Application of SUMOylation inhibitor in preparation of drugs for treating chronic myelogenous leukemia and TKI drug resistance of chronic myelogenous leukemia

Abstract

The invention discloses application of an SUMO inhibitor 2-D08 in preparation of drugs for treating chronic myelogenous leukemia (CML) and drug resistance of a tyrosine kinase inhibitor (TKI) of the SUMO inhibitor 2-D08. It is revealed for the first time that SUMOylation modification drives disease progression by stabilizing BCR-ABL protein, 2-D08 promotes BCR-ABL protein degradation by inhibiting the modification, and leukemia cell proliferation is remarkably inhibited. In-vitro experiments prove that the compound effectively inhibits the activity of TKI drug-resistant cells, and in-vivo experiments show that 5 mg / kg of the compound can improve liver and spleen infiltration of CML mice. The inhibitor provides a new scheme for overcoming drug resistance, and fills the application blank of SUMOylation modification in CML treatment.

Description

Technical Field

[0001] The present invention belongs to the field of drugs for blood diseases, and particularly relates to the application of SUMOylation inhibitors in the preparation of drugs for treating chronic myeloid leukemia and its TKI resistance. Background Art

[0002] Chronic Myeloid Leukemia (CML) is a myeloproliferative neoplasm characterized by the translocation of the ABL1 gene on chromosome 9 and the BCR gene on chromosome 22, t(9;22)(q34;q11), forming the BCR-ABL fusion gene. The BCR-ABL protein encoded by the BCR-ABL fusion gene is the core driver of CML. Through its constitutive tyrosine kinase activity with abnormal activation, it forms a multi-pathway synergistic carcinogenic network, including the PI3K / AKT / mTOR pathway, the RAS / MAPK pathway, and the JAK / STAT pathway, etc., leading to uncontrolled cell proliferation, apoptosis inhibition, and genomic instability, driving the malignant phenotype of leukemia cells.

[0003] Tyrosine kinase inhibitors (TKIs) that target and inhibit the tyrosine kinase activity of BCR-ABL are the first choice for the treatment of CML, such as imatinib, nilotinib, dasatinib, etc. Although they are highly effective in treating patients in the chronic phase of CML, their efficacy in the accelerated phase and blast crisis is poor, and drug resistance and recurrence are common problems during the treatment process. Once drug resistance occurs, the prognosis of patients deteriorates significantly.

[0004] The reasons for CML drug resistance are divided into two categories. One is the BCR-ABL kinase-dependent mechanism, and the other is the mechanism independent of BCR-ABL kinase activity. Mutations in the BCR-ABL kinase domain are the main cause of drug resistance, which inhibits the binding of TKIs to the BCR-ABL protein. In addition, gene amplification of BCR-ABL leading to overexpression of the fusion protein is also a cause of drug resistance. The effect of TKIs on leukemia stem cells in CML patients is not obvious, which leaves a hidden danger for recurrence after TKI treatment. This type of drug resistance is independent of BCR-ABL kinase activity. The mechanism of action of TKIs is to inhibit the activation of tyrosine kinases, but it does not affect the overall protein expression of BCR-ABL, so it cannot completely eliminate abnormal leukemia cells. Therefore, exploring and discovering regulatory molecules that directly target the expression of BCR-ABL protein is of great significance for analyzing new targets and new mechanisms for the treatment of CML.

[0005] SUMOylation is a key post-translational protein modification that dynamically regulates protein structure, interactions, and activity by covalently attaching small ubiquitin-like modifier (SUMO) to lysine residues of target proteins. In tumors, SUMOylation affects cell proliferation, apoptosis, metabolism, and genomic stability by regulating oncogenes, tumor suppressor genes, and signaling pathways. However, its role in chronic myeloid leukemia has not been fully elucidated, especially the regulatory mechanism of the core driver BCR-ABL remains a research gap. In particular, the potential role of the SUMOylation inhibitor 2-D08 in CML treatment remains unclear and warrants further investigation. Summary of the Invention

[0006] Existing TKIs cannot effectively eliminate BCR-ABL protein and are prone to drug resistance. There is an urgent need to develop a new treatment strategy that directly targets the expression of BCR-ABL protein.

[0007] To address the above technical deficiencies, the present invention provides the use of a SUMOylation inhibitor in the preparation of a drug for the treatment of chronic myeloid leukemia and its TKI resistance. The present invention for the first time reveals that SUMOylation modification drives the progression of CML by stabilizing BCR-ABL protein and verifies the therapeutic value of SUMOylation inhibitors.

[0008] The present invention is achieved by the following technical solutions:

[0009] The present invention provides the use of a SUMOylation inhibitor in the preparation of a drug for the treatment of chronic myeloid leukemia and its tyrosine kinase inhibitor (TKI) resistance. The SUMOylation inhibitor promotes the degradation of BCR-ABL protein by inhibiting the SUMOylation modification of BCR-ABL protein, thereby downregulating the expression level of BCR-ABL protein and inhibiting the proliferation of leukemia cells.

[0010] Preferably, the SUMOylation inhibitor is compound 2-D08 with the following structural formula:

[0011]

[0012] Preferably, the effective dose of compound 2-D08 in the drug is: 5 - 50 μM for in vitro application and 5 mg / kg for in vivo application.

[0013] Preferably, the drug is used to treat chronic myeloid leukemia resistant to TKI.

[0014] The present invention provides a drug for the treatment of chronic myeloid leukemia, comprising compound 2-D08 as claimed in claim 2 and a pharmaceutically acceptable carrier. The drug is an injection or a tablet.

[0015] The beneficial effects of the present invention are as follows: The present invention provides the application of SUMOylation inhibitors in the preparation of drugs for treating chronic myeloid leukemia and its TKI resistance. It provides a new strategy for patients with TKI resistance. Different from the previous pharmacodynamic mechanism of tyrosine kinase inhibitors, it can directly target and inhibit the protein expression of the pathogenic fusion gene BCR-ABL, thus potentially curing the disease fundamentally.

[0016] In vitro and in vivo experiments have confirmed that 2-D08 significantly downregulates the expression level by inhibiting the SUMOylation modification of BCR-ABL protein, thereby inhibiting the proliferation of leukemia cells and the progression of the disease in the mouse model. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 a-d show the cell viability of CML cell lines treated with 2-D08 at concentration gradients (0, 5, 10, 25, 50 μM) and time gradients (0, 6, 12, 24, 48 hours) detected by the CCK-8 method. Figure 1 e-f show the proliferation of CML cell lines after treatment with 50 μM 2-D08 for 24 hours detected by EdU.

[0018] Figure 2 a-b show the enlargement of the liver and spleen after intraperitoneal injection of PBS (control group) or 2-D08 into CML mice. Figure 2 c-d show the infiltration of leukemia cells in the liver and spleen detected by immunohistochemical staining.

[0019] Figure 3 a shows the cell viability of drug-resistant cells treated with different concentrations of 2-D08 (0, 5, 10, 25, 50 μM) for 24 hours detected by the CCK-8 method. Figure 3 b shows the proliferation of drug-resistant cells after treatment with 50 μM 2-D08 for 24 hours detected by EdU. Figure 3 c shows the cell viability of primary cells after treatment with 50 μM 2-D08 for 24 hours detected by the CCK-8 method.

[0020] Figure 4 a-b show the enlargement of the liver and spleen after intraperitoneal injection of PBS (control group) or 2-D08 into the mouse model of primary cell xenografts. Figure 4 c-d show the infiltration of leukemia cells in the liver and spleen of the mouse with primary cell xenografts detected by immunohistochemical staining.

[0021] Figure 5 a-b show the protein expression of BCR-ABL and the activation of its downstream molecules in CML cell lines after treatment with 50 μM 2-D08 for 24 hours detected by Western blot. DETAILED DESCRIPTION OF THE INVENTION

[0022] Example 1

[0023] 1. Materials and methods:

[0024] 1.1 Cell culture

[0025] The human leukemia cells K562 and MEG-01 were from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. The K562 / G01 cells were a gift from Professor Liu Jing of Nanchang University. The K562 and MEG-01 cells used in this study were cultured in RPMI 1640 medium containing 10% fetal bovine serum, and the K562 / G01 cells were cultured in IMDM medium containing 10% fetal bovine serum at 37 °C in an incubator with 5% CO 2 . Bone marrow mononuclear cells were obtained from chronic myeloid leukemia patients by density gradient centrifugation using lymphocyte separation medium.

[0026] 1.2 CCK-8 assay

[0027] An appropriate amount of cells was seeded in a 96-well plate to ensure uniform cell distribution and avoid differences between wells, and pre-cultured in an incubator at 37 °C with 5% CO 2 . 10 μL of CCK-8 solution was added to each well (ensuring that the final volume was 10% of the medium volume), and the culture plate was gently shaken to evenly distribute the reagent and avoid generating bubbles, followed by incubation in the incubator for 1 - 4 hours. Observe the color change: the higher the cell activity, the deeper the orange-yellow color. The absorbance was measured at a wavelength of 450 nm using a microplate reader.

[0028] 1.3 EdU assay

[0029] EdU reagent was added to the cell medium and incubated for 2 - 4 hours. The cells were plated on slides, fixed with 4% paraformaldehyde for 30 minutes, and then stained with the Cell Light TM EDU Apollo488 imaging kit. A fluorescence microscope was used for image capture and ImageJ for analysis.

[0030] 1.4 Immunohistochemical staining

[0031] Paraffin-embedded slides were dewaxed, dehydrated, and antigen retrieval was performed, followed by adding an appropriate amount of endogenous peroxidase blocker. Incubate with 3% BSA solution for 30 minutes, add an appropriate amount of diluted primary antibody, and incubate overnight at 4 °C. After incubation with the secondary antibody, DAB staining reagent was used for color development. Counterstain with hematoxylin and mount the slides. After air-drying, observe and analyze using a microscope.

[0032] 1.5 Protein sample extraction

[0033] Collect the cells after drug addition treatment, add an appropriate amount of RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors, vortex and mix well, and lyse on ice for 30 min. After centrifugation, take the supernatant. After detecting the protein concentration by the BCA method, add 6×loading buffer (1 / 5 volume of the supernatant) and boil for 10 minutes to obtain the protein sample.

[0034] 1.6 Protein immunoblotting

[0035] Prepare SDS-PAGE gels with different concentrations according to the required molecular weight, fix them in the electrophoresis tank, fill it with electrophoresis buffer, add an appropriate volume of protein sample, and perform electrophoresis on the electrophoresis apparatus. The voltage is 80 V for the first stage for 40 minutes and 120 V for the second stage. When it runs to the appropriate position, end the electrophoresis and perform membrane transfer. Cut the membrane and filter paper according to the size of the gel, assemble the membrane transfer sandwich, place the membrane transfer tank in an ice bath, add membrane transfer buffer, 300 mA, 60 min. After the membrane transfer is completed, block it with 5% skim milk for 1 hour, incubate with the appropriate concentration of primary antibody at 4 °C overnight. Incubate with the secondary antibody for 1 hour and expose it with the ECL color development kit.

[0036] 1.7 In vivo experiments in mice

[0037] 6-week-old NOD-SCID or NOG male mice were purchased from Vital River Laboratory Animal Technology Co., Ltd. K562 cells or bone marrow mononuclear cells derived from CML patients (4×10 6 ) were injected into mice via the tail vein to construct a CML mouse model. The CML mice were randomly divided into two groups: the control group and the 2-D08 group, and were given PBS and 2-D08 (5 mg / kg) respectively, administered every other day, and the mice were sacrificed after four administrations to observe the sizes of the liver and spleen. Detect the infiltration of leukemia cells by immunohistochemical staining.

[0038] Structural formula of compound 2-D08:

[0039]

[0040] 2. Experimental results:

[0041] ① To study the effect of the SUMOylation inhibitor 2-D08 on the activity of CML cells, using the CML leukemia cell lines K562 and MEG-01, different concentrations of 2-D08 (0, 5, 10, 25, 50 μM) were added to the cell culture medium and treated for 24 hours, or after treating with 50 μM 2-D08 for different times (0, 6, 12, 24, 48 hours), the cell activity was detected by the CCK-8 method. The CCK-8 results showed that when the SUMOylation inhibitor 2-D08 was used to treat the K562 and MEG-01 cell lines according to the concentration gradient and time gradient respectively, the cell activity was significantly reduced ( Figure 1 a-d).

[0042] ② Add 50 μM 2-D08 and treat for 24 h, and detect cell proliferation by the EdU method. The EdU experiment found that 2-D08 treatment significantly inhibited the proliferation activity of K562 and MEG-01 cell lines ( Figure 1 e-f).

[0043] ③ To study the effect of the SUMOylation inhibitor 2-D08 on the disease progression of CML mice, 6-week-old NOD-SCID male mice were purchased from Vital River Laboratory Animal Technology Co., Ltd. Inject K562 cells (4×10 6 ) into mice via the tail vein to establish a CML mouse model. Randomly divide the CML mice into two groups: the control group and the 2-D08 group, and give PBS and 2-D08 (5 mg / kg) respectively, administer the drug every other day, sacrifice the mice after administering the drug four times, and observe the size of the liver and spleen. Detect the infiltration of leukemia cells by immunohistochemical staining. The in vivo experiment of mice found that the size and weight of the liver and spleen in the 2-D08 group of mice were significantly reduced ( Figure 2 a-b). Immunohistochemical detection of the cell surface marker CD45 of K562 cells found that the infiltration of K562 cells in the 2-D08 group was significantly improved ( Figure 2 c-d).

[0044] ④ Use the drug-resistant cell line K562 / G01 to study the effect of 2-D08 on the activity of imatinib-resistant cells. Add different concentrations of 2-D08 (0, 5, 10, 25, 50 μM) to the cell culture medium and treat for 24 hours, and detect cell activity by the CCK-8 method. The CCK-8 results showed that the SUMOylation inhibitor 2-D08 treated the K562 / G01 cell line in a concentration gradient, and the cell activity was significantly reduced ( Figure 3 a). To study the effect of the SUMOylation inhibitor 2-D08 on the proliferation ability of drug-resistant cells, add 50 μM 2-D08 and treat for 24 h, and detect cell proliferation by the EdU method. The EdU experiment found that 2-D08 treatment significantly inhibited the proliferation activity of the K562 / G01 cell line ( Figure 3 b).

[0045] ⑤ Further explore the effect of the SUMOylation inhibitor 2-D08 on bone marrow mononuclear cells of CML patients. Treat primary cells with 50 μM 2-D08 for 24 hours, and detect cell activity by the CCK-8 method. The CCK-8 results showed that 2-D08 significantly inhibited the activity of primary cells ( Figure 3 c).

[0046] ⑥ Establish a xenograft mouse model of primary cells derived from CML patients: 6-week-old NOG male mice were purchased from Vital River Laboratory Animal Technology Co., Ltd. Inject bone marrow mononuclear cells derived from CML patients (4×10 6) It was injected into mice via the tail vein. The CML mice were randomly divided into two groups: the control group and the 2-D08 group, and were given PBS and 2-D08 (5 mg / kg) respectively, administered every other day. After four administrations, the mice were sacrificed and the sizes of the liver and spleen were observed. The infiltration of leukemia cells was detected by immunohistochemical staining. In the in vivo experiment of mice, it was found that the sizes and weights of the liver and spleen in the 2-D08 group of mice were significantly reduced ( Figure 4 a-b). By immunohistochemically detecting the surface marker CD45 of leukemia cells, it was found that the infiltration of leukemia cells in the 2-D08 group was significantly improved ( Figure 4 c-d).

[0047] ⑦ To explore the specific mechanism by which 2-D08 affects the progression of CML, after treating CML cells with 50 μM 2-D08 for 24 hours, Western blot was used to detect the activation of BCR-ABL and downstream molecules. The results showed that the treatment with 2-D08 inhibited the protein level of BCR-ABL and the activation of downstream related molecules ( Figure 5 a-b).

[0048] 3. Experimental conclusion: The above results prove that the SUMOylation modification inhibitor directly targets the BCR-ABL protein expression and participates in regulating the progression of CML disease. The SUMOylation modification inhibitor reduces the proliferation activity of CML cells and the progression of CML mice, providing new strategies and new targets for the treatment and drug resistance of CML.

[0049] The above description is only the preferred embodiment of this patent. It should be noted that for those of ordinary skill in the art, without departing from the principle of this patent technology, several improvements and substitutions can be made, and these improvements and substitutions should also be regarded as the protection scope of this patent.

Claims

1. Use of a SUMOylation inhibitor in the preparation of a drug for treating chronic myeloid leukemia and its tyrosine kinase inhibitor (TKI) resistance, characterized in that: The SUMOylation inhibitor promotes protein degradation by inhibiting SUMOylation modification of BCR-ABL protein, thereby downregulating the expression level of BCR-ABL protein and inhibiting the proliferation of leukemia cells.

2. The use according to claim 1, characterized in that: The SUMOylation inhibitor is compound 2-D08 having the following structural formula:

3. The use according to claim 2, characterized in that: The effective dose of compound 2-D08 in the drug is 5-50 μM for in vitro application and 5 mg / kg for in vivo application.

4. The use according to any one of claims 1 to 3, characterized in that: The drug is used to treat TKI-resistant chronic myeloid leukemia.

5. A drug for treating chronic myeloid leukemia, characterized in that: The drug comprises the compound 2-D08 according to claim 2 and a pharmaceutically acceptable carrier, wherein the drug is an injection or a tablet.

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