New application of compound Dinaciclib

Abstract

The invention belongs to the field of biological medicine, and particularly relates to novel application of a compound Dinaciclib. The invention finds that Dinaciclib not only can selectively inhibit the DNA replication of tumor cells to kill the tumor cells, but also can reduce immunosuppressive myeloid cell subgroups by inhibiting the expression of a trigger receptor 2 (TREM2) expressed by myeloid cells, promote the amplification of cytotoxic T lymphocytes to remodel a tumor microenvironment and activate immune response to indirectly kill the tumor cells. The invention discloses Dinaciclib which has immunoregulation and chemical treatment functions at the same time and can achieve the purpose of chemical immunotherapy on tumors. Therefore, the technical problems that pure chemical treatment and immunotherapy are poor in anti-tumor effect and many in toxic and side effects, and an existing chemical immunotherapy method is tedious in step and lacks a single small-molecule chemical immunotherapy agent are solved.

Description

Technical Field

[0001] This application belongs to the field of biomedicine, and particularly relates to a new application of the compound Dinaciclib. Background Art

[0002] Cancer is a major cause threatening human health and causing global population deaths. Immunotherapy exerts an anti-tumor effect by regulating the systemic immune system and is considered the most promising cancer treatment method. However, since tumor cells inhibit their immunogenic characteristics through immune checkpoints and induce an immunosuppressive tumor microenvironment (TME), the T cells are damaged by immunosuppressive myeloid cells, resulting in the current immunotherapy drugs being effective for only less than a quarter of cancer patients.

[0003] Myeloid cells are an important part of the tumor microenvironment and inhibit T cell responses through multiple mechanisms. Tumor-infiltrating myeloid cells include immune-stimulating and immunosuppressive subsets. Immune-stimulating myeloid cells include M1 macrophages and type 1 dendritic cells (DC1s). Suppressive myeloid cells include M2 macrophages and myeloid-derived suppressor cells (MDSCs). Immunosuppressive myeloid cells are considered the main obstacle to cancer immunotherapy and can disrupt the immune attack of cytotoxic T lymphocytes (CTLs) on tumor cells. Triggering receptors expressed on myeloid cells 2 (TREM2) expressed by myeloid cells is a new potential anti-tumor treatment target, which is highly expressed on the vast majority of primary and metastatic tumor-infiltrating macrophages, inhibits the activity of T cells, and is a marker of tumor-infiltrating macrophages. Blocking TREM2 will reduce the level of tumor-infiltrating suppressive macrophages, inhibit the accumulation of regulatory myeloid cells, and enhance the anti-tumor activity of CD8 + T cells, which is beneficial to the clearance or inhibition of tumors. Therefore, regulating the level of TREM2 can reshape the tumor microenvironment, eliminate suppressive myeloid cells or induce myeloid cells with immune-stimulating characteristics, and enhance the anti-tumor activity of T cells, which is an anti-tumor immunotherapy strategy with important application prospects. In addition, certain chemical drugs may induce immunogenic cell death (ICD) in tumor cells, which is characterized by the migration of calreticulin (CRT) from inside the dead cells to the plasma membrane surface, the release of high-mobility group protein B1, ATP, and IFN to the extracellular space, etc. The exposure of CRT on the cell membrane surface can promote the maturation of dendritic cells, process and present tumor-associated antigens, and recognize and phagocytose tumor cells, thereby activating a series of specific anti-tumor immune responses in the body. Therefore, inducing ICD is also an important anti-tumor immunotherapy strategy for stimulating DC maturation, activating effector T cells, and clearing tumor cells.

[0004] Chemotherapy immunotherapy is a treatment method that combines chemotherapy with immunomodulatory therapy. Chemotherapy immunotherapy has the characteristics of synergistic treatment. On the one hand, it directly kills tumor cells through the chemotherapy mechanism. On the other hand, it indirectly kills tumor cells by activating the immune response and prevents tumor recurrence and metastasis by remodeling the tumor microenvironment. It can reduce the dosage of drugs and enhance the therapeutic effect, and is a promising tumor treatment method. The chemotherapeutic immunotherapeutic agent OPA is a new type of platinum-based anticancer complex composed of oxaliplatin (OP) and artesunate (ART), which has significant antitumor activity. It can not only directly kill cancer cells by damaging DNA and inhibiting DNA repair, but also promote the maturation and proliferation of immune cells by inhibiting the expression of TREM2, reducing the number of immunosuppressive myeloid cell subsets, thereby improving the chemotherapy efficiency of platinum drugs.

[0005] However, as a chemotherapy drug, common side effects of oxaliplatin (OP) include peripheral neuropathy, nausea, vomiting, myelosuppression, etc., and these problems may be caused by long-term use. Although artesunate (ART) has good anti-cancer effects, it may also cause side effects such as hepatotoxicity and gastrointestinal discomfort in some cases. The combined use of the two may increase the toxicity burden. However, common implementation schemes of chemotherapy immunotherapy are the combination of immunocyte therapy and chemotherapy, or the combination of an immunomodulator (such as an immune checkpoint inhibitor) and a chemotherapeutic drug. They all act independently, with different pharmacokinetic properties and in vivo distributions, inconsistent action targets, asynchronous action times, and uncontrollable drug ratios at the tumor site. Therefore, serious systemic side effects often occur, affecting the synergistic anti-tumor effect.

[0006] Therefore, there is still a need for a chemotherapeutic immunotherapeutic agent with good safety that can damage tumor cell DNA and inhibit the activity of TREM2 expression. Summary of the Invention

[0007] In view of this, the present application discloses a new application of the compound Dinaciclib. The present application discovers that Dinaciclib has the functions of inhibiting the expression of the TREM2 gene and inhibiting DNA replication, and can be used as a single-molecule chemotherapeutic immunotherapeutic agent for anti-tumor.

[0008] The first aspect of the present application discloses the application of the compound Dinaciclib in the preparation of a TREM2 inhibitor, and the structural formula of the compound Dinaciclib is shown as (I); 。

[0009] The second aspect of the present application discloses the use of the compound Dinaciclib as a TREM2 inhibitor in the preparation of a medicament for treating tumors; the structural formula of the compound Dinaciclib is shown as (I); 。

[0010] The third aspect of the present application discloses the use of the compound Dinaciclib as a TREM2 inhibitor and a DNA replication inhibitor in the preparation of a medicament for treating tumors; the structural formula of the compound Dinaciclib is shown as (I); 。

[0011] Preferably, the tumors include prostate cancer, lung cancer, colorectal cancer, ovarian cancer, gastric cancer, pancreatic cancer, breast cancer, liver cancer, head and neck squamous cell carcinoma, glioma, melanoma, bladder cancer, cervical cancer, gallbladder cancer, osteoma, osteosarcoma, thyroid cancer, salivary gland cancer, esophageal cancer, glioblastoma, thymoma or endometrial cancer.

[0012] Furthermore, preferably, the treatment of tumors includes: triggering and / or enhancing anti-tumor immune responses as a TREM2 inhibitor, and inhibiting tumor cell DNA replication through chemical action.

[0013] Furthermore, preferably, the enhancement of anti-tumor responses is achieved through one or several of the following aspects: 1) Inhibiting the expression of the TREM2 gene in macrophages derived from bone marrow; 2) Inducing apoptosis in tumor cells such as RM1, MC38, LLC, etc., and inhibiting tumor cell proliferation and migration; 3) Inducing the polarization of tumor-promoting M2-type tumor-associated macrophages in the tumor microenvironment into a tumor-suppressive M1 phenotype; 4) Reducing the level of myeloid-derived suppressor cells in the tumor microenvironment; 5) Increasing the infiltration of CD8+ T lymphocytes into the tumor; 6) Enhancing the secretion of anti-tumor granzyme B and perforin by immune-stimulatory CD8 + T lymphocytes; 7) Remodeling or activating the tumor immune microenvironment.

[0014] Preferably, the myeloid-derived suppressor cells include Mo-MDSC and PMN-MDSC.

[0015] The fourth aspect of the present application discloses the use of the compound Dinaciclib as a TREM2 inhibitor in the preparation of a medicament for treating prostate cancer.

[0016] The fifth aspect of the present application discloses the use of the compound Dinaciclib as a TREM2 inhibitor in the preparation of a drug for treating colon cancer.

[0017] The sixth aspect of the present application discloses the use of the compound Dinaciclib as a TREM2 inhibitor in the preparation of a drug for treating lung cancer.

[0018] The present invention discovers that: Dinaciclib can directly kill tumor cells by inhibiting DNA replication, and can also indirectly kill tumor cells by inhibiting the expression of Triggering receptors expressed on myeloid cells 2 (TREM2) in myeloid cells, reducing immunosuppressive myeloid cell subsets, promoting the expansion of cytotoxic T lymphocytes, remodeling the tumor microenvironment and activating the immune response. That is, Dinaciclib has both immunomodulatory and chemotherapeutic functions, and can achieve the purpose of chemoimmunotherapy for tumors. Thus, it solves the technical problems of poor anti-tumor effects and many toxic and side effects in simple chemotherapy and immunotherapy, and the cumbersome steps of existing chemoimmunotherapy methods, and the lack of a single small molecule chemoimmunotherapy agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art.

[0020] Figure 1 Showing the chemical structural formula of the compound Dinaciclib; Figure 2 Showing the inhibitory effect (48 h) of Dinaciclib (10 nM, 20 nM, 30 nM, 40 nM, 50 nM / 20 nM) on the expression of TREM2 and its downstream signaling pathway key molecule p-Syk in bone marrow-derived macrophages (BMDM), (A) Immunoblotting; (B) RT-qPCR; (C) and (D) Immunofluorescence; Figure 3 Showing the inhibitory effect of Dinaciclib (40 mg / kg) on the expression of TREM2 in tumor tissues of RM1 prostate cancer, MC38 colon cancer, and LLC lung adenocarcinoma mouse models; Figure 4 Showing the cytotoxic effect of Dinaciclib on various cancer cells and normal cells; Figure 5 Showing the binding effect of Dinaciclib (20 nM) to TREM2 in BMDM, cell thermal shift assay (A - B); Figure 6Show the effects of Dinaciclib (10 nM, 20 nM, 30 nM) on apoptosis of RM1 prostate cancer cells (A - B), MC38 colon cancer cells (C - D), and LLC lung adenocarcinoma cells (E - F) after 24 - hour culture; Figure 7 Show the effects of Dinaciclib (10 nM, 20 nM, 30 nM) on cell proliferation of RM1 prostate cancer cells (A, B), MC38 colon cancer cells (A, C), and LLC lung adenocarcinoma cells (A, D) after 10 - day culture; Figure 8 Show the inhibitory effects of Dinaciclib (40 mg / kg) on RM1 prostate cancer (A - B), MC38 colon cancer (C - D), and LLC lung adenocarcinoma (E - F) in C57BL / 6 mice; Figure 9 Show the effects of Dinaciclib (40 mg / kg) on subsets of M1 macrophages (A - B), M2 macrophages (C - D), Mo - MDSC (E - F), and PMN - MDSC (G - H) in the RM1 prostate cancer xenograft tumor tissues of C57BL / 6 mice; Figure 10 Show the immunostimulatory effects of Dinaciclib (40 mg / kg) on CD8 + T cells in the RM1 prostate cancer xenograft tumor tissues of C57BL / 6 mice. Detailed implementation manners

[0021] This application provides a new application of the compound Dinaciclib, and provides a chemotherapeutic agent with good safety that can both damage tumor cell DNA and inhibit the activity of TREM2 expression.

[0022] The technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this application, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in this application without creative efforts shall fall within the protection scope of this application.

[0023] Among them, the compound Dinaciclib used in the following examples was purchased from Shanghai TargetMol Co., Ltd., and the chemical structural formula of Dinaciclib is as Figure 1 .

[0024] Example 1 This example tests the inhibitory ability of Dinaciclib on the expression of TREM2 and its downstream signaling pathway key molecule p-Syk in bone marrow-derived macrophages (BMDMs). The specific steps are as follows: 1. Collection of test samples: After BMDMs are treated with different concentrations of Dinaciclib for 48 h, proteins, mRNAs, and cells are collected for testing. The specific steps are as follows: (1) Inoculate BMDMs (1×10 5 cells / well) into 6-well plates, culture at 37 °C for 12 h, add Dinaciclib (10 nM, 20 nM, 30 nM, 40 nM, 50 nM), incubate at 37 °C for 48 h, lyse the cells with protein lysate to collect proteins, and add 5×SDS loading buffer to boil and denature. Perform SDS-PAGE electrophoresis, transfer the proteins to a PVDF membrane, block with 5% milk, incubate with TREM2 antibody (Anti-TREM-2, clone 78, MABN755, Merck millipore), P-SYK antibody (Anti-Syk(phospho Y352) antibody [EPR26232-39], ab300398, Abcam), and actin antibody (MonoclonalAnti-β-Actin antibody, A5316, Sigma-Aldrich) overnight at 4 °C; the next day, add the corresponding secondary antibody and incubate at room temperature for 1.5 h, and observe the exposure through a LAS500 ultrasensitive chemiluminescence imager (GE ImageQuant, USA). The results are as Figure 2 shown in A of

[0025] (2) Inoculate BMDMs (5×10 4 cells / well) into 12-well plates, culture at 37 °C for 12 h, add Dinaciclib (20 nM), incubate at 37 °C for 48 h, collect total RNA with TRIZOL, extract RNA by chloroform extraction method, and detect the transcriptional level of TREM2 by reverse transcription quantitative real-time PCR (RT-qPCR). The results are as Figure 2 shown in B of

[0026] (3) Inoculate BMDMs (1×10 5Cells (at a density of [X] cells / well) were seeded into confocal dishes and cultured at 37 °C for 12 h. Then, Dinaciclib (20 nM) was added and the cells were incubated at 37 °C for 48 h. The cells were fixed and blocked with 0.5% BSA. The primary antibody against TREM2 (Anti-TREM-2, clone 78, MABN755, Merck millipore) was incubated overnight at 4 °C. The next day, the corresponding secondary antibody was added and incubated for 1 h. Fluorescence observation was performed using a LSM-710 (Zeiss, Germany) confocal microscope equipped with DAPI and AF594 filter sets. The results are shown in Figure 2 C and D in

[0027] 2. Experimental results: Figure 2 The WB results in A of show that Dinaciclib has a concentration-dependent inhibitory effect on the expression of macrophage TREM2 and the key downstream signaling molecule P-Syk. The expression of TREM2 and P-Syk can be inhibited at a concentration of 10 nM and above. Figure 2 The RT-qPCR results in B of show that Dinaciclib (20 nM) inhibits the transcriptional level of TREM2 by nearly 70%. Figure 2 The immunofluorescence results in C and D of show that Dinaciclib (20 nM) inhibits the protein level of TREM2 by approximately 70%, which is close to the TREM2KO level. This indicates that Dinaciclib has the activity to inhibit TREM2 and is a key pharmacophore.

[0028] Example 2 This example tests the inhibitory effect of Dinaciclib on the expression of TREM2 in tumor tissues of mouse models (RM1 prostate cancer, MC38 colon cancer, LLC lung adenocarcinoma).

[0029] 1. Collection of test samples, specifically including the following steps: (1) PBS suspensions of RM1 prostate cells, MC38 colon cancer cells, and LLC lung adenocarcinoma cells were subcutaneously injected into the groin of mice at a density of 5×10 5 cells per mouse. When the tumor cells grew to 50 mm 3 in diameter 5 days after implantation, the mice were randomly grouped. Dinaciclib (40 mg / kg) was administered intraperitoneally every 2 days until the tumor diameter of the mice approached 20 mm and the experiment was terminated.

[0030] (2) After the administration, immunohistochemical analysis was used to evaluate the effect of Dinaciclib on the expression level of TREM2 in tumor tissues of mouse models of RM1 prostate cancer, MC38 colon cancer, and LLC lung adenocarcinoma. The results are shown in Figure 3 .

[0031] 2. Experimental results: For RM1 prostate cancer without Dinaciclib treatment ( Figure 3 A in Figure 3 ), MC38 colon cancer ( Figure 3 B in Figure 3 ), and LLC lung adenocarcinoma ( Figure 3 C in Figure 3 ), the tumor tissues were rich in macrophages overexpressing TREM2, indicating an immunosuppressive microenvironment that is conducive to tumor growth. After treatment with Dinaciclib, the brown regions of TREM2 in the tumor tissues of RM1 prostate cancer (

[0032] Example 3 This example tested the cytotoxicity of Dinaciclib against various cancer cells and normal cells.

[0033] 1. Collection of test samples: iBMDM (immortalized BMDM) cells, RM1 prostate cancer cells, MC38 colon cancer cells, and LLC lung adenocarcinoma cells after treatment with different concentrations of Dinaciclib for 48 h.

[0034] 2. Detection method, specifically including the following steps: (1) Inoculate iBMDM cells, RM1 prostate cancer cells, MC38 colon cancer cells, and LLC lung adenocarcinoma cells (5×10 4 cells / well, resuspended in 100 μL PBS) into 6-well plates and culture at 37°C for 12 h until adherent.

[0035] (2) Add Dinaciclib (10 nM, 20 nM, 30 nM, 50 nM, 100 nM) to iBMDM cells, and add Dinaciclib (10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 80 nM, 100 nM) to RM1 prostate cancer cells, MC38 colon cancer cells, and LLC lung adenocarcinoma cells, and incubate at 37°C for 48 h.

[0036] (3) Add 10 μL of CCK8 reagent to each well, continue to culture at 37°C for 4 h, and then measure the absorbance of the solution at 450 nm using a microplate reader. The results are as Figure 4 .

[0037] 3. Experimental results: Dinaciclib at a concentration of 20 nM and above had an effect on RM1 prostate cancer cells ( Figure 4in B), LLC lung adenocarcinoma cells ( Figure 4 in C), MC38 colorectal adenocarcinoma cells ( Figure 4 in D) all had strong toxicity, while at concentrations of 10 nM and 20 nM, they had no obvious effect on the activity of macrophages ( Figure 4 in A). That is, Dinaciclib has high selectivity and strong killing effect on tumor cells, and low toxicity to normal cells.

[0038] Example 4 This example tests the binding ability of Dinaciclib to TREM2 in macrophages.

[0039] 1. Collection of test samples: The protein of BMDM cells after being treated with Dinaciclib (20 nM) for 3 h.

[0040] 2. Detection method, which specifically includes the following steps: (1) Inoculate BMDM (1×10 5 cells / well) into a 6-well plate, culture at 37 °C for 12 h, add Dinaciclib (20 nM), incubate at 37 °C for 3 h, digest, wash with PBS, and then resuspend in cold PBS (1.2 mL) supplemented with protease inhibitor.

[0041] (2) Each group of cells is divided into 11 groups (100 μL of cell suspension in each group), and one group is not treated and placed on ice (as the initial value, untreated). The remaining 10 groups are heat-treated at 37 °C, 41 °C, 45 °C, 49 °C, 53 °C, 57 °C, 61 °C, 65 °C, 69 °C, and 73 °C for 3 minutes respectively, and then restored to room temperature for 3 min.

[0042] (3) Subsequently, perform three cycles of freezing and thawing in liquid nitrogen. After each thawing, shake well. Place the lysed cells on ice. Shake well, transfer the cell lysate to a 1.5 mL centrifuge tube, centrifuge at 4 °C and 20000×g for 20 min, and place the centrifuged sample on ice.

[0043] (4) Collect the supernatant, add 5×SDS loading buffer, boil for denaturation. Perform SDS-PAGE electrophoresis, transfer the protein to a PVDF membrane, block with 5% milk, incubate with TREM2 antibody (Anti-TREM-2, clone 78, MABN755, Merckmillipore) overnight at 4 °C; the next day, add the corresponding secondary antibody and incubate at room temperature for 1.5 h, and observe the exposure through a LAS500 ultrasensitive chemiluminescence imager (GE ImageQuant, USA). The results are as Figure 5 shown.

[0044] 3. Experimental results: Figure 5 From the results of the cellular thermal shift assay, it can be seen that Dinaciclib treatment of BMDM cells effectively altered the thermal stability of TREM2, indicating that Dinaciclib has the ability to bind to the TREM2 protein in cells.

[0045] Example 5 This example tests the effect of Dinaciclib on apoptosis of various cancer cells.

[0046] 1. Collection of test samples: RM1 prostate cancer cells, LLC lung adenocarcinoma cells, and MC38 colon cancer cells treated with different concentrations of Dinaciclib.

[0047] 2. Detection method, specifically including the following steps: (1) Inoculate RM1 prostate cancer cells, LLC lung adenocarcinoma cells, and MC38 colon cancer cells at a density of 2×10 5 cells per well in a 6-well plate and culture at 37°C for 12 h until they adhere. Treat the cells with Dinaciclib (10 nM, 20 nM, 30 nM) and incubate at 37°C for 48 h.

[0048] (2) Collect the cells by trypsin digestion and wash them with PBS. Centrifuge and precipitate at 500 g for 5 min, discard the supernatant, resuspend the cells in buffer (500 μL), stain them with PI and Annexin V in the dark for 30 min, and analyze apoptosis by flow cytometry. The results are as Figure 6 shown.

[0049] 3. Experimental results: Dinaciclib induced 26.04%, 11.18%, and 15.23% of RM1 prostate cancer cells ( Figure 6 A - B in Figure 6 ), LLC lung adenocarcinoma cells ( Figure 6 C - D in Figure 6 ), and MC38 colon cancer cells ( Figure 6 E - F in Figure 6 ) to enter early and late apoptosis at 20 nM; and induced 74.0%, 44.2%, and 40.8% of RM1 prostate cancer cells ( Figure 6 A - B in Figure 6 ), LLC lung adenocarcinoma cells ( Figure 6 C - D in

[0050] Example 6 This example tests the effect of Dinaciclib on the proliferation of various cancer cells.

[0051] 1. Collection of test samples: RM1 prostate cancer cells, MC38 colon cancer cells, and LLC lung adenocarcinoma cells treated with different concentrations of Dinaciclib.

[0052] 2. Detection method, specifically including the following steps: (1) Inoculate RM1 prostate cancer cells, MC38 colon cancer cells, and LLC lung adenocarcinoma cells in a 6-well plate at a density of 500 cells per well and culture at 37 °C for 12 h until they adhere. Treat the cells with Dinaciclib (10 nM, 20 nM, 30 nM) and culture at 37 °C for 10 d. Change the medium every 3 days and observe the cell status during the process until the number of cells in each clone is greater than 50.

[0053] (2) After cloning, take pictures of the cells under a microscope, then wash them once with PBS, add 1 mL of 4% paraformaldehyde to each well and fix for 30 - 60 min, and wash once with PBS.

[0054] (3) Add 1 mL of crystal violet staining solution to each well and stain the cells for 10 - 20 min.

[0055] (4) Wash the cells several times with PBS, air dry, and take pictures with a digital camera (take pictures of the entire 6-well plate and each well separately), and the results are as Figure 7 shown.

[0056] 3. Experimental results: Dinaciclib at 10 nM, 20 nM, and 30 nM can significantly inhibit the colony formation of RM1 prostate cancer cells ( Figure 7 A and B in), MC38 colon cancer cells ( Figure 7 A and C in), and LLC lung adenocarcinoma cells ( Figure 7 A and D in). The inhibition rate is about 50% at 20 nM and about 80% at 20 nM, confirming that Dinaciclib has the ability to inhibit the proliferation of tumor cells.

[0057] Example 7 This example tests the anti-tumor activity of Dinaciclib in mice.

[0058] 1. Collection of test samples: Tumor tissues of RM1 prostate cancer, MC38 colon cancer, and LLC lung adenocarcinoma mouse models treated with Dinaciclib (40 mg / kg).

[0059] 2. Detection method, specifically including the following steps: (1) The PBS suspensions of RM1 prostate cancer cells, MC38 colon cancer cells, and LLC lung adenocarcinoma cells were subcutaneously injected into the inguinal regions of wild-type (WT) and TREM2 knockout (TREM2 KO) mice at a dose of 5×10 5 cells per mouse. Five days after the implantation of tumor cells, when the tumors grew to 50 mm 3 , the mice were randomly grouped: Dinaciclib (40 mg / kg) (RM1 prostate cancer mice were first castrated by CTX) was administered intraperitoneally every 2 days until the tumor diameter of the mice approached 20 mm and the experiment was terminated.

[0060] (2) After the administration, the mice were sacrificed, and the tumor tissues were dissected, photographed with a digital camera, and weighed. The results are shown as follows. Figure 8 Shown.

[0061] 3. Experimental results: Dinaciclib can effectively inhibit the weights of prostate cancer ( Figure 8 A-B in), colorectal cancer ( Figure 8 C-D in), and lung cancer tumors ( Figure 8 E-F in) in WT mice to 0.20 g, 0.30 g, and 0.25 g, while the average tumor weights of the control groups were 0.77 g, 0.84 g, and 0.94 g. In TREM2 KO mice, Dinaciclib can further inhibit the weights of prostate cancer, colorectal cancer, and lung cancer tumors to 0.06 g, 0.11 g, and 0.08 g, while the average tumor weights of the control groups were 0.53 g, 0.48 g, and 0.58 g. In castration-resistant prostate cancer mice, Dinaciclib can effectively inhibit the tumor weight of WT mice to 0.10 g, while the tumor weight of the enzalutamide (ENZA) administration group was 0.40 g; in TREM2 KO mice, Dinaciclib further inhibited the tumor weight of mice to 0.03 g, while the tumor weight of the ENZA administration group was 0.12 g. It shows that Dinaciclib has a significant inhibitory effect on prostate cancer, colorectal cancer, and lung cancer, and the combination of TREM2 knockout and Dinaciclib further enhances the anti-tumor effect. Moreover, the anti-tumor effect of Dinaciclib on castration-resistant prostate cancer is stronger than that of ENZA.

[0062] Example 8 This example tests the effect of Dinaciclib on the tumor immune microenvironment of mice.

[0063] 1. Collection of test samples: Tumor tissues of RM1 prostate cancer mouse models after treatment with Dinaciclib (40 mg / kg).

[0064] 2. Detection method, specifically including the following steps: (1) Inject the PBS suspension of RM1 prostate cancer cells subcutaneously into the inguinal regions of wild-type (WT) and TREM2 knockout (TREM2 KO) mice at a dose of 5×10 5 cells per mouse. When the tumor cells grow to 50 mm3 5 days after implantation, randomly divide the mice into groups: Dinaciclib (40 mg / kg) (RM1 prostate cancer mice are first subjected to castration surgery CTX) is administered intraperitoneally every 2 days until the tumor diameter of the mice approaches 20 mm and the experiment is terminated.

[0065] (2) After the administration is completed, sacrifice the mice, collect the tumor tissues, and prepare single-cell suspensions by mechanical grinding. Subsequently, collect the cell suspensions by centrifugation at a speed of 1500 rpm for 5 min at low temperature, add RBC lysis buffer at room temperature for 5 min to remove red blood cells, wash the cells twice with cold PBS, and resuspend them in 100 μL of PBS suspension.

[0066] (3) Specific antibody staining reaction: The 100 μL reaction system contains: 1×10 5 - 1×10 7 cells of single tumor tissue cells, 100 μL of PBS, CD45 antibody (PEcy7-anti-mouse CD45, 100 tests, Biolegend), isotype control antibody IgG (IgG1 Iso Control-Pacific Blue / IgG1 Iso Control-PE, 100 tests, Biolegend) 3 μL, CD11B antibody (FITC-anti-mouse CD11B, 100 tests, Biolegend) 2 μL, F4 / 80 antibody (APC-anti-mouse F4 / 80, 100 tests, Biolegend) 2 μL, CD86 antibody (FITC-anti-mouseCD86, 100 tests, Biolegend) 2 μL, CD206 antibody (Pecy7-anti-mouse CD206, 100 tests, Biolegend) 2 μL, Ly6C antibody (PC5.5-anti-mouse Ly6C, 100 tests, Biolegend) 2 μL, Ly6G antibody (APCcy7-anti-mouse Ly6G, 100 tests, Biolegend) 2 μL. Incubate the above reaction system on ice (0 - 4 °C) in the dark for 30 min, add 1 mL of PBS, and centrifuge at 1500 rpm for 5 min; (4) Resuspend the cells by adding 500 μL of PBS and detect using a flow cytometer; (5) Result determination: Use the isotype control antibody as the negative control to judge non-specific staining. If there is no obvious staining effect, it means that non-specific staining can be ignored, and then count the percentage of positive cells.

[0067] 3. Experimental results: The results are as Figure 9 ( Figure 9 In A - B in Figure 9 the proportion of M1 macrophages, Figure 9 in C - D in Figure 9 the proportion of M2 macrophages, + in E - F in + the proportion of Mo - MDSC cells, + and in G - H in + the proportion of PMN - MDSC cells). Dinaciclib can effectively reduce the proportions of M2 macrophages (CD11B + Ly6C + ), Mo - MDSC (CD11B + Ly6G + ), PMN - MDSC (CD11B + CD86 + in the tumor - infiltrating cells of prostate cancer in WT mice to 63.0%, 18.5%, 19.0%, while those in the WT group are 89.1%, 31.4%, 34.4% respectively. In TREM2 KO mice, Dinaciclib can further inhibit the proportions of M2 macrophages, Mo - MDSC, PMN - MDSC in the tumor - infiltrating cells of prostate cancer to 48.1%, 10.1%, 12.4%, while those in the TREM2 KO group are 70.5%, 21.5%, 21.1% respectively. In castration - resistant prostate cancer mice, Dinaciclib can effectively inhibit the proportions of M2 macrophages, Mo - MDSC, PMN - MDSC in the tumor - infiltrating cells to 50.4%, 11.6%, 16.0%, while those in the ENZA - administered group are 75.6%, 24.3%, 23.6%; in TREM2 KO mice, Dinaciclib further inhibits the proportions of M2 macrophages, Mo - MDSC, PMN - MDSC in the tumor - infiltrating cells to 39.1%, 8.31%, 10.8%, while those in the ENZA - administered group are 51.0%, 13.9%, 15.0%. In addition, Dinaciclib can effectively increase the proportion of M1 macrophages (CD11BThe proportion reached 17.0% in the Dinaciclib group, while it was 9.14% in the WT group. In TREM2 KO mice, Dinaciclib further increased the proportion of tumor-infiltrating M1 macrophages in prostate cancer to 29.2%, while it was 14.0% in the TREM2 KO group. In castration-resistant prostate cancer mice, Dinaciclib effectively increased the proportion of tumor-infiltrating M1 macrophages to 27.6%, while it was 10.6% in the ENZA-treated group; in TREM2 KO mice, Dinaciclib further increased the proportion of tumor-infiltrating M1 macrophages to 40.3%, while it was 16.9% in the ENZA-treated group. This indicates that Dinaciclib improved the tumor immunosuppressive microenvironment, specifically manifested as Dinaciclib reducing M2 macrophages and increasing M1 macrophages in tumor tissues, while also reducing Mo-MDSC and PMN-MDSC in tumor tissues, which will decrease the immunosuppressive activity against CD8 + cytotoxic T cells.

[0068] Example 9 This example tested the anti-tumor function of Dinaciclib on tumor-infiltrating CD8 + T cells in mice.

[0069] 1. Collection of test samples: Tumor tissues of RM1 prostate cancer mouse models after treatment with Dinaciclib (40 mg / kg).

[0070] 2. Detection method, specifically including the following steps: (1) Inject a PBS suspension of RM1 prostate cancer cells subcutaneously into the inguinal regions of wild-type (WT) and TREM2 knockout (TREM2 KO) mice at a dose of 5×10 5 cells per mouse. When the tumor cells grew to 50 mm after 5 days of implantation, the mice were randomly grouped: Dinaciclib (40 mg / kg) (RM1 prostate cancer mice were first subjected to castration surgery CTX) was administered by intraperitoneal injection every 2 days until the tumor diameter of the mice approached 20 mm and the experiment was terminated. 3

[0071] (2) After the administration ended, the mice were sacrificed, the tumor tissues were collected, and single-cell suspensions were prepared by mechanical grinding. Subsequently, the cell suspensions were collected by low-speed centrifugation at 1500 rpm for 5 min, RBC lysis buffer was added at room temperature for 5 min to remove red blood cells, and the cells were washed twice with cold PBS and resuspended in 100 μL of PBS suspension.

[0072] (3) Specific surface antibody staining reaction: The 100 μL reaction system contained: 1×10 5 ~1×10​7 Cells, 100 μL of PBS, 3 μL of CD45 antibody (PEcy7-anti-mouse CD45, 100 tests, Biolegend), 3 μL of isotype control antibody IgG (IgG1 Iso Control-Pacific Blue / IgG1 Iso Control-PE, 100 tests, Biolegend), 2 μL of CD3 antibody (Pacific Blue-anti-mouse CD3, 100 tests, Biolegend), and 2 μL of CD8 antibody (FITC-anti-mouse CD8, 100 tests, Biolegend). Incubate the above reaction system on ice (0 - 4 °C) in the dark for 30 min, add 1 mL of PBS, and centrifuge at 1500 rpm for 5 min; (4) Incubate the cells with surface staining completed with a fixation and permeabilization solution (eBioscience™ Flow Cytometry Intracellular Fixation Buffer) at 4 °C for 50 min, add 1 mL of PBS, and centrifuge at 2000 rpm for 5 min; (5) Specific intracellular antibody staining reaction: The 100 μL reaction system contains: single cells from tumor tissue 5 ~1×10 7 cells, 100 μL of PBS, 2 μL of Granzyme B antibody (PE-anti-mouse Granzyme B, 100 tests, Biolegend), and 2 μL of Perforin antibody (APC-anti-mouse Perforin, 100 tests, Biolegend). Incubate the above reaction system on ice (0 - 4 °C) in the dark for 45 min, add 1 mL of PBS, and centrifuge at 2000 rpm for 5 min; (6) Resuspend the cells with 500 μL of PBS and detect with a flow cytometer; (7) Result determination: Use the isotype control antibody as negative to judge non-specific staining. If there is no obvious staining effect, it means that non-specific staining can be ignored, and then count the percentage of positive cells.

[0073] 3. Experimental results: The results are as Figure 10 ( Figure 10 where A - B in Figure 10 represents the proportion of Granzyme B in different groups, and A - B in +The proportions of Granzyme B and Perforin secreted by T cells reached 16.0% and 10.4%, while those in the WT group were 4.93% and 5.71%, respectively. In TREM2 KO mice, Dinaciclib could further increase the proportion of prostate cancer tumor-infiltrating CD8 + T cells secreting Granzyme B and Perforin to 19.0% and 14.3%, while those in the TREM2 KO group were 10.0% and 9.42%, respectively. In castration-resistant prostate cancer mice, Dinaciclib could effectively increase the proportion of tumor-infiltrating CD8 + T cells secreting Granzyme B and Perforin to 20.9% and 15.1%, while those in the ENZA-administered group were 11.2% and 9.85%; in TREM2 KO mice, Dinaciclib further increased the proportion of tumor-infiltrating CD8 + T cells secreting Granzyme B and Perforin to 31.0% and 19.5%, while those in the ENZA-administered group were 13.9% and 11.3%. This indicates that Dinaciclib improved the tumor immunosuppressive microenvironment, specifically manifested as Dinaciclib increasing the proportion of Granzyme B and Perforin secreted by CD8 + T cells and increasing CD8 + The anti-tumor immune activity of cytotoxic T cells.

[0074] In summary, the present invention provides a TREM2 inhibitor for tumor chemoimmunotherapy, namely Dinaciclib, which can not only directly kill tumor cells through chemical action, but also indirectly kill tumor cells by reducing immunosuppressive myeloid cell subsets through TREM2 expression, promoting the maturation and expansion of immune-stimulating cells, remodeling the tumor microenvironment, and activating the immune response. That is, the TREM2 inhibitor provided by the present invention has both immunomodulatory and chemotherapeutic functions, and can achieve the purpose of single-molecule chemoimmunotherapy for tumors.

[0075] The above are only the preferred embodiments of the present application. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present application, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present application.

Claims

1. Use of the compound Dinaciclib in the preparation of a TREM2 inhibitor, characterized in that the structural formula of the compound Dinaciclib is as shown in (I); 。 2. Use of the compound Dinaciclib as a TREM2 inhibitor in the preparation of a medicament for treating tumors; the structural formula of the compound Dinaciclib is as shown in (I); 。 3. Use of the compound Dinaciclib as a TREM2 inhibitor and a DNA replication inhibitor in the preparation of a medicament for treating tumors; the structural formula of the compound Dinaciclib is as shown in (I); 。 4. The application according to claim 2 or 3, characterized in that, the tumors include prostate cancer, lung cancer, colorectal cancer, ovarian cancer, gastric cancer, pancreatic cancer, breast cancer, liver cancer, head and neck squamous cell carcinoma, glioma, melanoma, bladder cancer, cervical cancer, gallbladder cancer, osteoma, osteosarcoma, thyroid cancer, salivary gland cancer, esophageal cancer, glioblastoma, thymoma or endometrial cancer.

5. The application according to claim 4, characterized in that, The treatment of tumors includes: triggering and / or enhancing an anti-tumor immune response as a TREM2 inhibitor, and inhibiting tumor cell DNA replication through chemical action.

6. The application according to claim 5, wherein The enhancement of the anti-tumor response is achieved through one or more of the following aspects 1) to 7): 1) Inhibiting the expression of the TREM2 gene in macrophages derived from bone marrow; 2) Inducing apoptosis in tumor cells such as RM1, MC38, LLC, etc., and inhibiting the proliferation and migration of tumor cells; 3) Inducing the polarization of tumor-promoting M2-type tumor-associated macrophages in the tumor microenvironment into a tumor-suppressive M1 phenotype; 4) Reducing the level of myeloid-derived suppressor cells in the tumor microenvironment; 5) Increase CD8 + T lymphocytes infiltrate into the tumor; 6) Enhanced immunostimulatory CD8 + T lymphocytes secrete the anti-tumor granzyme B and perforin; 7) Remodeling or activating the tumor immune microenvironment.

7. The application according to claim 6, wherein The myeloid-derived suppressor cells include Mo-MDSC and PMN-MDSC.

8. Use of the compound Dinaciclib as a TREM2 inhibitor in the preparation of a medicament for treating prostate cancer.

9. Use of the compound Dinaciclib as a TREM2 inhibitor in the preparation of a medicament for treating colon cancer.

10. Use of the compound Dinaciclib as a TREM2 inhibitor in the preparation of a medicament for treating lung cancer.

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