Use of a composition of a TACC2 inhibitor and a CDK2 inhibitor in the preparation of a medicament for preventing or treating tumors

Abstract

The present invention discloses the application of a composition of a TACC2 inhibitor and a CDK2 inhibitor in the preparation of a drug for preventing or treating tumors. Through TCGA, GEO public databases, and in vitro and in vivo functional experiments, the present invention identifies that TACC2 is lowly expressed in various tumors, and tumor cells and tissues with low TACC2 expression are extremely sensitive to CDK2 inhibitors. Therefore, based on the expression level of TACC2 in tumors, it can be used as a diagnostic and treatment target for tumor prognosis judgment, and can effectively guide the targeted treatment of CDK2 inhibitors in tumor patients, thereby performing individualized treatment, prolonging the survival period of patients, and improving the quality of life of patients.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of a composition of a TACC2 inhibitor and a CDK2 inhibitor in the preparation of a drug for preventing or treating tumors. Background Art

[0002] With the development of society, changes in people's living environment and eating habits, etc., malignant tumors have become one of the top killers of human health. In recent years, the number of patients has been increasing globally, bringing a heavy burden to the development of the global economy and human society. Precision cancer treatment is the current development trend of cancer diagnosis and treatment. Currently, molecular markers are used for diagnosis and as therapeutic targets in various tumors, benefiting cancer patients.

[0003] Cell cycle disorder is one of the main characteristics of tumor malignancy. Currently, CDK4 / 6 inhibitors have been well applied in various tumors. A large number of studies have shown that the activation of CDK2 in various tumors is one of the main reasons for the malignant progression of tumors. However, there is currently a lack of effective diagnostic indicators for CDK2-targeted therapy. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of a composition of a TACC2 inhibitor and a CDK2 inhibitor in the preparation of a drug for preventing or treating tumors.

[0005] The technical solution adopted by the present invention is as follows:

[0006] In the first aspect of the present invention, there is provided the application of a composition of a TACC2 inhibitor and a CDK2 inhibitor in the preparation of a drug for preventing or treating tumors.

[0007] In some embodiments of the present invention, the TACC2 inhibitor is a substance that inhibits or reduces the transcription or translation of the TACC2 gene, or a substance that can inhibit the normal biological function of the TACC2 protein.

[0008] In some embodiments of the present invention, the TACC2 inhibitor is selected from one or more of dsRNA, shRNA, microRNA, siRNA, an inhibitor of the gene promoter, a compound, a peptide, and an antibody.

[0009] In some embodiments of the present invention, the CDK2 inhibitor is a substance that inhibits or reduces the transcription or translation of the CDK2 gene, or a substance that can inhibit the normal biological function of the CDK2 protein.

[0010] In some embodiments of the present invention, the CDK2 inhibitor is selected from one or more of dsRNA, shRNA, microRNA, siRNA, an inhibitor of the gene promoter, a compound, a peptide, and an antibody.

[0011] In some embodiments of the present invention, the CDK2 inhibitor is preferably Dinaciclib, NU2508, K03861, SU9516, JNJ-7706621.

[0012] In some embodiments of the present invention, the tumor is clear cell renal carcinoma, papillary renal cell carcinoma, low-grade glioma, squamous cell carcinoma of the lung, soft tissue cancer, endometrial cancer, esophageal cancer, preferably esophageal cancer.

[0013] In a second aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating a tumor, the active ingredients of which include a TACC2 inhibitor and a CDK2 inhibitor;

[0014] In some embodiments of the present invention, the TACC2 inhibitor is a substance that inhibits or reduces the transcription or translation of the TACC2 gene, or a substance that can inhibit the normal biological function of the TACC2 protein.

[0015] In some embodiments of the present invention, the TACC2 inhibitor is selected from one or more of dsRNA, shRNA, microRNA, siRNA, an inhibitor of a gene promoter, a compound, a peptide, and an antibody.

[0016] In some embodiments of the present invention, the CDK2 inhibitor is a substance that inhibits or reduces the transcription or translation of the CDK2 gene, or a substance that can inhibit the normal biological function of the CDK2 protein.

[0017] In some embodiments of the present invention, the CDK2 inhibitor is selected from one or more of dsRNA, shRNA, microRNA, siRNA, an inhibitor of a gene promoter, a compound, a peptide, and an antibody.

[0018] In some embodiments of the present invention, the CDK2 inhibitor is preferably Dinaciclib, NU2508, K03861, SU9516, JNJ-7706621.

[0019] In some embodiments of the present invention, the tumor is clear cell renal carcinoma, papillary renal cell carcinoma, low-grade glioma, squamous cell carcinoma of the lung, soft tissue cancer, endometrial cancer, esophageal cancer, preferably esophageal cancer.

[0020] In a third aspect of the present invention, there is provided the use of a TACC2 inhibitor in the preparation of a drug for enhancing the anti-tumor effect of a CDK2 inhibitor.

[0021] In a fourth aspect of the present invention, there is provided a pharmaceutical preparation for synergistically enhancing the anti-tumor effect of a CDK2 inhibitor, and the active ingredient of the pharmaceutical preparation comprises a TACC2 inhibitor.

[0022] In some embodiments of the present invention, the above-mentioned drug can be introduced into the body, such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue, by injection, spraying, nasal dropping, eye dropping, osmosis, absorption, physical or chemical mediated methods; or can be introduced into the body after being mixed or encapsulated with other substances.

[0023] When needed, one or more pharmaceutically acceptable carriers can also be added to the above-mentioned drug. The carriers include conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, etc. in the pharmaceutical field.

[0024] The drug can be made into various forms such as injection solutions, tablets, powders, granules, capsules, oral liquids, ointments, creams, etc. The drugs in the above various dosage forms can all be prepared according to the conventional methods in the pharmaceutical field.

[0025] In a fifth aspect of the present invention, there is provided a TACC2 inhibitor, and the inhibitor is siRNA, and the sequences are as shown in SEQ ID NO.1 and SEQ ID NO.2.

[0026] In a sixth aspect of the present invention, there is provided a product, and the product comprises the TACC2 inhibitor described in the seventh aspect of the present invention.

[0027] In some embodiments of the present invention, the product is a drug.

[0028] In a seventh aspect of the present invention, there is provided the use of TACC2 as a target in the preparation of a detection reagent for the sensitivity of tumors to drugs, and the drug is a CDK2 inhibitor.

[0029] In some embodiments of the present invention, the tumor is clear cell renal carcinoma, papillary renal cell carcinoma, low-grade glioma, lung squamous cell carcinoma, soft tissue carcinoma, endometrial carcinoma, esophageal carcinoma, preferably esophageal carcinoma.

[0030] In an eighth aspect of the present invention, there is provided the use of a reagent for detecting TACC2 in the preparation of a detection reagent for the sensitivity of tumors to drugs, and the drug is a CDK2 inhibitor.

[0031] The beneficial effects of the present invention are:

[0032] Through public databases such as TCGA and GEO, as well as in vitro and in vivo functional experiments, the present invention identified that TACC2 is lowly expressed in various tumors, and tumor cells and tissues with low TACC2 expression are extremely sensitive to CDK2 inhibitors. Therefore, based on the expression level of TACC2 in tumors, it can be used as a diagnostic and treatment target for tumor prognosis judgment, and can effectively guide the targeted treatment of CDK2 inhibitors in tumor patients, thereby enabling individualized treatment, prolonging the survival period of patients, and improving the quality of life of patients. Brief Description of the Drawings

[0033] Figure 1 TACC2 is lowly expressed in various tumors.

[0034] Figure 2 Low expression of TACC2 is associated with poor tumor prognosis. Figure 2 A shows the expression of TACC2 in esophageal cancer identified by immunohistochemistry; Figure 2 B shows that low expression of TACC2 is significantly correlated with poor overall survival and poor disease-free survival in patients with esophageal cancer; Figure 2 C shows that low expression of TACC2 is associated with poor prognosis in various tumors (clear cell renal carcinoma, papillary renal cell carcinoma, low-grade glioma, lung squamous cell carcinoma, soft tissue carcinoma, endometrial carcinoma).

[0035] Figure 3 Sensitivity of low TACC2 expression to the CDK2 inhibitor Dinaciclib. Figure 3 A shows that the expression level of TACC2 in various tumors is correlated with the sensitivity to the CDK2 inhibitor Dinaciclib; Figure 3 B shows that the low TACC2 expression group (KYSE140, KYSE410, KYSE180, and KYSE510) is more sensitive to the CDK2 inhibitor Dinaciclib; Figure 3 C& Figure 3 E shows that interfering with TACC2 increases the sensitivity of cells to the inhibitor Dinaciclib; Figure 3 D& Figure 3 F shows that expressing TACC2 reduces the sensitivity to the inhibitor Dinaciclib.

[0036] Figure 4 Cell experiments found that low TACC2 expression is sensitive to various CDK2 inhibitors. Among them Figure 4 The CDK2 inhibitor in A is Dinaciclib; Figure 4 The CDK2 inhibitor in B is SU9516; Figure 4 The CDK2 inhibitor in C is NU2058; Figure 4 The inhibitor in D is JNJ-7706621; Figure 4 The inhibitor in E is K03861.

[0037] Figure 5 Animal experiments confirmed that low expression of TACC2 is sensitive to the CDK2 inhibitor Dinaciclib. Figure 5 A shows the effects of intratumoral injection (ITI) of TACC2-siRNA and intraperitoneal injection (IP) of the CDK2 inhibitor dinaciclib on esophageal cancer cells in a subcutaneous tumor model; Figure 5 B shows the inhibitory effect of dinaciclib on the tumor volume of esophageal cancer treated with TACC2-siRNA; Figure 5 C shows the morphological diagram of the inhibitory effect of dinaciclib on the tumors of esophageal cancer treated with TACC2-siRNA;

[0038] Figure 5 D shows the inhibitory effect of dinaciclib on the tumor weight of esophageal cancer treated with TACC2-siRNA; Figure 5 E shows the inhibitory effect of dinaciclib on the cell proliferation of esophageal cancer treated with TACC2-siRNA. Detailed implementation manners

[0039] The following will clearly and completely describe the concept and technical effects of the present invention in combination with embodiments to fully understand the purpose, features, and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts shall fall within the scope of protection of the present invention.

[0040] Example 1

[0041] The applicant analyzed the expression data of various tumors in the TCGA (The Cancer Genome Atlas) database and performed bioinformatics analysis, and found that TACC2 was significantly lowly expressed in various tumors ( Figure 1 ), where BLCA: bladder cancer, BRCA: breast cancer, COAD: colon adenocarcinoma, HNSC: head and neck squamous cell carcinoma, KIRC: renal clear cell carcinoma, LUAD: lung adenocarcinoma, LUSC: lung squamous cell carcinoma, PRAD: prostate adenocarcinoma, READ: rectal adenocarcinoma, SKCM: cutaneous melanoma, THCA: thyroid cancer, UCEC: endometrial cancer, UCS: uterine carcinosarcoma.

[0042] Example 2

[0043] The applicant identified the expression of TACC2 in esophageal cancer by immunohistochemistry ( Figure 2A), 184 esophageal cancer patients were divided into a TACC2 low-expression group (102 cases) and a TACC2 high-expression group (82 cases). Combining the prognostic information of esophageal cancer patients, survival prognosis analysis was performed. The results showed that in esophageal cancer patients, low expression of TACC2 was significantly correlated with poor overall survival and poor disease-free survival ( Figure 2 B).

[0044] Further analyzing the data of the TCGA (The Cancer Genome Atlas) database, the results showed that low expression of TACC2 was associated with poor prognosis in multiple tumors (clear cell renal carcinoma, papillary renal cell carcinoma, low-grade glioma, lung squamous cell carcinoma, soft tissue carcinoma, endometrial carcinoma). Figure 2 C).

[0045] The specific steps for detecting TACC2 expression by immunohistochemistry are as follows:

[0046] 1) Obtain paraffin-embedded tissue samples of esophageal cancer;

[0047] 2) Cut 4-μm paraffin-embedded tissue sections, and successively bake the sections in an oven at 60 °C for 3 h, dewax with xylene and gradient alcohol, and remove endogenous peroxidase with hydrogen peroxide; immerse in a citric acid antigen retrieval solution (pH 6.0) and boil in a microwave for 2.5 minutes;

[0048] 3) After blocking with 5% BSA at 37 °C for 30 minutes, add the TACC2 antibody (diluted at a concentration of 1:1000, Proteintech, catalog number 11407-1-AP) and incubate overnight at 4 °C;

[0049] 4) After washing 3 times with PBST, add the secondary antibody (DAKO, Santa Clara, CA, USA) to the sections, place in a wet box, and incubate in an incubator at 37 °C for 30 minutes;

[0050] 5) After washing 3 times with PBST, develop color with 3,3’-diaminobenzidine (DAB) for 2 minutes;

[0051] 6) Terminate with water, counterstain with hematoxylin, differentiate with hydrochloric acid alcohol, and rinse with water for 2 hours, then dehydrate at 37 °C;

[0052] 7) Finally, mount the sections with a neutral balsam mounting medium and take pictures;

[0053] 8) The TACC2 expression score was independently evaluated by two pathologists. The scoring rules are as follows: a) Proportion of positive cells: 0 (none), 1 (1%-10%), 2 (11%-50%), 3 (51%-75%), and 4 (76%-100%); b) Fluorescence intensity of positive cells: 0 (none), 1 (weak), 2 (medium), and 3 (strong); Then, the proportion and intensity are multiplied to obtain the total score (0-12). The average score of the two pathologists is the final score. The final score of 0-6 indicates low TACC2 expression; a score of 7-12 indicates high TACC2 expression.

[0054] 9) Through the follow-up department, the medical records and prognostic information of the patients corresponding to the specimens were queried, and R and SPSS software were used for analysis.

[0055] Example 3

[0056] The applicant analyzed the CCLE public database and found that the expression level of TACC2 was correlated with the sensitivity to the CDK2 inhibitor Dinaciclib in multiple tumor cells (esophageal cancer, pancreatic cancer, melanoma, head and neck tumors, osteosarcoma) ( Figure 3 A); According to the TACC2 expression level, esophageal cancer cell lines were divided into two groups. The relatively high TACC2 expression group (EC18, KYSE30, and ECA109) and the low TACC2 expression group (KYSE140, KYSE410, KYSE180, and KYSE510) were more sensitive to the CDK2 inhibitor Dinaciclib ( Figure 3 B). By interfering with TACC2 with siRNA in breast cancer cell BT549 and pancreatic cancer cell PANC1, and detecting the sensitivity to the CDK2 inhibitor Dinaciclib, it was found that compared with the control (siNC target sequence: UCUCCGAACGUGUCACGUU (SEQ ID NO.3)), interfering with TACC2 (siTACC2#1 target sequence: CGAAUGUUUUGGACUUAAC (SEQ ID NO.1); siTACC2#2 target sequence: GCCGACCUCUUCAGAAGAU (SEQ ID NO.2)) increased the sensitivity of cells to the inhibitor Dinaciclib ( Figure 3 C&E); Overexpressing TACC2 in melanoma cell A375, melanoma cell SK-MEL-110, pancreatic cancer cell PANC1, and pancreatic cancer cell CAPAN2, and detecting the sensitivity to the CDK2 inhibitor Dinaciclib, it was found that overexpressing TACC2 decreased the sensitivity to the inhibitor Dinaciclib ( Figure 3 D&F).

[0057] Among them, the cell experiment for the sensitivity of the CDK2 inhibitor:

[0058] 1) Seed 3×10 3 cells / 200 μL into a 96-well plate;

[0059] 2) Add CDK2 inhibitor in gradients;

[0060] 3) Incubate for 96 hours;

[0061] 4) Finally, add 20 μL of MTT reagent to each well and incubate at 37 °C for 4 hours;

[0062] 5) Aspirate the culture medium, add 200 μL of DMSO to each well to dissolve for 15 minutes, and finally detect the value with an ELISA reader at OD = 490;

[0063] 5) Statistically analyze the sensitivity of cells to CDK2 inhibitor.

[0064] Example 4

[0065] Furthermore, by using siRNA to interfere with TACC2 in esophageal cancer cells KYSE30 and ECA109, it was found that after knocking down TACC2, and then adding CDK2 inhibitor, a drug sensitivity test was carried out. The MTT results showed that interfering with TACC2 made esophageal cancer cells more sensitive to a variety of CDK2 inhibitors( Figure 4 ), and the CDK2 inhibitors used included Dinaciclib( Figure 4 A), SU9516( Figure 4 B), NU2058( Figure 4 C), JNJ-7706621( Figure 4 D), K03861( Figure 4 E), and had the same effect. Among them, Dinaciclib is a CDK1 / 2 / 5 / 9 inhibitor, NU2058 is a CDK1 / 2 inhibitor, K03861 is a CDK2 inhibitor, SU9516 is a CDK1 / 2 / 4 inhibitor, and JNJ-7706621 is a CDK1 / 2, aurora-A / B inhibitor.

[0066] Example 5

[0067] To explore the therapeutic effect of CDK2 inhibitor on TACC2-low-expressing tumors at the in vivo animal level, the applicant used a subcutaneous tumor model and subcutaneously inoculated KYSE30 cells into nude mice. Considering that siRNA may have high clinical translation potential, the applicant studied the effect of intratumoral injection (ITI) of TACC2-siRNA and intraperitoneal injection (IP) of CDK2 inhibitor dinaciclib on esophageal cancer cells in a subcutaneous tumor model( Figure 5A). The applicant observed that compared with the siNC control group, the growth and tumor weight of xenograft tumors treated with TACC2-siRNA ITI increased; in addition, the inhibitory effect of dinaciclib on esophageal cancer tumors treated with TACC2-siRNA was much higher than that on xenografts treated with control-siRNA( Figure 5 B & C & D). The applicant also found that in the treatment group using dinaciclib and TACC2-siRNA ITI in combination, the TUNEL fluorescence staining was the strongest in all groups, indicating the highest apoptosis rate of tumor cells( Figure 5 E). These data suggest that the combination therapy of TACC2-siRNA and dinaciclib may benefit cancer patients with ESCC.

[0068] Among them, the animal experiment on CDK2 inhibitor sensitivity:

[0069] 1) Resuspend the cells at a concentration of 2×10 6 cells per 100 μL in serum-free DMEM medium containing 20% Matrigel (BD Biosciences);

[0070] 2) Subcutaneously inject 100 μL (2×10 6 cells) of tumor cells into nude mice;

[0071] 3) Starting from the 5th day, inject siNC (UCUCCGAACGUGUCACGUU (SEQ ID NO.3)) or siTACC2 (siTACC2#1: CGAAUGUUUUGGACUUAAC (SEQ ID NO.1); siTACC2#2: GCCGACCUCUUCAGAAGAU (SEQ ID NO.2)) modified with 2OMe + 5Chol intratumorally every 2 days;

[0072] 4) Starting from the 5th day, inject the CDK2 inhibitor dinaciclib (16 mg / kg / ) intraperitoneally once a day;

[0073] 5) Measure the tumor size every day and end the experiment on the 21st day. Weigh the tumors, take pictures of the tumors, statistically analyze the tumor weights, and use GraphPad to plot the tumor growth curve.

[0074] In summary, through the TCGA, GEO public databases, and in vitro and in vivo functional experiments, the applicant identified that TACC2 is lowly expressed in various tumors, and tumor cells and tissues with low TACC2 expression are highly sensitive to CDK2 inhibitors.

[0075] Therefore, TACC2 is lowly expressed in various tumors, and the combination of low expression of TACC2 and CDK2 inhibitor achieves a synthetic lethal effect, which can be used for precision cancer treatment.

[0076] The above specific embodiments have described the present invention in detail. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those of ordinary skill in the art, various changes can be made without departing from the gist of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of a composition of a TACC2 inhibitor and a CDK2 inhibitor in the preparation of a drug for preventing or treating tumors; The TACC2 / CDK2 inhibitor is a substance that inhibits or reduces the transcription or translation of the TACC2 / CDK2 gene, or a substance that can inhibit the normal biological function of the TACC2 / CDK2 protein; The TACC2 inhibitor is siRNA, and the sequences are as shown in SEQ ID NO.1 and SEQ ID NO.2; The tumor is esophageal cancer; The CDK2 inhibitor is Dinaciclib.

2. A pharmaceutical composition, characterized in that Its active ingredients include a TACC2 inhibitor and a CDK2 inhibitor; The TACC2 inhibitor is siRNA, and the sequences are as shown in SEQ ID NO.1 and SEQ ID NO.2; The CDK2 inhibitor is Dinaciclib.

3. Use of a TACC2 inhibitor in the preparation of a drug for enhancing the anti-tumor effect of a CDK2 inhibitor; The TACC2 inhibitor is siRNA, and the sequences are as shown in SEQ ID NO.1 and SEQ ID NO.2; The tumor is esophageal cancer; The CDK2 inhibitor is Dinaciclib.

4. A pharmaceutical preparation for synergistically enhancing the anti-tumor effect of a CDK2 inhibitor, characterized in that The active ingredients of the pharmaceutical preparation include a TACC2 inhibitor; The TACC2 inhibitor is siRNA, and the sequences are as shown in SEQ ID NO.1 and SEQ ID NO.2; The CDK2 inhibitor is Dinaciclib; The tumor is esophageal cancer.

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