Application of GAC as target spot in enhancing treatment of chronic myelogenous leukemia by imatinib

Abstract

The invention belongs to the technical field of biotechnology, and particularly relates to application of GAC as a target spot in enhancing treatment of chronic myelogenous leukemia by imatinib. According to the application of the GAC in preparation of a product target for preventing and / or treating the chronic myelogenous leukemia and the application of the GAC inhibitor 968 in preparation of a medicine for preventing and / or treating the chronic myelogenous leukemia, through the pharmacological effect of the GAC inhibitor 968, the GAC inhibitor 968 takes the GAC as the target in the CML, and it is prompted that the GAC inhibitor 968 can be used for treating the CML disease; gAC is taken as a target to play a role in remarkably reducing the number and proportion of bone marrow LSK cells, and further experiments find that when the GAC inhibitor 968 and a classical CML treatment medicine IM are combined for use, neutrophils in peripheral blood are also reduced to a certain extent, and the treatment effect of the GAC inhibitor 968 is enhanced, so that the CML disease progress caused by malignant proliferation of stem cells is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biotechnology, and specifically relates to an application of GAC as a target in enhancing imatinib treatment of chronic myeloid leukemia. Background Art

[0002] Chronic myeloid leukemia (CML) is a blood cancer that originates from the malignant proliferation of bone marrow hematopoietic stem cells. The global incidence rate is 0.6 to 2.0 per 100,000 people, and the incidence rate gradually increases with age. Compared with Western patients, Chinese CML patients are younger. Currently, the preferred treatment for CML is taking tyrosine kinase inhibitors (TKI), such as the classic drug imatinib mesylate (IM), which can significantly improve the survival rate of patients in the early stages of treatment.

[0003] The main clinical pathological features of CML patients are bone marrow myeloid cell hyperplasia, significant increase in neutrophils, and granulocyte infiltration in multiple organs. Studies have shown that leukemia stem cells and drug-resistant cells rely on mitochondrial metabolism and are sensitive to oxidative phosphorylation. This indicates that when CML develops, the metabolism of energy substances in the body will be regulated accordingly. Glutamine, as another major energy source for cancer cells besides glucose, plays an important role in the occurrence and development of tumors. After being transported to the mitochondria, glutamine is converted into α-ketoglutarate by glutaminase (GLS) and indirectly participates in the TCA cycle, which can continuously provide raw materials for the synthesis of biological macromolecules. In order to reproduce, cancer cells need to continuously and rapidly synthesize macromolecules, so hindering glutamine metabolism has become a widely watched cancer treatment target. Among them, glutaminase inhibitors have high selectivity and good pharmacological activity and are often used in cancer treatment.

[0004] There are two main types of glutaminase in the human body: kidney-type glutaminase (GLS1) and liver-type glutaminase (GLS2). Tumor cells overactivate kidney-type glutaminase (GLS1) to achieve rapid and massive proliferation. There are two transcriptional splicing mutants in GLS1, the longer one is called KGA and the other is called GAC. GLS2 mainly acts on non-cancerous cells and catalyzes the metabolism of glutamine. The metabolic regulation mechanism of glutamine in CML patients is still unclear, so there are challenges and opportunities in the study of regulating glutamine metabolism to affect the development of CML disease. Summary of the invention

[0005] In view of the above problems, the object of the present invention is to provide an application of GAC as a target in enhancing imatinib treatment of chronic myeloid leukemia.

[0006] The technical contents of the present invention are as follows:

[0007] The present invention provides an application of GAC as a target for preparing a product for preventing and / or treating chronic myeloid leukemia, wherein the GAC is glutaminase C, which is a splice of renal glutaminase GLS1;

[0008] The product includes a kit or a separately packaged pharmaceutical composition or a mixed packaged pharmaceutical composition;

[0009] The present invention also provides the use of a substance that inhibits or reduces the expression and / or activity of the GAC gene in the preparation of a product for inhibiting chronic myeloid leukemia;

[0010] The substances that inhibit or reduce the expression and / or activity of the GAC gene include GAC inhibitor 968, GAC siRNA and GAC gRNA.

[0011] The present invention also provides a use of GAC inhibitor 968 in the preparation of a drug for preventing and / or treating chronic myeloid leukemia;

[0012] The GAC inhibitor 968 is a Glutaminase C allosteric inhibitor, also known as C-IN-1, with a molecular weight of 475.42 and a molecular formula of C 27 H 27 Bn 2 O, CAS No. 311795-38-7, structural formula is as follows:

[0013]

[0014] The present invention also provides the use of GAC inhibitor 968 in combination with imatinib (IM) in the preparation of a product for inhibiting chronic myeloid leukemia.

[0015] The present invention also provides a product, comprising GAC inhibitor 968 and imatinib (IM);

[0016] The product has at least one of the following functions:

[0017] 1) Treatment of chronic myeloid leukemia;

[0018] 2) Inhibit the growth of peripheral blood neutrophils in chronic myeloid leukemia;

[0019] 3) Inhibit the growth of bone marrow LSK cells in chronic myeloid leukemia;

[0020] 4) Inhibit GLS1 gene expression or activity.

[0021] The beneficial effects of the present invention are as follows:

[0022] The present invention discloses an application of GAC as a target in the preparation of a product for preventing and / or treating chronic myeloid leukemia, and an application of GAC inhibitor 968 in the preparation of a drug for preventing and / or treating chronic myeloid leukemia. Through the pharmacological action of GAC inhibitor 968, GAC inhibitor 968 uses GAC as a target in CML, suggesting that it can be used to treat CML disease; GAC is used as a target to significantly reduce the number and proportion of bone marrow LSK cells. Through further experiments, it is found that when compound 968 is used in combination with IM, a classical CML therapeutic drug, the survival period of mice is further prolonged, and the peripheral blood neutrophils are also reduced to a certain extent. When used in combination with IM, its therapeutic effect is significantly enhanced, thereby improving CML disease progression caused by malignant proliferation of stem cells, and providing a new method and new idea for the clinical treatment of CML. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The results of the glucose, glutamine, glutamate and lactic acid levels in the serum of CML model mice when the disease was induced;

[0024] Figure 2 The results of the lactic acid levels in the liver, kidney and spleen tissues of CML model mice when the disease was induced;

[0025] Figure 3 The results of glucose levels in liver, kidney and spleen tissues of CML model mice when the disease was induced;

[0026] Figure 4 This is the result of the expression level of GLS2 protein in the liver of CML model mice when the disease is induced;

[0027] Figure 5 This is the result diagram of the protein expression levels of GLS1-related genes GAC, KGA and GLS2 in the femur when the CML model mice were induced to develop the disease;

[0028] Figure 6 This is the result diagram of the mRNA expression level of GLS-related genes in the liver tissue of CML model mice when the disease was induced;

[0029] Figure 7 This is the result diagram of the mRNA expression level of GLS related genes in the kidney tissue of mice when the disease was induced in CML model mice;

[0030] Figure 8 The results of the expression of GLS-related gene mRNA in bone marrow leukocytes of CML model mice when the disease was induced;

[0031] Fig. 9The results of the mRNA expression level of GLS-related genes in sca-1 cells sorted from bone marrow leukocytes when the disease was induced in CML model mice;

[0032] Fig.10 This is the result diagram of the mRNA expression level of GLS related genes in the spleen leukocytes of CML model mice when the disease was induced;

[0033] Fig.11 This is the result diagram of the mRNA expression level of GLS related genes in peripheral blood leukocytes when the disease was induced in CML model mice;

[0034] Fig.12 This is the survival status of CML mice after drug withdrawal after treatment, and the results were observed until the 90th day after the end of treatment;

[0035] Fig.13 The results of the results of the serum energy substance levels including glucose, glutamate, glutamine and lactate in CML model mice after treatment;

[0036] Fig.14 The results of the protein expression level of GLS2 in the liver of CML model mice after treatment;

[0037] Fig.15 The results of the protein expression levels of GLS1-related genes GAC, KGA and GLS2 in the femur of CML model mice after treatment;

[0038] Fig.16 This is a graph showing the mRNA expression level of GLS-related genes in liver tissue of CML model mice after treatment;

[0039] Fig.17 This is a graph showing the mRNA expression level of GLS-related genes in the kidney tissue of CML model mice after treatment;

[0040] Fig.18 The results of the expression of GLS-related gene mRNA in bone marrow leukocytes of CML model mice after treatment;

[0041] Fig.19 This is the result diagram of the mRNA expression level of GLS related genes in spleen leukocytes of CML model mice after treatment;

[0042] Fig. 20 The results of the mRNA expression level of GLS-related genes in sca-1 cells obtained by sorting spleen leukocytes in CML model mice after treatment;

[0043] Fig.21This is a diagram showing the proportion and number of LSK cells in the bone marrow of CML model mice after treatment;

[0044] Fig. 22 This is the survival status of CML mice after the start of treatment. The drug was administered continuously for 21 days and the situation was observed until the 300th day;

[0045] Fig.23 This is a diagram showing the proportion of neutrophils in the peripheral blood of CML model mice after treatment;

[0046] In the above figures, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001; ns indicates no significant difference. DETAILED DESCRIPTION

[0047] The present invention is further described in detail below through specific implementation cases and accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. After reading the present invention, various equivalent forms of modifications to the present invention by those skilled in the art are all within the scope of the claims attached to this application.

[0048] Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents from the conventional market.

[0049] Example 1

[0050] Study the effects of CML disease on the metabolic levels of glucose, lactate, glutamine and glutamate and the expression of GLS based on CML mouse model

[0051] 1) During CML, the levels of glucose, lactate, glutamine, and glutamate metabolism in the body are regulated

[0052] The SCL-tTA / BCR-ABL double transgenic mice (hereinafter referred to as CML mice) constructed by hybridization of SPF-grade FVB.Cg-Tg(Tal1-tTA)19Dgt / J transgenic mice (introduced from Nanjing Institute of Biomedicine and maintained, bred and expanded by our laboratory, hereinafter referred to as SCL-tTA mice) and FVB / N-Tg(tetO-BCR-ABL1)2Dgt / J transgenic mice (introduced from Nanjing Institute of Biomedicine and maintained, bred and expanded by our laboratory, hereinafter referred to as BCR-ABL mice) were used as the CML mouse model and randomly divided into two groups: a control group (control) in which BCR-ABL gene expression was inhibited by feeding with doxycycline and a CML group (in which BCR-ABL gene expression was induced by feeding without doxycycline).

[0053] After the two groups were fed doxycycline normally or stopped feeding doxycycline for four weeks, serum, liver tissue, spleen tissue, and kidney tissue were collected respectively, and metabolic kits were used to detect four indicators, namely glucose and its metabolite lactate, and glutamine and its metabolite glutamate (metabolic detection kits were all purchased from Promega). The obtained RLU value (relative light unit) is proportional to the concentration, thereby reflecting the changes in the content of these four substances and evaluating the metabolism of glucose and glutamine during the development of CML disease.

[0054] The results showed that Figure 1 As shown in the figure, after the onset of CML model mice, the levels of energy substances including glutamine in the serum and various tissues of the body changed significantly. The glucose content in the serum decreased significantly, the glutamine content increased significantly, and the glutamate and lactic acid contents did not change significantly. Figure 2 and Figure 3 As shown, the lactic acid content in the liver increased significantly, the glucose content in the spleen increased significantly, while the lactic acid in the spleen and kidney and the glucose in the liver and kidney did not change significantly.

[0055] The above results suggest that CML mice undergo metabolic reprogramming after onset of the disease.

[0056] 2) When CML develops, the expression level of GLS-related genes in the body is regulated

[0057] Immunohistochemistry was used to detect the expression of GLS2 in the liver tissues of mice in the control group and CML group (GLS2 antibody was purchased from Thermo Fisher Scientific). Figure 4 As shown, it was found that the expression of GLS2 protein in the liver of mice in the CML group was significantly downregulated;

[0058] Multiple immunofluorescence was used to detect the expression of GLS-related genes in the femurs of the two groups of mice (the antibodies GAC and KGA (two mutants of GLS1, GAC and KGA) were purchased from Proteintech, and GLS2 was purchased from Thermo Fisher). The results are as follows Figure 5 As shown, it was found that GAC expression was upregulated, while KGA and GLS2 expression was not obvious;

[0059] qPCR was used to detect liver tissue, kidney tissue, collected spleen leukocytes, peripheral blood leukocytes, bone marrow leukocytes and sca-1 cells separated by magnetic beads from the two groups of mice (qRT-PCR kit was purchased from TaKaRa, probe was synthesized by Thermo Fisher Scientific, and magnetic beads were purchased from Miltenyi Biotec). It was found that after the onset of CML in mice, the expression levels of GLS-related genes in various tissues and cells were regulated to varying degrees.

[0060] Beta-actin (Actb) was used as the internal reference gene for probe quantitative PCR (qPCR) detection, relative expression level = 2^ -(ΔΔCt) , ΔΔCt = (Ct target gene - Ct reference gene) experimental group - (Ct target gene - Ct reference gene) control group;

[0061] like Figure 6 As shown, the expression level of GLS2 mRNA in mouse liver tissue was upregulated, the expression level of GAC mRNA was downregulated, and the expression level of KGA mRNA was upregulated;

[0062] like Figure 7 As shown, GAC mRNA expression was downregulated in the kidney;

[0063] like Figure 8 As shown, GAC mRNA expression in bone marrow leukocytes was downregulated, and KGA expression did not change significantly;

[0064] like Fig. 9 As shown, in sca-1 cells sorted from bone marrow leukocytes, KGA mRNA expression was significantly upregulated, GAC expression was downregulated, and GLS2 expression was almost non-existent;

[0065] like Fig.10 As shown, GAC mRNA expression in spleen leukocytes did not change significantly, and KGA expression was slightly upregulated (e.g. Fig.10 );

[0066] like Fig.11 As shown, in peripheral blood leukocytes, GAC and KGA mRNA expression was downregulated, and GLS2 was almost not expressed;

[0067] The above experimental results show that GLS1 gene expression is correlated with the onset of CML.

[0068] Example 2

[0069] In vivo experiments demonstrate that glutaminase inhibitor compound 968 effectively prolongs the survival of CML mice

[0070] 6-8 week old CML mice were randomly divided into two groups: Compound 968 group (10 mg / kg / d) (Compound 968 purchased from MCE) and solvent control group (VEH group).

[0071] After the CML mice were grouped, they stopped feeding doxycycline. Two weeks after the onset of the disease, the treatment began. The drug was administered once a day for five consecutive days a week for a total of three weeks by intraperitoneal injection. After the treatment, the drug was stopped and the survival curve was observed. The observation period was 90 days after the end of treatment, and a survival curve was prepared.

[0072] Depend on Fig.12(968: 10 mg / kg / d compound 968 treatment group, sample size n=13; VHE: solvent control treatment group, sample size n=10) The results show that compared with the VEH group, compound 968 can significantly prolong the survival of CML mice, which indicates that intervening in glutamine metabolism is indeed beneficial to improving the survival of CML.

[0073] Example 3

[0074] Study on the effect of compound 968 on the metabolic levels of glucose, lactate, glutamine and glutamate and on the expression of GLS based on CML mouse model

[0075] 1) Compound 968 effectively regulates the utilization of energy substances in CML mice

[0076] like Fig.13 As shown in the figure, compared with the VEH group, the serum glucose, glutamine and glutamate levels of CML mice treated with 968 were significantly decreased, while the lactate content did not change significantly. These results indicate that intervening in glutamine metabolism changes the utilization pattern of energy substances in the body, causing CML mice to use more glutamine as an energy substance.

[0077] 2) Compound 968 effectively regulates GLS gene expression in CML mice

[0078] Immunohistochemistry was used to detect the expression of GLS2 in the liver of mice in the VEH group and the 968 group. Fig.14 As shown, it was found that GLS2 expression was upregulated in the liver of CML mice treated with 968 compared with the VEH group;

[0079] Multiple immunofluorescence was used to detect the expression of GLS-related genes in the femurs of the two groups of mice. Fig.15 As shown, it was found that GAC expression in the femur of CML mice treated with 968 was significantly downregulated compared with the VEH group, while KGA and GLS2 expressions were not significant;

[0080] qPCR was used to detect the liver tissue, kidney tissue, collected spleen leukocytes, and spleen sca-1 cells and bone marrow sca-1 cells obtained by magnetic bead sorting in the two groups of mice. It was found that the expression levels of GLS-related genes in various tissues and cells were regulated to varying degrees;

[0081] Relative expression = 2^ -(ΔΔCt) , ΔΔCt = (Ct target gene - Ct reference gene) experimental group - (Ct target gene - Ct reference gene) control group;

[0082] like Fig.16As shown, compared with the VEH group, the expression level of GLS2 mRNA in the liver tissue of CML mice treated with 968 was upregulated, the expression level of KGA mRNA was upregulated, and the expression of GAC mRNA did not change significantly;

[0083] like Fig.17 As shown, the expression of GAC and KGA in kidney was not significantly changed;

[0084] like Fig.18 As shown, in sca-1 cells sorted from bone marrow leukocytes, the expression of KGA and GAC did not change significantly, and GLS2 was almost not expressed;

[0085] like Fig.19 As shown, the expression of GAC in spleen leukocytes did not change significantly, while the expression of KGA was significantly upregulated;

[0086] like Fig. 20 As shown, in sca-1 cells obtained by splenic leukocyte sorting, GAC expression was significantly decreased, and KGA expression was upregulated to a certain extent but not significantly;

[0087] These results suggest that intervention in glutamine metabolism alters the gene expression of GLS in CML mice.

[0088] Example 4

[0089] Study on the effect of compound 968 on bone marrow LSK cells based on CML mouse model

[0090] After the treatment of mice in the 968 group and the VEH group, bone marrow cells were collected and flow cytometry was used to detect bone marrow LSK cells (LSK, Lin - Sca-1 + c-Kit + ) ratio (the flow cytometry antibody was purchased from Thermo Fisher Scientific). Fig.21 It can be seen that compared with the solvent control group (VEH), compound 968 can significantly reduce the proportion and number of bone marrow LSK cells in CML mice, suggesting that the therapeutic effect of 968 on CML mice may be due to the inhibitor's ability to reduce bone marrow LSK cells, thereby exerting a therapeutic effect.

[0091] Example 5

[0092] In vivo experiments demonstrated that the combination of GLS1 inhibitor compound 968 and IM effectively prolonged the survival of CML mice

[0093] 6-8 week old CML mice were randomly divided into four groups: Compound 968 group (10 mg / kg / d) (Compound 968 purchased from MCE), IM group (50 mg / kg / d) (IM purchased from selleck), 968+IM combined group, and solvent control group (VEH group). After grouping, CML mice stopped feeding doxycycline, and treatment began 30 days after induction of disease. Compound 968 was administered once a day for five consecutive days a week, for a total of three weeks by intraperitoneal injection; IM was administered once a day for 21 consecutive days of gavage. After the end of treatment, the drug was stopped and the survival curve was observed. The results are shown in the figure. Fig. 22 (968: 10 mg / kg / d compound 968 treatment group, sample size n=7; IM: 50 mg / kg / d imatinib treatment group, sample size n=8; 968+IM: compound 968 and imatinib combination treatment group, sample size n=8; VHE: solvent control treatment group, sample size n=8), the observation period was 300 days after the start of treatment, and a survival curve was made.

[0094] Example 6

[0095] In vivo experiments demonstrated that the combination of GLS1 inhibitor compound 968 and IM effectively reduced peripheral blood neutrophils

[0096] During the treatment, peripheral blood samples were collected from the compound 968 group, IM group, 968+IM combined group and solvent control group (VEH group) to detect changes in the proportion of neutrophils (flow cytometry antibodies purchased from Biolegend). Fig.23 (968: 10 mg / kg / d compound 968 treatment group, sample size n=3; IM: 50 mg / kg / d imatinib treatment group, sample size n=3; 968+IM: compound 968 and imatinib combination treatment group, sample size n=3; VHE: solvent control treatment group, sample size n=3) as shown in the results, compared with the solvent control group (VEH), the 986 group and the 968+IM group could reduce the proportion of peripheral blood neutrophils during the experiment.

[0097] The in vivo test results of the present invention confirm that the glutaminase inhibitor compound 968 can effectively reduce the LSK cells in the bone marrow of CML mice, regulate the metabolism of energy substances in CML mice, change the expression level of the GLS1 gene in the body, and is beneficial to improving the development of CML disease and significantly prolonging the survival of CML mice when used in combination with IM.

Claims

1. A use of GAC as a target for preparing a product for preventing and / or treating chronic myeloid leukemia, characterized in that: The GAC is glutaminase C, which is a splice form of kidney-type glutaminase GLS1.

2. The use of GAC according to claim 1 as a target for preparing a product for preventing and / or treating chronic myeloid leukemia, characterized in that: The product includes a kit or a separately packaged pharmaceutical composition or a mixed packaged pharmaceutical composition.

3. Use of substances that inhibit or reduce GAC gene expression and / or activity in the preparation of products for inhibiting chronic myeloid leukemia.

4. Use of the substance for inhibiting or reducing GAC gene expression and / or activity according to claim 3 in the preparation of a product for inhibiting chronic myeloid leukemia, characterized in that: The substances that inhibit or reduce the expression and / or activity of the GLS1 gene include GAC inhibitor compound 968, GAC siRNA and GAC gRNA.

5. A use of a GAC ​​inhibitor 968 in the preparation of a drug for preventing and / or treating chronic myeloid leukemia.

6. Use of the GAC inhibitor 968 according to claim 5 in the preparation of a drug for preventing and / or treating chronic myeloid leukemia, characterized in that: The GAC inhibitor 968 is a Glutaminase C allosteric inhibitor, also known as C-IN-1, with a molecular weight of 475.42 and a molecular formula of C 27 H 27 BrN2O, CAS No. 311795-38-7, has the following structural formula:

7. Use of the GAC inhibitor 968 according to claim 6 in combination with imatinib (IM) in the preparation of a product for inhibiting chronic myeloid leukemia.

8. A product, characterized in that The composition of the product includes the GAC inhibitor 968 and IM as described in claim 6.

9. The product according to claim 8, characterized in that The product has at least one of the following functions: 1) Treatment of chronic myeloid leukemia; 2) Inhibit the growth of peripheral blood neutrophils in chronic myeloid leukemia; 3) Inhibit the growth of bone marrow LSK cells in chronic myeloid leukemia; 4) Inhibit GLS1 gene expression or activity.

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