Use of vk4 in the preparation of a medicament for treating leukemia

Abstract

The application discloses application of VK4 in preparation of a drug for treating leukemia, and belongs to the leukemia treatment field. The application research finds that VK4 can inhibit proliferation of leukemia cells through cycle arrest and induction of apoptosis, and VK4 can inhibit proliferation of HL60 cells and K562 cells through blocking of a JAK / STAT signal path. The results of Wright's staining, immunohistochemical staining and H&E staining of a mouse leukemia model show that VK4 also has an anti-leukemia effect in vivo, and has no obvious liver and kidney damage, and can provide a new drug selection for clinical treatment of leukemia.

Description

Technical Field

[0001] This invention belongs to the field of leukemia treatment, specifically involving the application of VK4 in the preparation of drugs for treating leukemia. Background Technology

[0002] Leukemia is the most common malignant clonal disease of hematopoietic stem cells, characterized by the massive accumulation and proliferation of clonal leukemic cells in the bone marrow and other hematopoietic tissues. The mechanisms include uncontrolled proliferation, impaired differentiation, and inhibited apoptosis. Clonal leukemic cells not only infiltrate other non-hematopoietic tissues and organs but also suppress normal hematopoietic function. High morbidity, high mortality, and high relapse rates are the main challenges in leukemia treatment.

[0003] Currently, the main treatment methods for most types of leukemia in clinical practice include chemotherapy, radiotherapy, hematopoietic stem cell transplantation, targeted therapy, and immunotherapy. Chemotherapy has largely alleviated the progression of leukemia, but drug resistance, the toxicity of chemotherapy drugs, and multi-organ dysfunction caused by weakened immunity after chemotherapy remain challenges that need to be addressed. Radiotherapy also damages normal tissues, and the side effects are often unbearable. The high cost of transplantation and long-term postoperative care limits the use of allogeneic hematopoietic stem cell transplantation to some extent. The high cost of molecularly targeted drugs and the potential for multi-systemic side effects have prevented the widespread application of this treatment. Although chimeric antigen receptor-T-cell immunotherapy has shown potential in hematologic malignancies, cytokine release syndrome (the most common side effect) also limits its application. Over the past few decades, the first complete remission rate and overall survival rate of leukemia have improved with these treatment methods, but the persistently high relapse rate remains a challenge for modern medicine. Therefore, there is an urgent need to develop new therapeutic drugs to alleviate the difficulties in treating leukemia.

[0004] Vitamin K (VK) refers to a class of structurally similar compounds with a 2-methyl-1,4-naphthoquinone ring and a variable aliphatic chain, including vitamin K1 (VK1), vitamin K2 (VK2), vitamin K3 (VK3), and vitamin K4 (VK4). Menadiol diacetate (VK4) is a water-soluble synthetic derivative of VK2, initially used as a clinical hemostatic agent. Recent studies have found that VK4 exhibits good antitumor activity in solid tumors. In 2013, Jiang et al. first demonstrated the anticancer activity of VK4 in prostate cancer PC-3 cells, and in 2017, Di et al. found that VK4 can inhibit the proliferation of U2OS osteosarcoma cells and induce apoptosis through mitochondrial dysfunction. Although VK4 has been shown to have anti-tumor effects in solid tumors, there are no studies on VK4's anti-leukemia effects. The purpose of this study is to investigate the effects of VK4 on the proliferation and apoptosis of human leukemia cells and to explore the molecular mechanism of VK4's action, aiming to provide new ideas and treatment options for the clinical treatment of leukemia. Summary of the Invention

[0005] In view of the content mentioned in the background art, the present invention provides the application of VK4 in the preparation of drugs for treating leukemia. The purpose is to study the effect of VK4 on the proliferation and apoptosis of human leukemia cells and explore the molecular mechanism of VK4 action, aiming to provide new ideas and treatment options for clinical treatment of leukemia.

[0006] This invention provides the application of VK4 in the preparation of drugs for treating leukemia.

[0007] Furthermore, VK4 inhibits leukemia cell proliferation through the JAK / STAT signaling pathway.

[0008] Furthermore, the leukemia is any one of human acute myeloid leukemia, human acute lymphoblastic leukemia, or human chronic myeloid leukemia.

[0009] Furthermore, the leukemia mentioned is human acute myeloid leukemia.

[0010] This invention also provides the use of VK4 in the preparation of drugs that induce cell cycle arrest in leukemia cells or promote apoptosis in leukemia cells.

[0011] This invention also provides the use of VK4 in the preparation of JAK / STAT inhibitors for leukemia.

[0012] A drug for the treatment of leukemia, the drug comprising VK4 and pharmaceutically acceptable excipients.

[0013] Furthermore, the excipients include at least one of diluents, fillers, excipients, binders, humectants, disintegrants, surfactants, lubricants, and flavorings.

[0014] Furthermore, the dosage form of the drug is an injection.

[0015] Furthermore, the dosage of the injection is 2.5-10 mg / kg.

[0016] This invention involves treating HL60, Jurkat, and K562 cells in the logarithmic growth phase with different concentrations of vitamin K4 for corresponding time periods, then using the CCK-8 assay to detect cell viability and calculate IC50. 50 The study investigated the changes in cell cycle distribution, apoptosis, and mitochondrial membrane potential levels in leukemia cells treated with VK4 using flow cytometry. Western blotting (WB) was used to detect the expression of cell cycle and apoptosis-related proteins and pathway-related proteins in leukemia cells after VK4 treatment. The association between VK4 and the JAK / STAT signaling pathway was studied using the JAK2 / STAT3 small molecule inhibitor AG490, and CCK-8 assays, flow cytometry, and WB were used to verify whether VK4 exerts its anti-leukemic effect through the JAK / STAT signaling pathway. Finally, a leukemia model was established in Balb / c nude mice by tail vein injection of HL60 cells, and the in vivo anti-leukemic effect of VK4 and drug toxicity were verified and assessed using Wright staining, immunohistochemistry, and H&E staining.

[0017] CCK-8 assay results showed that VK4 significantly inhibited the proliferation of HL60, Jurkat, and K562 leukemia cells, with an IC50 value of [missing information]. 50 The concentrations were 5.35 μM, 8.44 μM, and 11.59 μM, respectively. Flow cytometry results showed that VK4 induced S-phase cell cycle arrest in HL60 and Jurkat cells, and G2 / M-phase cell cycle arrest in K562 cells. VK4 induced apoptosis in HL60 and K562 cells via the endogenous mitochondrial pathway, and inducing apoptosis in Jurkat cells via both endogenous and exogenous pathways. Combined results from CCK-8, flow cytometry, and Western blotting showed that VK4 inhibited the proliferation of HL60 and K562 cells through the JAK / STAT signaling pathway.

[0018] The beneficial effects of this invention are:

[0019] This invention reveals that VK4 can inhibit leukemia cell proliferation through cell cycle arrest and apoptosis induction. VK4 inhibits the proliferation of HL60 and K562 cells by blocking the JAK / STAT signaling pathway. Wright staining, immunohistochemistry, and H&E staining results in a mouse leukemia model show that VK4 also has anti-leukemic activity in vivo without significant liver or kidney damage, suggesting it may be a potential therapeutic agent for leukemia. Attached Figure Description

[0020] Figure 1 In the diagram: A represents the chemical structural formula of Menadiol Diacetate (VK4); B and E represent the effects of VK4 on the proliferation activity of leukemia cell lines HL60, Jurkat, K562, and normal human PBMCs, respectively.

[0021] Figure 2 In the figure: AF represents the cell cycle arrest induced by different concentrations of VK4 in different leukemia cells; GI represents the expression of cyclin in different leukemia cells after treatment with different concentrations of VK4.

[0022] Figure 3 In the middle: A represents the apoptosis of different leukemia cells after treatment with different concentrations of VK4; BD represents the expression of Caspase 3 protein in different leukemia cells after treatment with different concentrations of VK4.

[0023] Figure 4 In the middle: AC represents the changes in the expression levels of proteins related to the endogenous apoptosis pathway in leukemia cells after treatment with different concentrations of VK4; D represents the changes in the mitochondrial membrane potential level in leukemia cells after treatment with different concentrations of VK4.

[0024] Figure 5 In the table: A shows the changes in the expression levels of JAK / STAT signaling pathway-related proteins in HL60 and K562 cells after treatment with different concentrations of VK4; B shows the changes in cell viability of HL60 and K562 cells after pretreatment with the JAK2 / STAT3 inhibitor AG490; C shows the changes in apoptosis in HL60 and K562 cells after pretreatment with AG490; and D shows the changes in the expression levels of apoptosis-related proteins in HL60 and K562 cells after pretreatment with AG490.

[0025] Figure 6 In the middle: A represents the construction of a mouse leukemia model; B represents the changes in the proportion of leukemia cells in peripheral blood smears of mice 28 days after intraperitoneal injection of different doses of VK4. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with embodiments. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0028] Example

[0029] Experimental Materials and Methods

[0030] 1. Cell Culture and Processing

[0031] Human acute myeloid leukemia (AML) cell line HL60, human acute lymphoblastic leukemia (ALL) cell line Jurkat, and human chronic myeloid leukemia (CML) cell line K562 were all purchased from the Cell Bank of the Chinese Academy of Sciences. All three cell lines were cultured in RPIM-1640 medium containing 10% fetal bovine serum in a cell culture incubator at 37°C and 5% CO2. VK4 and AG490 cells were dissolved using dimethyl sulfoxide (DMSO). Different concentrations of VK4 were used to treat cells under different experimental conditions, with DMSO concentration <0.1% serving as the control group.

[0032] 2. Experimental reagents

[0033] VK4 and AG490 were purchased from MCE; antibodies Cyclin A2, Cyclin B1, Cyclin E2, CDK4, PARP, Cleaved-Caspase 3, and Cleaved-Caspase 9 were purchased from Cell Signaling Technology; antibodies CDK1, Caspase 3, Caspase 9, and GAPDH were purchased from Proteintech; antibodies JAK2, Phospho-JAK2, STAT3, Phospho-STAT3, STAT5, and Phospho-STAT5 were purchased from Abmart; and antibody CDK2 was purchased from Abcam.

[0034] 3 Experimental Methods

[0035] 3.1 Preparation of peripheral blood mononuclear cells from normal healthy individuals

[0036] After collecting 3 mL of venous blood from healthy volunteers, the cells were allowed to stand at room temperature for 1-2 h. Then, 3 mL of human lymphocyte separation medium was added, and the cells were centrifuged at 400 g for 30 min at room temperature. After centrifugation, the liquid in the tube separated into four layers. The second layer, a ring of milky white lymphocytes, was transferred to another centrifuge tube. An appropriate amount of 1× erythrocyte lysis buffer was added, and the cells were gently mixed. After lysis at 4°C for 10 min, the cells were centrifuged at 300 g for 5 min at room temperature, and the supernatant was discarded. The cells were washed three times with washing buffer (1× PBS), and the supernatant was discarded. The cells were resuspended in RPIM-1640 medium containing 10% fetal bovine serum and cultured in a 37°C, 5% CO2 incubator. This research protocol was approved by the Research Ethics Committee of the First Affiliated Hospital of Gannan Medical University, ethics number LLSC-2024 No. 283.

[0037] 3.2 Effect of VK4 on the proliferation activity of leukemia cells and PBMCs as detected by CCK-8 assay

[0038] The effect of VK4 on the proliferative activity of leukemia cells and PBMCs was detected using the Cell Counting Kit-8 (CCK-8) enhanced assay kit. HL60, Jurkat, and K562 cells that had entered the logarithmic growth phase were collected. HL60, K562 cells and PBMCs were seeded at 10,000 cells / well, and Jurkat cells at 15,000 cells / well in 96-well plates. Jurkat and K562 cells were treated with 0 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM VK4 for 24 h. HL60 cells were treated with 0 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, and 8 μM VK4 for 24 h. PBMCs were treated with 0 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, and 40 μM VK4 for 24 h. Paclitaxel was used as a positive control. After 24 h, discard 100 μL of culture medium per well, add 10 μL / well of CCK-8, and incubate at 37℃ for another 4 h. After incubation, perform dual-wavelength assays using a continuous-spectrum multi-mode microplate reader, with a detection wavelength of 450 nm and a reference wavelength of 630 nm. Data processing: Calculate cell viability (OD). 实验孔 -OD 空白孔 ) / (OD 对照孔 -OD 空白孔() × 100%, using Graphpad Prism 10.0 to process and analyze the data and calculate IC. 50 IC50 values ​​for each cell line. 50 The values ​​are shown in Table 1, where Taxol was used as a positive control drug in the cytotoxic activity experiment.

[0039] Figure 1 In the diagram, A represents the chemical structural formula of Menadiol Diacetate (VK4). For example... Figure 1 As shown in the figure below, after treatment of leukemia cells with different concentrations of VK4, VK4 significantly inhibited the proliferation activity of HL60 cells, Jurkat cells, and K562 cells in a dose-dependent manner, with an IC50 value of [missing information]. 50 The values ​​were 5.35 μM, 8.44 μM, and 11.59 μM, respectively. We then evaluated the cytotoxicity of VK4 on normal human peripheral blood mononuclear cells (PBMCs). CCK-8 results showed that the cell viability of PBMCs did not change significantly after 24 h of VK4 treatment. Figure 1 The results (E) indicate that VK4 has no significant toxicity to normal healthy human PBMCs. These results suggest that the antileukemic effect of VK4 is selective and dose-dependent.

[0040] Table 1. IC50 of VK4 in different leukemia cells 50 (Mean±SD)

[0041]

[0042] 3.3 Flow cytometry analysis of changes in cell cycle and apoptosis levels after VK4 treatment

[0043] 3.3.1 Cell cycle detection

[0044] Cell cycle analysis was performed using propidium iodide (PI) staining. HL60 cells and Jurkat cells were 5 × 10⁸ cells per cell line. 5 Hole / hole, K562 fine 2×10 5 Cells were evenly seeded per well in 6-well plates, treated with control solvent (<0.1% DMSO) and different concentrations of VK4 for 12 h, and then collected. Cells were washed with pre-chilled PBS, resuspended in PBS, fixed with 70% anhydrous ethanol, and incubated overnight at -20°C. The next day, after centrifugation at 4°C, the supernatant was discarded, the ethanol was evaporated, and the cells were resuspended in PBS. RNase A enzyme was added, and the mixture was gently mixed and incubated at 37°C for 30 min. Then, PI solution (working concentration 10 mg / mL) was added, and the cells were incubated at room temperature in the dark for 20 min. After filtration through a 300-mesh nylon membrane, the cells were analyzed using a BD FACSCalibur flow cytometer. Flowjo 10.8.1 was used to analyze cell cycle distribution and plot the data.

[0045] Cell proliferation is regulated by the cell cycle. To investigate how VK4 inhibits leukemia cell proliferation, the effect of VK4 on the cell cycle distribution of leukemia cells was detected by PI staining and flow cytometry. The results showed that most HL60 and Jurkat cells arrested in the S phase after VK4 treatment. However, in K562 cells, VK4 treatment significantly arrested cells in the G2 / M phase. Figure 2 (AF). Further analysis was conducted to examine the expression of related proteins at different stages of the cell cycle after VK4 treatment. (e.g., AF). Figure 2 As shown in the midline GI, Western blotting results revealed that in HL60 and Jurkat cells, treatment with different concentrations of VK4 for 12 h resulted in a dose-dependent reduction in the expression of cyclins CDK1, CDK2, Cyclin A2, Cyclin B1, and Cyclin E2. In K562 cells, treatment with different concentrations of VK4 significantly decreased the expression of key regulatory proteins of the G2 / M phase, including CDK1, CDK4, and Cyclin B1. These results indicate that VK4 can induce cell cycle arrest in leukemia cells and downregulate the expression of corresponding cyclins in a dose-dependent manner.

[0046] 3.3.2 Apoptosis Detection

[0047] Apoptosis was detected using the Annexin V-Alexa Fluor 488 / PI kit. All three cell lines were 5 × 10⁻⁶ cells. 5 Cells were seeded per well in 6-well plates, treated with control solvent (<0.1% DMSO) and different concentrations of VK4 for 24 h, and then collected. Cells were washed with pre-chilled PBS, resuspended in 1× Binding buffer, and the cell concentration was adjusted to (1~5)×10⁶ cells / well. 6 / mL. 100 μL of cell suspension was transferred to each sample into a flow cytometry tube. The voltage was adjusted for the negative control tubes (Annexin V- / PI-), and the compensation was adjusted for the single-stain tubes (Annexin V+ / PI- and Annexin V- / PI+). 5 μL of Annexin V was added to each tube, and the cells were incubated at room temperature in the dark for 5 min. Then, 10 μL of PI dye was added, followed by 400 μL of PBS, and the cells were analyzed using a BD FACSCalibur flow cytometer. Data were analyzed and plotted using Flowjo 10.8.1.

[0048] Flow cytometry was used to detect apoptosis to determine whether the antiproliferative effect of VK4 is achieved by inducing apoptosis. Figure 3As shown in Figure A, after treatment with different concentrations of VK4 for 24 h, the proportion of apoptotic cells in the three cell lines increased in a dose-dependent manner. Western blotting also revealed that the expression level of the key apoptosis-executing molecule Caspase 3 splice also increased with increasing VK4 concentration. Figure 3 (BD), the above results indicate that VK4 can induce apoptosis in leukemia cells.

[0049] 3.3.3 Detection of mitochondrial membrane potential levels

[0050] Changes in intracellular mitochondrial membrane potential (MMP) levels were detected using the JC-1 fluorescent probe. Cell plating and drug treatment times were consistent with those for apoptosis. After 24 h, experimental group cells and negative and positive control cells were collected (cell numbers adjusted to (2~5)×10⁻⁶). 5 (Number of cells). After washing the cells twice with PBS, resuspend the cells in 500 μL / sample 1×JC-1 staining working solution and incubate at 37℃ for 30 min. After incubation, centrifuge at 4℃ and discard the supernatant. Wash the cells twice more with PBS, and finally resuspend the cells in 500 μL PBS. Analyze the data using flow cytometry and plot the data using Flowjo 10.8.1.

[0051] Changes in intracellular MMPs in leukemia cells after VK4 treatment were detected by flow cytometry to determine whether the antiproliferative effect of VK4 is achieved through the endogenous apoptosis pathway. Figure 4 As shown in Figure D, after treatment with different concentrations of VK4 for 24 h, the ratio of JC-1 monomers to JC-1 polymers in the three cell lines increased in a dose-dependent manner, indicating that VK4 treatment could significantly reduce MMP.

[0052] 3.4 Western blot for protein immunoblotting

[0053] The expression of cell cycle and apoptosis-related proteins was determined using Western blotting. The procedure is briefly described below: Cell cycle and apoptotic cell treatment were as described previously. After treatment, cells were collected, washed with PBS, and lysis buffer was added. Cells were then sonicated and lysed on ice for 30 min. After lysis, 2 μL of each sample was taken for protein concentration determination. The remaining sample was added to 1× loading buffer, incubated in a 100℃ boiling water bath for 5 min, and then annealed on ice. An equal volume of protein sample was added, and the protein was separated by polyacrylamide gel electrophoresis and transferred to a PVDF membrane. A 5% skim milk blocking buffer was prepared using PBS-Tween 20 (PBST), and the membrane was blocked at room temperature for 2 h. After blocking, the PVDF membrane was washed with PBST. The PVDF membrane was then placed in a prepared specific primary antibody solution (concentration according to the antibody manufacturer's instructions) and incubated overnight at 4℃ on a shaker. After washing again with PBST, the PVDF membrane was placed in a prepared non-specific secondary antibody solution (secondary antibody concentration 1:5000) and incubated at room temperature for 2 h. The PVDF membrane was washed again with PBST and then immersed in chemiluminescent solution. Protein expression was detected using the Chemi Dox XRS chemiluminescence imaging system. Image J software was used to analyze the gray values ​​of the protein bands and normalize the gray values ​​to calculate the relative protein expression level (relative protein expression level = target protein gray value / internal reference gray value).

[0054] Western blot analysis was used to detect changes in the expression levels of apoptosis-related proteins in three cell lines after treatment with different concentrations of vitamin K4. The results showed that after 24 h of treatment with different concentrations of vitamin K4, compared with the control group, the expression of cleaved-caspase 9, cleaved-caspase 3, and cleaved-PARP in the three cell lines was significantly increased. Figure 4 The results suggest that VK4 can induce apoptosis in HL60, K562, and Jurkat cells through the endogenous mitochondrial apoptosis pathway.

[0055] Simultaneously, the association between VK4 and the JAK / STAT signaling pathway was investigated using the JAK2 / STAT3 small molecule inhibitor AG490, and CCK-8, flow cytometry, and Western blotting were used to verify whether VK4 exerts its anti-leukemic effect through the JAK / STAT signaling pathway. Figure 5 As shown in Figure A, Western blot results indicated that VK4 treatment alone significantly reduced the expression of p-JAK2, p-STAT3, and p-STAT5 in HL60 and K562 cells. CCK-8 and flow cytometry results showed that pretreatment with AG490 (for 12 h) partially rescued the inhibitory effect of VK4 on HL60 and K562 cells, and partially antagonized the apoptosis-inducing effect of VK4 on HL60 and K562 cells. Figure 5 (Chinese BC). Finally, as... Figure 5 As shown in Figure D, Western blotting results indicated that AG490 could prevent the activation of Caspase 3, Caspase 9, and PARP in HL60 and K562 cells after VK4 treatment. These results suggest that VK4 exerts its anti-leukemic effect by inhibiting the JAK / STAT signaling pathway, suppressing the proliferation of HL60 and K562 cells, and inducing apoptosis in both cells.

[0056] 3.5 Animal Experiments

[0057] Balb / c nude mice (3-4 weeks old, 15-20 g) were used in the experiment and purchased from Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd., production license number: SCXK(Su)2020-0009. Mice were pretreated with cyclophosphamide 100 mg / kg for 2 consecutive days. On the third day, HL60 cells in the logarithmic growth phase were collected, and each mouse was slowly injected with 5 × 10⁶ sterile HL60 cells via the tail vein. 6 On days 7 and 14 post-transplantation, blood was collected from the tail vein of mice for peripheral blood smears and Wright-Giemsa staining to determine the success of the leukemia model. After successful model establishment, mice were randomly divided into a control group (without VK4 as a solubilizer) and an experimental group (low, medium, and high doses of VK4). The control group solution and different doses (2.5 / 5.0 / 10.0 mg / kg) of VK4 solution were administered intraperitoneally for 28 consecutive days. Mice were sacrificed immediately after drug treatment. Before sacrifice, peripheral blood smears were collected again from the tail vein for staining and observation under an oil immersion microscope to determine if the proportion of leukemia cells decreased after drug treatment. Mice were euthanized by cervical dislocation under anesthesia, arranged according to group, and photographed. Liver, spleen, and kidney tissues were dissected and stained with hematoxylin and eosin (H&E) for comparison with normal mice to determine whether organ damage and its severity occurred after VK4 treatment. This research protocol was approved by the Research Ethics Committee of the First Affiliated Hospital of Gannan Medical University, with ethics number LLSC-2024-078.

[0058] We also established a leukemia model in Balb / c nude mice to investigate the in vivo antitumor effect of VK4. The results showed that VK4 also had a significant anti-leukemia effect in vivo, and VK4 did not cause significant damage to the liver and kidneys of mice. Figure 6 )

[0059] The embodiments described above are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they are not intended to limit the present invention. It should be noted that various changes and modifications can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the concept and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of VK4 in the preparation of drugs for treating leukemia.

2. The application according to claim 1, characterized in that, VK4 inhibits leukemia cell proliferation through the JAK / STAT signaling pathway.

3. The application according to claim 1, characterized in that, The leukemia mentioned is any one of human acute myeloid leukemia, human acute lymphoblastic leukemia, or human chronic myeloid leukemia.

4. The application according to claim 1, characterized in that, The leukemia mentioned is acute myeloid leukemia.

5. Application of VK4 in the preparation of drugs that induce cell cycle arrest or promote apoptosis in leukemia cells.

6. Application of VK4 in the preparation of JAK / STAT inhibitors for leukemia.

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