tumors were formed in a volume of 1000 mg / kg. Mice were injected with 17 mg / kg TST and 0.2 mg / mouse anti-4-1BB antibody on days 1, 3, 5, and 7. Treatment of tumor-bearing mice with anti-4-1BB antibody or TST alone resulted in 38% and 48% inhibition of tumor growth, respectively, compared with the control group (Figure 8b). In particular, combination therapy with anti-4-1BB antibody and TST demonstrated significantly improved tumor growth inhibition, with tumor growth inhibited by 65% in tumor-bearing mice compared with the control group (p<0.001). After 9 days, no difference in body weight was observed between the experimental groups (Figure 8c).

[0111] Next, to examine cell death in the tumors, TUNEL staining of tumor sections was performed on day 9. The number of apoptotic tumor cells was 1.38-fold and 2.46-fold higher in the anti-4-1BB-treated group (p<0.05) and TST-treated group (p<0.05), respectively, than in the control group (Figure 8d). The combination of TST and anti-4-1BB antibody synergistically increased the number of apoptotic LLC-1 tumor cells by 4.92-fold and 2.76-fold (CDI=0.5) compared with the anti-4-1BB-treated group (p<0.001) or TST-treated group (p<0.001), respectively. Furthermore, immunohistochemical staining for CD3 in tumor sections revealed a significant increase in CD3 expression in anti-4-1BB-treated (p<0.05) and TST-treated (p<0.05) tumors. + The combination of TST and anti-4-1BB antibody significantly increased intratumoral CD3 T cells by 1.28-fold and 1.32-fold, respectively, compared to the control group (Fig. 8d). The combination of TST and anti-4-1BB antibody significantly increased intratumoral CD3 T cells compared to the anti-4-1BB antibody-treated group (p<0.001) or TST-treated group (p<0.001). + The T-cell frequency (CDI = 0.49) also increased by 2.72-fold and 2.63-fold, respectively (Figure 8e). These results indicate that the combined treatment of TST and anti-4-1BB antibody has a significant synergistic effect on immunogenicity and anti-tumor activity. In particular, the combined treatment of TST and anti-4-1BB antibody significantly delayed tumor growth and attracted T cells to the tumor area in the immune-resistant LLC1 lung tumor model. Furthermore ... 3When treatment was initiated with TST alone, the anti-4-1BB antibody and TST groups showed 36.6% and 63.1% tumor growth inhibition, respectively, while the TST and anti-4-1BB antibody combination group showed a 78% tumor growth inhibition, demonstrating a synergistic antitumor effect (CDI = 0.94) (Figure 9a). Furthermore, the anti-PD-1 antibody (0.2 mg / mouse) and TST groups showed 38.7% and 63.1% tumor growth inhibition, respectively, while the TST and anti-PD-1 antibody combination group showed a 78.4% tumor growth inhibition, demonstrating a synergistic antitumor effect (CDI = 0.95) (Figure 9b). These results suggest that TST, when used in combination with immune checkpoint inhibitors, is a clinically meaningful strategy for suppressing tumor growth and improving the efficacy of immunotherapy.

[0112] Example 7: Confirmation of the effect of TST on colon cancer cell lines 7.1. Cell Death, Viability, and Protein Expression Analysis (1) Reagents and cell cultures used in the experiments Dimethyl sulfoxide (DMSO) and TST (Sigma-Aldrich, St. Louis, MO, USA), Amicon centrifugal filter devices (MWCO 50 K, UFC805024, Merck Millipore), Sephadex G25 gel filtration columns (PD-10; #17-0851-01, GE Healthcare), anti-PD-1 (clone: RMP1-14, BP0146) and anti-4-1BB (clone: 17B5, BE0296) antibodies (BioXcell), anti-CD3 antibody (ab16669, Abcam), and anti-PD-L1 antibody (13684, Cell Signaling Technology).

[0113] Human colon cancer cell lines (HCT116, HCT8, and SW480) and primary human coronary artery smooth muscle cells (SMCs) were purchased from the American Type Culture Collection (Manassas, VA, USA). The MC38 mouse colon adenocarcinoma cell line was provided by Dr. Choi Beomgyu (National Cancer Center, Goyang, Korea). MC38 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS and 1% penicillin / streptomycin. SW480 and HCT8 cells were cultured in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% FBS and 1% penicillin / streptomycin. HCT116 cells were maintained in McCoy's 5A medium supplemented with 10% FBS and 1% penicillin / streptomycin. Cells were maintained at 37°C in a humidified incubator supplied with 5% CO2.

[0114] (2) Immunoblotting For immunoblotting analysis, protein concentration was measured using a bicinchoninic acid kit (Cat. 23225; Thermo Fisher Scientific, Carlsbad, CA, USA). Proteins were separated by SDS-PAGE electrophoresis. Primary antibodies against FOXM1 (#ab207298, Abcam), PD-L1 (#14-5982-82, Invitrogen), and glyceraldehyde-3-phosphate dehydrogenase (#sc-25778, Santa Cruz Biotechnology) were added and incubated overnight at 4°C. After binding the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody to the washed membrane, protein bands were detected using a chemiluminescence kit (Thermo Fisher Scientific, Carlsbad, CA, USA).

[0115] (3) Cell death analysis (annexin V analysis) MC38 cells (8.0×10 4) were seeded into 6-well plates and cultured for 24 hours. Cells were treated with TST (5 μM) for 48 hours, harvested, and analyzed by FACS-Verse using the BD FITC Annexin V Apoptosis Detection Kit I (Cat No. 556547; BD Biosciences, San Diego, CA, USA) according to the manufacturer's protocol.

[0116] (4) Cell viability analysis MC38 and SMC were cultured at 3 × 10 in a 96-well plate. 3 The cells were cultured at a density of 100 cells / well. After treating the cells with various concentrations of TST dissolved in the medium for 48 hours, WST-1 reagent (MK400; Takara) was added at 10 μL / well and the cells were cultured for 1 hour. The absorbance was measured at 450 nm for further analysis.

[0117] (5) PD-L1 expression analysis by TST treatment To confirm the dose-dependent downregulation of PD-L1 levels by the FOXM1 inhibitor TST, we performed live cell imaging of PD-L1 expression in MC38 cells. MC38 cells were cultured in a Lab-Tek II system and treated with various concentrations of TST (0, 1, 5, and 10 μM) for 48 hours. Subsequently, the cells were incubated with an anti-PD-L1 antibody (#14-5982-82; eBioscience, San Diego, CA, USA) followed by binding with an Alexa Fluor 594-conjugated secondary antibody (A21209; Thermo Fisher Scientific, Waltham, MA, USA). Nuclei were stained with Hoechst 33342 dye (Thermo Fisher Scientific, Waltham, MA, USA). The cells were washed three times with PBS (pH 7.4, 10 mM MgCl, 136 mM NaCl), and the medium was replaced with fresh complete medium. Nuclei (λ) were stained. ex 359 nm, λ em 457 nm) and PD-L1 (λ ex 590 nm, λ em618 nm) fluorescence images were acquired using a confocal scanning laser microscope (LSM780; Carl Zeiss, Oberkochen, Germany).

[0118] 7.2. Effect of TST on PD-L1 expression in colon cancer cells We analyzed the effect of TST on PD-L1 expression in human and mouse colon cancer cells. Treatment with 5 μM TST significantly reduced both FOXM1 and PD-L1 levels in human colon cancer cells (HCT8, HCT116, and SW480) (Figure 10). TST treatment also dose-dependently reduced FOXM1 and PD-L1 expression in MC38 mouse colon cancer cells (Figure 11a). Confocal fluorescence images of immunocytochemical analysis of PD-L1 expression showed a strong fluorescent signal on the cancer cell surface (Figure 11b). Plasma membrane PD-L1 levels were also dose-dependently reduced after TST treatment. Similar to how FOXM1 directly binds to the nuclear PD-L1 promoter and selectively upregulates PD-L1 expression, TST significantly reduced PD-L1 expression in human and mouse colon cancer cells through inhibition of FOXM1 by TST.

[0119] The in vitro cytotoxicity of TST against MC38 cancer cells and normal SMC cells was tested. The median inhibitory concentration (IC 50 ) was 3.8 μM in MC38 cancer cells, whereas no significant cell death was observed in normal SMC cells. Flow cytometry analysis showed that TST treatment significantly increased cell death in MC38 cancer cells (Figure ​(Figure11e).11e).

[0120] Example 8: Confirmation of the antitumor effect of ThioLipo 8.1. Preparation and Characterization of ThioLipos (1) Manufacturing method of ThioLipos Cholesterol, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) were purchased from Avanti Polar Lipids (Alabama, USA). DOPC, cholesterol, and DSPE-PEG2000 were added to a round-bottom flask at molar ratios of 55, 40, and 5, respectively. TST was dissolved in chloroform at a concentration of 1.5 mg / mL, and 1 mL of the solution was added to the lipid sample. The solution was dried using a rotary evaporator to leave a thin lipid film. PBS (1 mL) was added to the round-bottom flask and sonicated in a bath sonicator for 5 min at 25 °C. After removing the lipid from the flask, the solution was transferred to a drum vial and sonicated for 20 min using a probe sonicator. Unentrapped TST and other impurities were removed by centrifugation at 600 × g for 5 min at 25°C. Free TST was further removed using a PD-10 size-exclusion column. The size distribution (Z-average) and polydispersity index were characterized using dynamic light scattering (DLS; Malvern Zetasizer). The concentration of TST encapsulated in nanoliposomes was analyzed by dissolving the nanoliposomes in DMSO and measuring the absorbance at 300 nm using a UV-Vis spectrophotometer (DU730, Beckman Coulter, Brea, CA). For transmission electron microscopy (TEM) of ThioLipos, a hydrophilic grid (Quantifoil, R1.2 / 1.3, 200 mesh, EMS) was prepared using a glow discharge system (PELCO easiGlow, Ted Pella). 4 μL of sample was applied to the grid and blotted for 1.5 s at 100% humidity and 4°C. The samples were then flash-frozen in liquid ethane for vitrification in a Vitrobot Mark IV (FEI) and analyzed using a Talos L120C (FEI) at 120 kV at the NanoBioImaging Center (Seoul National University, Korea).

[0121] (2) Characterization of ThioLipos

[0206] TST is a hydrophobic compound with low solubility in water, and liposome encapsulation was employed as a system for effectively delivering these hydrophobic drugs to tumors (Figure 12a). The hydrodynamic size of the fabricated ThioLipos was 192 nm, and TEM images of the ThioLipos showed a rounded tomographic structure (Figure 12c). The stability of the ThioLipos was also tested. There was no significant change in the size distribution over 15 days (Figure 12d). The encapsulation efficiency of TST in nanoliposomes was 63 ± 3%, and the TST-to-lipid ratio in the nanoliposomes was 0.37 ± 0.018.

[0122] We also investigated whether ThioLipo could improve blood TST concentrations in vivo. Due to the hydrophobic nature of TST, free TST was injected intraperitoneally, while LipoThio was injected intravenously. Because no sensitive method for detecting TST in blood or other tissues has been established, we analyzed TST concentrations using a TripleTOF 5600 ultra-performance liquid chromatography system. Mice treated with ThioLipo had significantly higher blood TST concentrations 24 hours later (Figure 13).

[0123] 8.2. Checking the effectiveness of ThioLipo (1) Syngeneic colon cancer model All animal experiments were approved by the Institutional Animal Care and Use Committee of the National Cancer Center (NCC-21-615). C57BL / 6 mice (6 weeks old; Orient Bio, Korea) were inoculated with MC38 cells (5 × 10 5 cells) were injected subcutaneously. The tumor size was approximately 150 mm 3When the mice reached 10 mg / kg, they were randomly assigned to treatment groups. Mice were treated with anti-4-1BB (10 mg / kg, n = 10) and anti-PD-1 (10 mg / kg, n = 9) antibodies by intraperitoneal injection on day 0 and every 2 days. ThioLipo was administered intravenously via the tail vein at a dose of 10 mg TST equivalent (TST) / kg on day 0 and every 2 days (n = 11). Mice in the anti-4-1BB + ThioLipo group (n = 10) were treated with anti-4-1BB antibody (10 mg / kg, intraperitoneal injection) and ThioLipo (10 mg TST equivalent / kg, intravenous injection) on day 0 and every 2 days. Mice in the anti-PD-1 + ThioLipo group (n = 8) were treated with anti-PD-1 antibody (10 mg / kg, intraperitoneal injection) and ThioLipo (10 mg TST equivalent / kg, intravenous injection) on day 0 and every 2 days. Control group mice (n = 9) were intravenously injected with PBS solution (100 μL) on day 0 and every 2 days. The mice were treated with antibody and ThioLipo a total of four times. The tumor volume and body weight of the mice were measured daily until day 8. The tumor volume (V) was determined by AB. 2 / 2 (A and B are the long and short diameters (mm), respectively). The antitumor synergistic effect of combined administration was determined by calculating the drug interaction index (CDI). CDI was calculated using the following formula: CDI = AB / (A × B), where AB is the tumor size ratio of the control group to the combination group, and A and B are the tumor size ratios of each treatment group to the control group. CDI values <1, <0.7, =1, and >1 indicate synergy, significant synergy, additivity, and antagonism, respectively.

[0124] On day 8, mice were euthanized, and tumor tissues and normal vital organs (heart, lungs, kidneys, spleen, and liver) were collected and fixed in 4% formaldehyde solution for immunohistochemistry (IHC) and histopathological analysis. Tissues were paraffin-embedded, sectioned, and stained. IHC analysis of CD3 (1:150, ab16669; Abcam) and PD-L1 (1:100, #13684; Cell Signaling Technology) levels in tumor tissue was performed from paraffin sections using a Ventana Roche Discovery XT Immunostainer (Mannheim, Germany) according to the DAB-ChromoMap Discovery Research standard procedure. Antigen retrieval was initiated by heat-induced epitope unlabeling while the slides were immersed, according to the manufacturer's instructions. Sections were incubated with primary antibodies at 37°C, followed by application of secondary antibodies at room temperature. Following the copper-enhanced diaminobenzidine development method, the sections were detected using the DAB ChromoMap detection kit, followed by counterstaining with hematoxylin for 4 minutes. The sections were then manually dehydrated, cleared in xylene, and mounted on coverslips. Furthermore, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) analysis was performed to assess cell death in the treatment groups. Histopathological analysis was performed by staining tissue sections with hematoxylin and eosin (H&E), and imaging of stained tissue sections was performed using a Vectra Polaris system (Akoya Biosciences). Cell death in normal tissues of the treatment groups was analyzed using the TUNEL analysis kit, compared with the control group.

[0125] (2) Serum biochemical analysis To analyze the potential toxicity of each treatment, serum samples were collected from each group on the final day of the experiment. Albumin (ALB), creatinine (CREA), total protein (TP), blood urea nitrogen (BUN), glucose (GLU), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), creatine phosphokinase (CPK), total bilirubin (T-Bil), triglycerides (TG), phosphate (PHOS), calcium (CA), blood cholesterol (CHO), and albumin / globulin ratio (A / G) were measured by TNPbio (Gwangju, Gyeonggi-do, Korea).

[0126] In all statistical analyses, differences between groups were assessed using Student's t-test. Statistical significance was set at P<0.05, and data were expressed as mean ± standard error.

[0127] (3) Confirmation of the efficacy of combined administration of immune checkpoint inhibitors and ThioLipos In a syngeneic colon cancer model, tumor size was 150 mm 3 Treatment began on day 0 when tumor volume reached 100 μg / mL (Figure 14a). ThioLipo was administered intravenously, and antibody treatments were administered intraperitoneally. Each treatment group was treated with ThioLipo (10 mg TST equivalent / kg), anti-4-1BB antibody (10 mg / kg), or anti-PD-1 antibody (10 mg / kg). Treatment with anti-4-1BB antibody, anti-PD-1 antibody, or ThioLipo alone reduced tumor volume by 24% (p<0.05), 39% (p<0.05), and 31% (p<0.05), respectively, compared to the control group (Figure 14b and Figure 14c).

[0128] If the tumor size is small (80 mm 3 ) is expected to show better therapeutic results with ICI treatment alone, but to confirm the effect of ICI and ThioLipos combination therapy in a rapidly growing tumor model, 3The experiment was initiated with tumors measuring 100 μg / kg. Notably, combination therapy with the anti-4-1BB + ThioLipo and anti-PD-1 + ThioLipo groups demonstrated synergistic antitumor effects, suppressing tumor growth by 56% (p<0.001) and 74% (p<0.001) in tumor-bearing mice compared with the control group. The calculated CDI values for the anti-4-1BB + ThioLipo and anti-PD-1 + ThioLipo groups were 0.83 and 0.61, respectively, and no differences in body weight were observed between the treatment groups.

[0129] The enhanced therapeutic effect of ThioLipos was further analyzed by staining tumor sections using a TUNEL assay kit and IHC on day 8 (Figure 15). Cell death in tumors increased 3.2-fold, 8.1-fold, and 7.7-fold in the ThioLipo-treated group (P<0.05), anti-4-1BB-treated group (p<0.001), and anti-PD-1-treated group, respectively, compared with the control group. Cell death in the anti-4-1BB + ThioLipo and anti-PD-1 + ThioLipo groups increased 2.4-fold and 2.7-fold compared with the anti-4-1BB-treated group (p<0.001) and anti-PD-1-treated group (p<0.01), respectively. IHC staining of tumor sections revealed CD3 + The results showed that T cells were increased 3.8-fold, 3.4-fold, and 4.7-fold in the ThioLipo-treated group (p<0.01), anti-4-1BB-treated group (p<0.001), and anti-PD-1-treated group (p<0.001), respectively, compared with the control group. The tumor-infiltrating CD3 T cells in the anti-4-1BB + ThioLipo and anti-PD-1 + ThioLipo groups were significantly increased. +T cell counts were increased 2.3-fold and 3.4-fold compared with the anti-4-1BB-treated group (P<0.01) and anti-PD-1-treated group (P<0.001), respectively. Furthermore, IHC staining for PD-L1 protein was performed on tumor sections to measure changes in PD-L1 levels. As expected, PD-L1 levels in tumor sections were significantly downregulated in ThioLipo-treated tumors (47.7% inhibition compared with the control group, p<0.001). PD-L1 levels in the combination treatment group were also significantly lower than in the ICI-treated group. Although anti-PD-1 treatment increased PD-L1 expression, combined treatment with anti-PD-1 antibody and ThioLipos significantly suppressed PD-L1 levels in tumor tissue.

[0130] To confirm the in vivo effects of each treatment group on normal tissues, i.e., the side effects of the therapeutic regimen, vital organs (heart, spleen, lungs, kidneys, and liver) were collected and sectioned at the end of the experiment. H&E and TUNEL staining of the tissue sections was performed, and blood samples from the mice were also collected and analyzed. As previously mentioned, no differences in body weight were observed between the treatment groups. TUNEL-stained images of normal tissue sections showed no increase in cell death in any of the treatment groups compared to the control group (Figure 16a). Furthermore, no histopathological changes were observed in H&E-stained images of normal tissue sections. Serum biochemical analysis also showed no signs of toxic effects in the treatment groups compared to the control group (Figure 16b).

[0131] Example 9: Confirmation of the effects of FDI-6 and RCM1 on lung cancer, colon cancer, and brain cancer cell lines 9.1. Cell Death, Viability, and Protein Expression Analysis (1) Reagents and cell cultures used in the experiments Dimethyl sulfoxide (DMSO), FDI-6 (Sigma-Aldrich, St. Louis, MO, USA, CAS Number: 313380-27-7), RCM1 (R&D Systems, Minneapolis, MN 55413, USA, CAS No: 339163-65-4), Amicon centrifugal filter device (MWCO 50 K, UFC805024, Merck Millipore), Sephadex G25 gel filtration column (PD-10; #17-0851-01, GE Healthcare), anti-PD-1 (clone: RMP1-14, BP0146), anti-4-1BB (clone: 17B5, BE0296) antibodies (BioXcell), anti-CD3 antibody (ab16669, Abcam), and anti-PD-L1 antibody (13684, Cell Signaling Technology).

[0132] Human lung cancer cell lines (H1299, PC9), colon cancer cell lines (HCT116, SW480), and brain tumor cell line (U118) were purchased from the American Type Culture Collection (Manassas, VA, USA). H1299, PC9, U118, and SW480 cells were cultured in RPMI (Roswell Park Memorial Institute) medium supplemented with 10% FBS and 1% penicillin / streptomycin. HCT116 cells were cultured in McCoy's 5A medium supplemented with 10% FBS and 1% penicillin / streptomycin. Cells were maintained at 37°C in a humidified incubator with 5% CO2.

[0133] (2) Immunoblotting For immunoblotting analysis, protein concentration was measured using a bicinchoninic acid kit (Cat. 23225; Thermo Fisher Scientific, Carlsbad, CA, USA), and proteins were separated by SDS-PAGE electrophoresis. Primary antibodies against FOXM1 (#ab207298, Abcam), PD-L1 (#14-5982-82, Invitrogen), and glyceraldehyde-3-phosphate dehydrogenase (#sc-25778, Santa Cruz Biotechnology) were added and incubated overnight at 4°C. After washing, the membrane was incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody, and protein bands were detected using a chemiluminescence kit (Thermo Fisher Scientific, Carlsbad, CA, USA).

[0134] (3) Analysis of PD-L1 expression in lung cancer cell lines treated with RCM1 To confirm the dose-dependent downregulation of PD-L1 expression by the FOXM1 inhibitor RCM1, live cell imaging of PD-L1 expression in H1299 lung cancer cell line cells was performed. H1299 cells were cultured in a Lab-Tek II and treated with RCM1 (10 μM) for 48 hours. Subsequently, the cells were incubated with an anti-PD-L1 antibody (#14-5982-82; eBioscience, San Diego, CA, USA) followed by an Alexa Fluor 594-conjugated secondary antibody (A21209; Thermo Fisher Scientific, Waltham, MA, USA). Nuclei were stained with Hoechst 33342 dye (Thermo Fisher Scientific, Waltham, MA, USA). The cells were washed three times with PBS (pH 7.4, 10 mM MgCl, 136 mM NaCl), and the medium was replaced with fresh complete medium. Nuclei (λ) were stained. ex 359 nm, λ em 457nm) and PD-L1 (λ ex 590nm, λ em 618 nm) fluorescence images were acquired using a confocal scanning laser microscope (LSM780; Carl Zeiss, Oberkochen, Germany).

[0135] 9.2. Effect of FDI-6 and RCM1 on PD-L1 expression in lung cancer, colon cancer, and brain cancer cells We analyzed the effects of FDI-6 and RCM1 on PD-L1 expression in lung cancer, colon cancer, and brain tumor cell lines. FDI-6 treatment significantly reduced both FOXM1 and PD-L1 expression in lung cancer cell lines (PC9) and colon cancer cell lines (SW480) (Figures 17a and 17b). Furthermore, RCM1 treatment significantly reduced FOXM1 and PD-L1 expression in lung cancer cell lines (H1299), colon cancer cell lines (HCT116), and brain tumor cell lines (U118) (Figure 18a). Confocal fluorescence imaging analysis also confirmed that PD-L1 levels on the cancer cell surface were reduced after RCM1 treatment (Figure 18b). We confirmed that FOXM1 directly binds to the nuclear PD-L1 promoter and selectively upregulates PD-L1 expression, and that the FOXM1 inhibitors FDI-6 and RCM1 reduce PD-L1 expression by inhibiting FOXM1 in human lung cancer, colon cancer, and brain cancer cells.

[0136] From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting. The scope of the present invention should be interpreted as including all modifications and variations derived from the meaning and scope of the claims below, and equivalent concepts thereof, rather than the above detailed description.

Claims

1. A pharmaceutical composition for preventing or treating cancer, comprising a FOXM1 inhibitor and an immune checkpoint inhibitor as active ingredients.

2. 2. The pharmaceutical composition of claim 1, wherein the FOXM1 inhibitor is at least one selected from the group consisting of thiazolidinedione, diarylheptanoid, RCM-1, thiostrepton, honokiol, FDI-6, siomycin A, monensin, FOXM1 Apt, and peptide 9R-P201.

3. 2. The pharmaceutical composition of claim 1, wherein the immune checkpoint inhibitor is at least one or more selected from the group consisting of an anti-PD-L1 antibody, an anti-PD-1 antibody, an anti-CTLA4 antibody, an anti-PD-L2 antibody, an LTF2 regulatory antibody, an anti-LAG3 antibody, an anti-A2aR antibody, an anti-TIGIT antibody, an anti-TIM-3 antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-VISTA antibody, an anti-CD47 antibody, an anti-BTLA antibody, an anti-KIR antibody, an anti-IDO antibody, and an anti-4-1BB antibody.

4. The pharmaceutical composition of claim 1, wherein the composition reduces the expression of PD-L1.

5. 2. The pharmaceutical composition of claim 1, wherein the FOXM1 inhibitor is carried in a liposome.

6. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is for combined administration of a FOXM1 inhibitor and an immune checkpoint inhibitor.

7. The pharmaceutical composition according to claim 6, wherein the pharmaceutical composition is in the form of a mixture of the FOXM1 inhibitor and the immune checkpoint inhibitor, or the FOXM1 inhibitor and the immune checkpoint inhibitor are formulated separately and administered simultaneously or sequentially.

8. 2. The pharmaceutical composition of claim 1, wherein the cancer is lung cancer, pancreatic cancer, pharyngeal cancer, laryngeal cancer, esophageal cancer, gastric cancer, duodenal cancer, small intestine cancer, large intestine cancer, colon cancer, rectal cancer, breast cancer, ovarian cancer, prostate cancer, liver cancer, thyroid cancer, kidney cancer, uterine cancer, brain cancer, skin cancer, melanoma, malignant bone tumor, bladder cancer, or blood cancer.

9. An anti-cancer adjuvant containing a FOXM1 inhibitor and an immune checkpoint inhibitor as active ingredients.

10. 10. The anticancer adjuvant according to claim 9, wherein the FOXM1 inhibitor is at least one selected from the group consisting of thiazolidinedione, diarylheptanoid, RCM-1, thiostrepton, honokiol, FDI-6, siomycin A, monensin, FOXM1 Apt, and peptide 9R-P201.

11. 10. The anti-cancer adjuvant of claim 9, wherein the immune checkpoint inhibitor is at least one selected from the group consisting of anti-PD-L1 antibody, anti-PD-1 antibody, anti-CTLA4 antibody, anti-PD-L2 antibody, LTF2 regulatory antibody, anti-LAG3 antibody, anti-A2aR antibody, anti-TIGIT antibody, anti-TIM-3 antibody, anti-B7-H3 antibody, anti-B7-H4 antibody, anti-VISTA antibody, anti-CD47 antibody, anti-BTLA antibody, anti-KIR antibody, anti-IDO antibody, and anti-4-1BB antibody.

12. Use of a composition comprising a FOXM1 inhibitor and an immune checkpoint inhibitor for the prevention or treatment of cancer.

13. Use of a composition comprising a FOXM1 inhibitor and an immune checkpoint inhibitor for the manufacture of a medicament for the prevention or treatment of cancer.

14. A method for preventing or treating cancer, comprising administering to an individual a composition comprising a FOXM1 inhibitor and an immune checkpoint inhibitor.

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