Application of CD132 in treatment of AML

Abstract

The invention discloses an application of CD132 in treatment of AML (acute myeloid leukemia). The CD132 is differentially expressed in the AML, the expression level of the CD132 is related to the malignant degree of the AML and the total survival rate of AML patients, and CD132 knock-down can inhibit the proliferation capacity of AML cells, influence the cell cycle of the AML cells and promote cell apoptosis of the AML cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to the application of biomarkers in the treatment of AML. Background Art

[0002] AML (acute myeloid leukemia, AML) is the most common adult leukemia and seriously endangers human health and life safety. The treatment of AML usually includes induction chemotherapy, consolidation chemotherapy and hematopoietic stem cell transplantation. The purpose of induction chemotherapy is to enable the patient to achieve complete remission, that is, the proportion of primitive cells in the bone marrow is less than 5%, and clinical symptoms and signs disappear. Commonly used chemotherapy regimens include cytarabine combined with anthracyclines. For high-risk patients or patients with relapsed and refractory disease, hematopoietic stem cell transplantation may be an effective treatment option. In recent years, with the development of molecular biology and targeted therapy, some targeted drugs targeting specific gene mutations or fusion genes have also been gradually applied to the treatment of AML, such as FLT3 inhibitors, IDH1 / 2 inhibitors, etc.

[0003] The prognosis of AML varies depending on factors such as subtype, patient age, general condition, cytogenetics and molecular biological characteristics. In general, M3 leukemia has a relatively good prognosis after timely and effective treatment; while patients with complex chromosomal abnormalities and certain gene mutations (such as FLT3-ITD hypermutation, etc.) have a poor prognosis. Older patients with other diseases also often have a poor prognosis. The long-term survival rate of AML patients under 60 years old is 35-40%, while the long-term survival rate of patients 60 years old and above is only 5-15%. Acute myelomonocytic / monocytic leukemia (including French-American-British classification, i.e., M4 and M5 in FAB classification, accounting for 29.1% and 8.9% of AML, respectively) is an acute leukemia in which malignant clonal proliferation of both granulocytes and monocytes occurs simultaneously. The relapse rate is significantly higher than other AML subtypes. The incidence of bone marrow and extra-bone marrow relapse after hematopoietic stem cell transplantation is 34% and 50%, respectively. The 3-year disease-free survival rate of AML-M4 / 5 patients is only about 26%, so AML-M4 / 5 is the main population of relapsed and refractory patients (Blood. 2018 Jul 26; 132(4): 362-8.; Cancer discovery. 2020 Apr; 10(4): 536-51). Improving the long-term survival rate of AML patients has always been a clinical problem that needs to be solved urgently.

[0004] AML is a complex disease. Although some progress has been made in diagnosis, treatment and prognosis assessment, there are still many shortcomings. Further in-depth research is needed in the future to develop more accurate diagnostic methods, effective treatments and reliable prognostic assessment indicators to improve patients' survival rate and quality of life. Summary of the invention

[0005] In order to make up for the deficiencies of the prior art, the present invention provides a target that can achieve the treatment of AML.

[0006] To achieve the above object, the present invention provides the following technical solution:

[0007] The first aspect of the present invention provides the use of CD132 as a target in the preparation of a drug for treating AML.

[0008] Furthermore, the drug includes an inhibitor of CD132.

[0009] Furthermore, the inhibitor includes any substance that can reduce the expression of nucleic acid encoding CD132, reduce the level of CD132 protein, or inhibit the activity of CD132.

[0010] Furthermore, the inhibitor includes nucleic acid inhibitors, proteolytic enzymes, protein binding molecules or a combination thereof.

[0011] Furthermore, the nucleic acid inhibitors include interfering RNA, ribozymes, antisense oligonucleotides, zinc fingers, and gRNA.

[0012] Furthermore, the nucleic acid inhibitor is selected from interfering RNA.

[0013] Furthermore, the nucleic acid inhibitor is shRNA.

[0014] Furthermore, the shRNA is shown as SEQ ID NO.1 and SEQ ID NO.2.

[0015] Furthermore, the nucleic acid inhibitor is gRNA.

[0016] Furthermore, the sequence of the gRNA is shown in SEQ ID NO.3.

[0017] Furthermore, the nucleic acid inhibitor includes gRNA and Cas9 protein.

[0018] Furthermore, the protein binding molecule is selected from anti-CD132 antibodies.

[0019] Furthermore, the anti-CD132 antibody is REGN7257.

[0020] Furthermore, the AML includes M4 and / or M5 subtypes.

[0021] Furthermore, the drug inhibits the proliferation of AML cells, affects the cell cycle of AML cells, or promotes apoptosis of AML cells.

[0022] A second aspect of the present invention provides a drug for treating AML, wherein the drug comprises an inhibitor of CD132.

[0023] Furthermore, the inhibitor includes any substance that can reduce the expression of nucleic acid encoding CD132, reduce the level of CD132 protein, or inhibit the activity of CD132.

[0024] Furthermore, the inhibitor includes nucleic acid inhibitors, proteolytic enzymes, protein binding molecules or a combination thereof.

[0025] Furthermore, the nucleic acid inhibitors include interfering RNA, ribozymes, antisense oligonucleotides, zinc fingers, and gRNA.

[0026] Furthermore, the nucleic acid inhibitor is selected from interfering RNA.

[0027] Furthermore, the nucleic acid inhibitor is shRNA.

[0028] Furthermore, the shRNA is shown as SEQ ID NO.1 and SEQ ID NO.2.

[0029] Furthermore, the nucleic acid inhibitor is gRNA.

[0030] Furthermore, the sequence of the gRNA is shown in SEQ ID NO.3.

[0031] Furthermore, the nucleic acid inhibitor includes gRNA and Cas9 protein.

[0032] Furthermore, the protein binding molecule is selected from anti-CD132 antibodies.

[0033] Furthermore, the anti-CD132 antibody is REGN7257.

[0034] Furthermore, the drug also includes a pharmaceutically acceptable adjuvant.

[0035] Furthermore, the AML includes M4 and / or M5 subtypes.

[0036] The third aspect of the present invention provides the use of CD132 in screening candidate drugs for treating AML.

[0037] Furthermore, the method for screening candidate drugs for treating AML is as follows: treating a culture system expressing or containing the CD132 gene or a protein encoded by it with a substance to be screened; and detecting the expression or activity of the CD132 gene or a protein encoded by it in the system; wherein, when the substance to be screened inhibits the expression level or activity of the CD132 gene or a protein encoded by it, the substance to be screened is a candidate drug for treating AML.

[0038] Furthermore, the candidate drug inhibits the proliferation of AML cells, affects the cell cycle of AML cells, or promotes apoptosis of AML cells.

[0039] Furthermore, the AML is selected from M4 and / or M5 subtypes.

[0040] A fourth aspect of the present invention provides a method for screening candidate drugs for treating AML, the method comprising treating a culture system expressing or containing the CD132 gene or a protein encoded by it with a substance to be screened; and detecting the expression or activity of the CD132 gene or a protein encoded by it in the system; wherein, when the substance to be screened inhibits the expression level or activity of the CD132 gene or a protein encoded by it, the substance to be screened is a candidate drug for treating AML.

[0041] Furthermore, the method also includes a step of verifying the function of the candidate drug, such as verifying the effect of the candidate drug on AML cell morphology, cell proliferation, cell cycle, and cell apoptosis.

[0042] Furthermore, the candidate drug inhibits the proliferation of AML cells, affects the cell cycle of AML cells, or promotes apoptosis of AML cells.

[0043] Furthermore, the AML is selected from M4 and / or M5 subtypes.

[0044] A fifth aspect of the present invention provides a method for computer-assisted screening of candidate drugs for treating AML, the method comprising:

[0045] Obtain the domain structure of CD132,

[0046] Based on the spatial structure of the domain of CD132, substances that regulate CD132 are screened, and the substances that regulate CD132 are candidate drugs for treating AML.

[0047] Furthermore, the method also includes a step of verifying the function of the candidate drug, such as verifying the effect of the candidate drug on AML cell morphology, cell proliferation, cell cycle, and cell apoptosis.

[0048] Furthermore, the AML is selected from M4 and / or M5 subtypes.

[0049] The sixth aspect of the present invention provides the use of a candidate drug screened by the method described in the fourth aspect or the fifth aspect of the present invention in the preparation of a drug for treating AML.

[0050] The seventh aspect of the present invention provides the use of CD132 in constructing AML.

[0051] Furthermore, the method for constructing an AML model comprises administering a CD132 promoter.

[0052] Furthermore, the promoter includes any substance that can increase the expression of nucleic acid encoding CD132, increase the level of CD132 protein or promote the activity of CD132.

[0053] Furthermore, the promoter includes a CD132 overexpression system.

[0054] Furthermore, the CD132 overexpression system includes selecting an expression vector including a plasmid, a viral vector or a transposon vector, and constructing it by chemical transfection, physical transfection or viral infection.

[0055] Furthermore, the plasmid includes pCDNA series vectors, the viral vector includes a lentiviral vector, an adeno-associated virus (AAV) vector or an adenoviral vector, and the transposon vector includes a PiggyBac transposon vector or a Sleeping Beauty transposon vector.

[0056] Furthermore, the viral vector is selected from a lentiviral vector.

[0057] Furthermore, the chemical transfection method includes liposome transfection and cationic polymer transfection, and the physical transfection method includes electroporation and gene gun method.

[0058] Furthermore, the AML includes M4 and / or M5 subtypes.

[0059] An eighth aspect of the present invention provides a method for constructing an AML model, the method comprising administering an agent that regulates CD132.

[0060] Furthermore, the reagent includes a CD132 promoter.

[0061] Furthermore, the promoter includes any substance that can increase the expression of nucleic acid encoding CD132, increase the level of CD132 protein or promote the activity of CD132.

[0062] Furthermore, the promoter includes a CD132 overexpression system.

[0063] Furthermore, the AML model is a malignant model.

[0064] Furthermore, the AML malignant model includes one or more of changes in cell morphology, enhanced cell proliferation ability, cell cycle disorder, cell differentiation disorder, changes in cell adhesion and migration ability, metabolic changes and immune phenotype changes.

[0065] A ninth aspect of the present invention provides an AML cell model, wherein the cells overexpress CD132.

[0066] Furthermore, the AML cell model is constructed by the construction method described in the eighth aspect of the present invention.

[0067] The tenth aspect of the present invention provides the use of the AML cell model described in the ninth aspect of the present invention in screening drugs for treating AML or evaluating the efficacy of drugs for treating AML.

[0068] According to an eleventh aspect of the present invention, there is provided a method for treating AML, comprising administering to a subject an effective amount of a CD132 inhibitor.

[0069] The present invention has the following advantages and beneficial effects:

[0070] The present invention discovers for the first time a new target for the diagnosis, typing, prognosis evaluation and treatment of acute myeloid leukemia - CD132. The present invention finds that CD132 is significantly highly expressed in AML M4 and M5 subtypes, and the CD132 expression level is correlated with the malignancy of AML M4 / 5 subtypes and the overall survival rate of AML M4 / 5 subtype patients. CD132 knockdown can inhibit the proliferation ability of AML cells, affect the cell cycle of AML cells, and promote the apoptosis of AML cells, which confirms the effectiveness of CD132 in the prevention, treatment and / or diagnosis of AML. Moreover, the present target is a membrane surface protein, which is more conducive to targeted treatment of acute myeloid leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 The differential expression effect diagram of CD132 in AML, wherein 1A is the differential expression effect diagram of CD132 in normal CD34+ cells and AML cells analyzed by the data set of GEO database, 1B is the differential expression effect diagram of CD132 in AMLM4 / 5 subtype and healthy people in the training set detected by flow cytometry; 1C is the differential expression effect diagram of CD132 in AMLM4 / 5 subtype and healthy people in the validation set detected by flow cytometry; 1D is the differential expression effect diagram of CD132 in AML M4 / 5 subtype and AML non-M4 / 5 subtype in the training set detected by flow cytometry; 1E is the differential expression effect diagram of CD132 in AMLM4 / 5 subtype and AML non-M4 / 5 subtype in the training set detected by flow cytometry;

[0072] Figure 2 2A is the ROC curve of CD132 used to diagnose AML M4 / 5 subtypes and healthy subjects in the training set; 2B is the ROC curve of CD132 used to diagnose AML M4 / 5 subtypes and healthy subjects in the validation set; 2C is the ROC curve of CD132 used to diagnose AML M4 / 5 subtypes and AML non-M4 / 5 subtypes in the training set; 2D is the ROC curve of CD132 used to diagnose AML M4 / 5 subtypes and AML non-M4 / 5 subtypes in the validation set;

[0073] Figure 3This is a correlation analysis diagram between WT1 and CD132;

[0074] Figure 4 The following are the effects of CD132 on the prognosis prediction of AMLM4 / 5 subtypes. 4A is the effect diagram of the differential expression of CD132 in different stemness-strength acute monocytic leukemia (FAB-M4 / 5) subtype samples under the LSC17 (Nature 540, 433–437 (2016)) standard analyzed by the GEO database data set. 4B is the effect diagram of the correlation between CD132 and the long-term overall survival rate of AMLM4 / 5 subtypes analyzed by the OHSU database.

[0075] Figure 5 This is a diagram of the differential colony formation of AML cells with CD132 high / low;

[0076] Figure 6 6A is a graph showing the effect of knocking down CD132 on cell function in a human leukemia cell line, wherein 6B is a graph showing the effect on THP-1 cell proliferation, 6C is a graph showing the effect on THP-1 cell colony formation, 6D is a graph showing the effect on THP-1 cell apoptosis, 6E is a graph showing the effect on Molm-13 cell proliferation, 6F is a graph showing the effect on Molm-13 cell colony formation, 6G is a graph showing the effect on Molm-13 cell cycle, and 6H is a graph showing the effect on OCI-AML3 cell cycle;

[0077] Figure 7 7A is a diagram showing the effect of overexpression of CD132 on cell function in human leukemia cell lines, wherein 7A is a diagram showing the effect on THP-1 cell proliferation, and 7B is a diagram showing the effect on THP-1 cell cycle;

[0078] Figure 8 This is a diagram showing the effect of CD132 antibody on the leukemia cell cycle.

[0079] Note: *(p<0.05), **(p<0.01), ***(p<0.001) ****(p<0.0001); DETAILED DESCRIPTION

[0080] The following provides definitions of some terms used in this specification. Unless otherwise defined, all technical and scientific terms used in the present invention generally have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0081] The present invention provides the use of a reagent for detecting CD132 in preparing a product for diagnosing AML / predicting the prognosis of AML.

[0082] In the present invention, CD132 includes wild type, mutant or its fragment. The term covers full length, unprocessed CD132, and any form of CD132 processed from cells. The term covers naturally occurring variants (such as splice variants or allelic variants) of CD132. The term covers, for example, CD132 genes, human CD132 and CD132 from any other vertebrate sources, including mammals, such as primates and rodents (such as mice and rats). As a preferred embodiment, in the present invention, CD132 is a gene for people, and gene ID is 3561.

[0083] In the present invention, the term "diagnosis" refers to the discovery, judgment or cognition of the health status or condition of an individual based on one or more symptoms, data or other information related to the individual. Diagnosis can usually be performed between disease patients and normal human bodies, or between different disease subtypes of disease patients. In the present invention, the diagnosis of AML includes diagnosing individuals who do not suffer from AML and individuals who suffer from AML, or diagnosing the FAB typing of AML. The FAB typing of acute myeloid leukemia (AML) is mainly classified according to the morphological characteristics of leukemia cells and the results of cytochemical staining, as follows: M0 (acute myeloid leukemia microdifferentiation), M1 (acute myeloid leukemia undifferentiated type), M2 (acute myeloid leukemia partially differentiated type), M3 (acute promyelocytic leukemia), M4 (acute myelomonocytic leukemia), M5 (acute monocytic leukemia), M6 (erythroleukemia), M7 subtype (acute megakaryocytic leukemia) seven subtypes. In some embodiments of the present invention, CD132 is used to diagnose different FAB subtypes, such as diagnosing M0 / M1 / M2 and M4 / M5 subtype patients.

[0084] In the present invention, the term "prognosis" refers to determining or predicting the course of a disease or condition. The course of a disease or condition can be determined, for example, based on life expectancy (survival rate, survival period) or quality of life. "Prognosis" includes the determination of the time course of a disease or condition with or without treatment. In the case of considering treatment, prognosis includes determining the efficacy of the disease or condition. In the present invention, the prognosis prediction of AML includes predicting the long-term overall survival rate of AML patients.

[0085] The present invention provides a reagent for detecting CD132 selected from an oligonucleotide probe that specifically recognizes the CD132 gene, a primer that specifically amplifies the CD132 gene, or a binding agent that specifically binds to a protein encoded by the CD132 gene.

[0086] In the present invention, the term "probe" refers to a molecule that can bind to a specific sequence or subsequence or other part of another molecule. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe that can bind to another polynucleotide (often referred to as a "target polynucleotide") through complementary base pairing. Depending on the stringency of the hybridization conditions, the probe can bind to a target polynucleotide that lacks complete sequence complementarity with the probe. The probe can be labeled directly or indirectly. Hybridization methods include, but are not limited to: solution phase, solid phase, mixed phase or in situ hybridization assays.

[0087] In the present invention, the term "amplification primer" or "primer" refers to an oligonucleotide that can site-specifically anneal to an RNA or DNA region adjacent to a target sequence and serve as an initiator of DNA synthesis under suitable conditions, under which the synthesis of primer extension products is induced, for example, in the presence of nucleotides and a polymerization inducing agent such as a DNA-dependent DNA polymerase, and suitable temperature, pH, metal concentration, and salt concentration. Typically, a PCR reaction uses a pair of amplification primers, also referred to as a "primer pair," including an "upstream" or "forward" primer and a "downstream" or "reverse" primer that defines the region of RNA or DNA to be amplified.

[0088] In the present invention, the term "amplification" refers to a method of replicating a portion of a nucleic acid using, for example, any of a variety of primer extension reactions. Exemplary primer extension reactions include, but are not limited to, PCR. Unless otherwise specified, "amplification" refers to a single replication, or arithmetic, logarithmic, or exponential amplification.

[0089] In the present invention, the term "binding agent" refers to all or part of a protein (protein, protein-like or protein-containing) molecule that can bind to a membrane protein using specific intermolecular interactions. The binding agent of an albumen is, for example, a receptor of a protein, a lectin that binds to a protein, an antibody to a protein, a peptide body (peptidebody) to a protein, a bispecific dual binding agent or a bispecific antibody form. More specifically, the term "binding agent" refers to a polypeptide, more specifically a protein domain. Suitable protein domains are elements of the overall protein structure, which are self-stable and folded independently of the rest of the protein chain and are commonly referred to as "binding domains". The length of such binding domains varies from about 25 amino acids to 500 amino acids and more. Many binding domains can be classified as folded and are recognizable, identifiable, 3-D structures. Some folds are very common in many different proteins, so that they are given specific names.

[0090] The reagents for detecting CD132 provided in the present invention also include detectable labels. Detectable labels refer to compositions that can produce detectable signals indicating the presence of target polynucleotides in the assay sample. Suitable labels include, but are not limited to, radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, bioluminescent moieties. Therefore, labels are any compositions that can be detected by a device or method, including but not limited to spectroscopy, photochemistry, biochemistry, immunochemistry, electricity, optics, chemical detection devices or any other suitable devices. In some embodiments, labels can be detected visually without the aid of a device.

[0091] In the present invention, radioactive isotopes include but are not limited to 3 H. 14 C. 35 S. 125 I. 131 I. Enzymes include, but are not limited to, horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, acetylcholinesterase. Fluorescent molecules include, but are not limited to, FITC, rhodamine, and lanthanide phosphors.

[0092] The present invention can use any known method to detect the expression level of CD132 gene or protein, including but not limited to RT-PCR, qRT-PCR, biochip detection, Southern blotting, in situ hybridization, and immunoblotting.

[0093] In the present invention, "expression level" refers to the amount, accumulation or rate of a biomarker molecule or genome. The expression level can be represented, for example, by the amount or synthesis rate of a messenger RNA (mRNA) encoded by a gene, the amount or synthesis rate of a polypeptide or protein encoded by a gene, or the amount or synthesis rate of a biomolecule accumulated in a cell or biological fluid. The term "expression level" refers to the absolute amount of a molecule in a sample or the relative amount of the molecule determined under steady-state or non-steady-state conditions.

[0094] The product for diagnosing AML / predicting the prognosis of AML of the present invention can be provided in any form, including but not limited to a chip, a kit, a test paper or a nucleic acid membrane strip.

[0095] In the present invention, the term "chip", also referred to as "array", refers to a solid support comprising connected nucleic acid or peptide probes. The array typically comprises a plurality of different nucleic acid or peptide probes attached to the surface of a substrate at different known positions. These arrays, also referred to as "microarrays", can typically be produced using mechanical synthesis methods or light-guided synthesis methods that incorporate a combination of photolithography and solid phase synthesis methods. The array may comprise a flat surface, or may be a nucleic acid or peptide on a bead, gel, polymer surface, fiber such as an optical fiber, glass or any other suitable substrate. The array may be packaged in a manner that allows for diagnosis of a fully functional device or other manipulation.

[0096] In the present invention, the term "nucleic acid membrane strip" includes a substrate and an oligonucleotide probe fixed on the substrate; the substrate can be any substrate suitable for fixing the oligonucleotide probe, such as a nylon membrane, a nitrocellulose membrane, a polypropylene membrane, a glass sheet, a silica gel wafer, a micro-magnetic bead, etc.

[0097] In the present invention, the term "kit" refers to any delivery system for delivering materials, including kits for research and clinical applications.

[0098] The kit described in the present invention includes a reagent for detecting CD132 gene or protein, and one or more substances selected from the group consisting of: container, instruction manual, positive control, negative control, buffer, adjuvant, solvent, preservative or protein stabilizer. The components of the kit can be packaged in the form of an aqueous medium or in a lyophilized form. Suitable containers in the kit generally include at least one vial, test tube, flask, PET bottle, syringe or other container, in which a component can be placed, and preferably, can be appropriately divided. When there are more than one component in the kit, the kit will generally also include a second, third or other additional container, in which additional components are placed separately. However, components of different combinations may be contained in a vial. The kit of the present invention will generally also include a container for accommodating reactants, sealed for commercial sale. Such a container may include an injection molded or blow molded plastic container, in which the required vial can be retained.

[0099] The kits described in the present invention include but are not limited to qPCR kits, ELISA kits, immunoblotting detection kits, immunochromatography detection kits, immunohistochemistry detection kits, flow cytometry analysis kits, and electrochemiluminescence detection kits.

[0100] The chip, kit, test paper or nucleic acid membrane strip described in the present invention can be used to detect the expression level of multiple genes or proteins including CD132 gene or protein and their expression products (for example, multiple genes or proteins related to AML). Detecting multiple markers of AML simultaneously can greatly improve the accuracy of AML diagnosis or prognosis prediction.

[0101] The present invention provides the use of CD132 as a target in preparing a drug for treating AML.

[0102] In the present invention, the term "treatment" may refer to therapeutic treatment or preventive measures, wherein the goal is to prevent or slow down (mitigate) an undesirable physiological condition, disorder or disease, or to obtain a beneficial or desired clinical result. In the present invention, treatment may refer to both treatment and prevention. Beneficial or desired clinical results include, but are not limited to, relieving symptoms; alleviating the extent of a condition, disorder or disease; stabilizing (i.e., not worsening) the state of a condition, disorder or disease; delaying the onset of a condition, disorder or disease or slowing its progression; improving a condition, disorder or disease state; and alleviating (whether partial or complete) (whether detectable or undetectable) or improving or ameliorating a condition, disorder or disease. Treatment may include causing a clinically significant response without excessive side effects. Treatment also includes a prolonged survival period compared to the expected survival period when not receiving treatment.

[0103] In the present invention, the drug includes an inhibitor of CD132. An inhibitor refers to any substance that can inhibit the activity of CD132 protein, inhibit the stability of CD132 gene or protein, inhibit the expression level of CD132, inhibit the effective action time of CD132 protein, and inhibit the activity of CD132. As an embodiment of the present invention, the "inhibitor" is a substance that inhibits the expression level of CD132.

[0104] In the present invention, inhibitors include nucleic acid inhibitors, proteolytic enzymes, protein binding molecules, and combinations thereof.

[0105] In some embodiments, the nucleic acid inhibitor is selected from: an interfering molecule with CD132 or its transcript as a target sequence and capable of inhibiting CD132 gene expression or gene transcription, including but not limited to shRNA, siRNA, ribozyme, antisense oligonucleotide, dsRNA, microRNA, zinc finger, gRNA, or a construct that can express or form the shRNA, siRNA, ribozyme, antisense oligonucleotide, dsRNA, microRNA, zinc finger, gRNA. Protein inhibitors are selected from substances that can inhibit CD132 protein. Proteolytic enzymes are selected from enzymes that can catalyze CD132 proteolysis. Protein binding molecules are selected from substances that specifically bind to CD132 protein, such as antibodies or ligands that can inhibit CD132 protein activity.

[0106] In some embodiments, the nucleic acid inhibitor is selected from siRNA. siRNA may include partially purified RNA, substantially pure RNA, synthetic RNA or recombinantly produced RNA and RNA that is different from natural RNA by adding, deleting, replacing and / or changing one or more nucleotides. The changes may include adding non-nucleotide substances, such as adding to the end of the siRNA or one or more internal nucleotides of the siRNA; making the siRNA resistant to nuclease digestion (e.g., using 2'-substituted ribonucleotides or modifying the sugar phosphate backbone); or replacing one or more nucleotides in the siRNA with deoxyribonucleotides.

[0107] In some embodiments, the nucleic acid inhibitor is selected from shRNA. shRNA is a non-coding small RNA molecule that can form a hairpin structure. shRNA can inhibit gene expression through the RNA interference pathway. When shRNA is introduced into a cell, it will be recognized and cut into small interfering RNA (siRNA) of about 21 nucleotides by the nuclease Dicer in the cell. siRNA binds to a protein complex called RNA-induced silencing complex (RISC). The Argonaute protein in RISC uses the antisense strand of siRNA to recognize and bind to the target mRNA, and then degrades the target mRNA by cutting or inhibiting translation, thereby achieving inhibition of specific gene expression. In a specific embodiment of the present invention, the nucleic acid inhibitor is selected from shRNA.

[0108] In some embodiments, the nucleic acid inhibitor is selected from gRNA. In some embodiments, gRNA is a segment of nucleotides complementary to the target DNA sequence, which guides the Cas9 protein to a specific DNA site and then performs double-stranded DNA cleavage at the PAM sequence, so as a preferred embodiment, the nucleic acid inhibitor includes gRNA and Cas9 protein.

[0109] In some embodiments, the nucleic acid inhibitor is selected from ribozymes, a class of RNAs that can be engineered to enzymatically cleave and inactivate other RNA targets in a specific sequence-dependent manner. Ribozymes and methods of delivering them are well known in the art (Hendry et al., BMC Chem. Biol., 4(1): 1 (2004); Grassi et al., Curr. Pharm. Biotechnol., 5(4): 369-386 (2004); Bagheri et al., Curr. Mol. Med., 4(5): 489-506 (2004); Kashani-Sabet M., Expert Opin. Biol. Ther., 4(11): 1749-1755 (2004), each of which is incorporated herein by reference in its entirety. By cleaving the target RNA, ribozymes inhibit translation, thereby preventing expression of the target gene. Ribozymes can be chemically synthesized in the laboratory and structurally modified to increase their stability and catalytic activity using methods known in the art. Alternatively, the ribozyme gene can be introduced into the cell by gene delivery mechanisms known in the art.

[0110] In some embodiments, nucleic acid inhibitors are selected from antisense oligonucleotides. Antisense oligonucleotides (antisense nucleic acid sequences) may include nucleotide sequences complementary to sense nucleic acids encoding proteins (e.g., complementary to double-stranded cDNA molecule coding strands or complementary to CD132 mRNA). Antisense oligonucleotides and delivery methods are well known in the art (Goodchild, Curr. Opin. Mol. Ther., 6 (2): 120-128 (2004); Clawson et al., Gene Ther., 11 (17): 1331-1341 (2004)), which are incorporated into the present invention by reference in their entirety. Antisense oligonucleotides may be complementary to the complete coding strand of a target sequence, or only partially complementary to it. The length of antisense oligonucleotides may be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or more nucleotides.

[0111] In some embodiments, the inhibitor is selected from a protein binding molecule. A protein binding molecule refers to any molecule that binds to a protein, including but not limited to small molecule compounds, polypeptides, proteins, antibodies. In some embodiments, the binding molecule of the protein is selected from an antibody. The antibodies include but are not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired biological activity. "Antibody fragments" comprise a portion of a complete antibody, preferably comprising its antigen binding region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. In a specific embodiment, the antibody is selected from a monoclonal antibody such as REGN7257.

[0112] In some embodiments, the inhibitor is selected from proteolytic enzymes. Proteolytic enzymes are a class of enzymes that can catalyze the hydrolysis of proteins, including but not limited to serine proteases, thiol proteases, aspartic proteases, and metalloproteases.

[0113] The drug of the present invention can also be used in combination with other compounds for treating AML, and other compounds for treating AML can be administered simultaneously with the main active ingredient (e.g., an inhibitor of CD132), or even administered simultaneously in the same composition, and the two can be administered sequentially (e.g., before or after) or simultaneously in the same pharmaceutical formulation (i.e., together) or in different pharmaceutical formulations (i.e., alone). It is a single formulation in the same formulation, while it is non-single in different pharmaceutical formulations. Regarding the route of administration, the posology of other compounds for treating AML and the main active ingredient (e.g., an inhibitor of CD132) can also be different.

[0114] In some embodiments, the medicine includes pharmaceutically acceptable adjuvants, and the adjuvants include excipients, disintegrants, sweeteners, adhesives, coating agents, expanders, lubricants, glidants, flavoring agents, solubilizers, etc. For administration, in addition to active ingredients, the medicine of the present invention can preferably be prepared using at least one pharmaceutical carrier. When the composition is formulated as a liquid solution, it can contain at least one pharmaceutical carrier selected from the following: saline solution, sterile water, Ringer's solution, buffered saline, injectable albumin solution, glucose solution, maltodextrin solution, glycerol, ethanol and mixtures thereof. If necessary, other conventional additives can be added, including antioxidants, buffer, antibacterial agents, etc. In addition, diluents, dispersants, surfactants, adhesives and lubricants can be further added to prepare injectable preparations (such as aqueous solutions, suspensions or emulsions, etc.), pills, capsules, granules or tablets.

[0115] As an embodiment, diluents such as lactose, sodium chloride, glucose, urea, starch, water, etc.; adhesives such as starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methyl cellulose, carboxymethyl cellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, alginic acid and alginate, xanthan gum, hydroxypropyl cellulose and hydroxypropyl methylcellulose, etc.; surfactants such as polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, monostearate, cetyl alcohol, etc.; lubricants such as zinc stearate, monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolauric sucrose acid ester, sodium lauryl sulfate, magnesium lauryl sulfate, magnesium lauryl sulfate, etc.

[0116] The medicine of the present invention can be administered orally, parenterally, by inhalation spray, topical, rectal, nasal, buccal, vaginal or by implanted drug storage device. The medicine of the present invention can contain any commonly used non-toxic pharmaceutically acceptable carrier, adjuvant or excipient. In some cases, medicinal acid, alkali or buffer can be used to adjust the pH of the preparation to improve the stability of the compound or its dosage form. The term parenteral used in the present invention includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. As long as the target tissue can be reached, the medicine of the present invention can be given to the receptor by any route.

[0117] The present invention provides the use of CD132 in screening candidate drugs for the treatment of AML. In some embodiments, the candidate drug can regulate any substance of CD132, including but not limited to small molecule compounds, protein drugs, and nucleic acid drugs. The protein drugs include but are not limited to polypeptides and antibodies; the nucleic acid drugs include but are not limited to interfering RNA, ribozymes, antisense oligonucleotides, zinc fingers, and gRNA.

[0118] The present invention provides a method for screening candidate drugs for treating AML, the method comprising treating a culture system expressing or containing a CD132 gene or a protein encoded by it with a substance to be screened; and detecting the expression or activity of the CD132 gene or the protein encoded by it in the system; wherein, when the substance to be screened inhibits the expression level or activity of the CD132 gene or the protein encoded by it, the substance to be screened is a candidate drug for treating AML.

[0119] In some embodiments, the culture system includes (but is not limited to) a cell system, a subcellular system, a solution system, a tissue system, an organ system or an animal system (such as an animal model, preferably an animal model of a non-human mammal, such as a mouse, rabbit, sheep, monkey, etc.), etc.

[0120] The present invention provides the application of CD132 in constructing AML model. In some embodiments, AML model is constructed by administering a promoter of CD132. The term "promoter" refers to any substance that can increase the expression of nucleic acid encoding CD132, increase CD132 protein level or promote the activity of CD132, including CD132 overexpression system or CD132 agonist.

[0121] In some embodiments, the CD132 overexpression system comprises obtaining the CD132 gene by chemical synthesis, an organism acquisition method or a gene library screening method, selecting an expression vector including a plasmid or a viral vector or a transposon vector, and constructing it by chemical transfection, physical transfection or viral infection.

[0122] The chemical synthesis method includes directly chemically synthesizing the CD132 gene or synthesizing multiple short fragments and then splicing them into the CD132 gene. The organism acquisition method includes extracting total RNA and obtaining cDNA through reverse transcription, and then amplifying the CD132 gene using PCR technology or directly extracting the CD132 gene from genomic DNA. The gene library screening method includes screening from a genomic library and a cDNA library.

[0123] The term "vector" is a tool that can carry the target gene into the host cell and replicate and express it there. Common vectors include plasmids, viral vectors, transposon vectors, etc.

[0124] As one embodiment, the vector is selected from plasmids, non-limiting examples of which include pQE-12, pUC-series, pCDNA series (e.g., pcDNA1, pcDNA3 (Invitrogen), pcDNA3.1), pBluescript (Stratagene), pET-series expression vectors (Novagen) or pCRTOPO (Invitrogen), λgt11, pJOE, pBBR1-MCS series, pJB861, pBSMuL, pBC2, pUCPKS, pTACT1, pTRE, pCAL -n-EK, pESP-1, pOP13CAT, E-027pCAGKosak-Cherry (L45a) vector system, pREP (Invitrogen), pCEP4 (Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, pIZD35, Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pRc / CMV, pSPORT1 (GIBCO BRL), pGEMHE (Promega), pLXIN, pSIR (Clontech), pIRES-EGFP (Clontech), pEAK-10 (EdgeBiosystems) pTriEx-Hygro (Novagen) and pCINeo (Promega). Non-limiting examples of plasmid vectors suitable for Pichia pastoris include, for example, plasmids pAO815, pPIC9K, and pPIC3.5K (all Invitrogen). Another vector suitable for expressing proteins in Xenopus embryos, zebrafish embryos, and a variety of mammalian and avian cells is the multi-purpose expression vector pCS2+.

[0125] As an embodiment, the vector is selected from a viral vector such as a retroviral vector. The retroviral vector includes but is not limited to adenoviral vectors, lentiviral vectors, Sendai virus vectors, baculovirus vectors, Epstein-Barr virus vectors, papovavirus vectors, vaccinia virus vectors, herpes simplex virus vectors, hybrid vectors and adeno-associated virus (AAV) vectors. In a specific embodiment of the present invention, the vector is selected from a lentiviral vector.

[0126] The present invention provides a method for constructing an AML model, wherein the AML model is a malignant model.

[0127] In the present invention, the term "malignant model" is an experimental model used to study the malignant transformation of cells and the mechanism of tumor occurrence and development. Compared with the conventional AML model, the malignant model has the following changes in performance or characterization: changes in cell morphology, enhanced cell proliferation ability, cell cycle disorder, cell differentiation disorder, changes in cell adhesion and migration ability, metabolic changes and immune phenotype. In a specific embodiment of the present invention, the AML malignant model has the characteristics of cell cycle disorder and enhanced cell proliferation ability.

[0128] The present invention will be further described in detail below in conjunction with the accompanying drawings and examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0129] Example 1 Expression level of CD132 in AML cells

[0130] 1. Experimental methods

[0131] 1) Database analysis

[0132] The transcriptome differences between AML cells and normal CD34+ cells were analyzed using the R language (version 4.3.2) and the DESeq package in the GEO database dataset (GSE30029).

[0133] 2) Detection of CD132 and WT1 expression by flow cytometry

[0134] Bone marrow blood samples were collected from the subjects and divided into training and validation sets. The training set included 13 healthy donors, 16 AML patients with M0, M1, and M2 subtypes, and 21 AML patients with M4 and M5 subtypes. The validation set included 14 healthy donors, 16 AML patients with M0, M1, and M2 subtypes, and 22 AML patients with M4 and M5 subtypes.

[0135] The diagnostic criteria for AML refer to the WHO 2016 classification criteria for hematopoietic and lymphoid tumors: 1) ≥ 20% of primitive cells in peripheral blood or bone marrow; 2) but when the patient is confirmed to have clonal recurrent cytogenetic abnormalities t(8;21)(q22;q22), inv(16)(p13q22) or t(16;16)(p13;q22) and t(15;17)(q22;q12).

[0136] Preparation of primary AML patient / healthy donor bone marrow cells: about 3 mL of bone marrow blood was collected from the posterior superior iliac spine and anticoagulated in EDTA or heparin tubes. Density gradient centrifugation was used to extract bone marrow mononuclear cells; bone marrow mononuclear cells were placed in PBS; CD132-PE and AML surface antigen CD117-FITC / CD34-APC / CD33-BV421 (antibodies were purchased from BD Biosciences or biolegend) were labeled; incubated for 15 minutes; washed twice with PBS; flow cytometry analysis was performed on CD34+ cells and AML cell surface CD132 expression, and RT-qPCR was used to analyze WT1 expression, and ROC curve analysis was performed.

[0137] 3) The Pearson correlation coefficient analysis method was used to analyze the correlation between WT1 and CD132 in all samples.

[0138] 2. Experimental results

[0139] 1) The results are as follows: Figure 1 As shown in A, the expression level of CD132 in the transcriptome of AML cells was significantly higher than that in normal CD34+ cells, and the difference was statistically significant (P=0.0004).

[0140] 2) Flow cytometry was used to measure the expression of CD132. The results were as follows: Figure 1 As shown in B-1E, the expression level of CD132 in AML-M4 / 5 subtype was significantly higher than that in healthy donors and AML patients with non-M4 / 5 subtypes (M0 / M1 / M2 subtypes).

[0141] The ROC curve analysis results are as follows Figure 2 As shown, CD132 can effectively distinguish the M4 / 5 subtype from healthy subjects (training set: AUC is 0.8168, sensitivity is 100%, specificity is 66.67%, cutoff value is 42.82; validation set: AUC is 0.7727, sensitivity is 100%, specificity is 59.09%, cutoff value is 50.9), as well as distinguish the M4 / 5 subtype from non-M4 / 5 patients (training set: AUC is 0.7783, sensitivity is 87.5%, specificity is 61.9%, cutoff value is 48; validation set: AUC is 0.7642, sensitivity is 81.25%, specificity is 59.09%, cutoff value is 49.85).

[0142] 3) The results of correlation analysis are as follows Figure 3 As shown, WT1 does not associate with CD132.

[0143] Example 2 Application of CD132 in predicting the prognosis of AML cells

[0144] 1. Experimental methods

[0145] 1) In the dataset of GEO database (GSE14468), the acute monocytic leukemia (FAB-M4 / 5) subtype was analyzed for CD132 expression in different stemness samples according to the LSC17 scoring criteria (Nature. 2016 Dec 15; 540(7633): 433-437.);

[0146] 2) Perform survival analysis on the acute myeloid leukemia dataset BeatAML using logrank in the OHSU database.

[0147] 2. Experimental results

[0148] 1) The results are as follows: Figure 4 As shown in A, the expression level of CD132 in the top 50 samples with the strongest stemness was significantly higher than that in the top 50 samples with the weakest stemness (P=0.0077), indicating that CD132 is associated with the degree of stemness of AMLM4 / 5 subtype.

[0149] 2) Survival analysis was performed using the acute leukemia dataset from the OHSU database, with the diagnosis to 12 months of follow-up as the node, and landmark survival analysis was performed. The results are as follows Figure 4 As shown in B, the dotted line represents the overall survival rate before and after diagnosis to 12 months of follow-up. After 12 months, there was a significant difference in the survival of patients with high CD132 and low CD132 groups. High CD132 expression was significantly correlated with the long-term overall survival rate of AML (FAB-M4 / 5) patients. The overall survival of patients with high CD132 expression was significantly higher than that of AML (FAB-M4 / 5) patients with low CD132 expression. Therefore, CD132 can be used to predict the prognosis of AML (FAB-M4 / 5) patients.

[0150] Example 3 Effect of CD132 on AML cells

[0151] 1. Experimental methods

[0152] 1) Bone marrow cell colony formation assay

[0153] (1) Sorting CD132 high and CD132 low cells

[0154] Primary leukemia patient / healthy donor bone marrow cells: about 3 mL of bone marrow blood was collected from the posterior superior iliac spine, and anticoagulated in EDTA or heparin tubes. Density gradient centrifugation was used to extract bone marrow mononuclear cells; bone marrow mononuclear cells were placed in PBS; labeled with CD132-PE and AML surface antigen CD117-FITC / CD34-APC / CD33-BV421 (antibodies were purchased from BD Biosciences or biolegend); incubated for 15 minutes; washed twice with PBS; flow sorter was used to sort CD117 / CD34 / CD33+ and CD132high / low cells, and received in 15 ml centrifuge tubes; CFU was plated.

[0155] (2) Colony formation assay

[0156] Thaw stem cell culture medium for CFU assay at room temperature (3 mL of stem cell culture medium in each 15 mL centrifuge tube); resuspend and count cells, inoculate 5000 cells / well; take 1.1 mL of stem cell culture medium and add it to a 35 mm small dish, and make one duplicate well. Cover the dish lid. Add water or PBS to the third dish; put the three small dishes into a larger dish, cover the dish lid, and culture for 7-14 days; observe and count under a microscope.

[0157] 2) Functional experiments after CD132 silencing in cell lines

[0158] (1) Construction of CD132 silenced cell line

[0159] Human leukemia cell lines: THP-1, Molm-13, OCI-AML3, shCD132#1: 5'-CAGCTGGACTGAACAATCAGTG GAT-3' (SEQ ID NO.1); shCD132#2: 5'-CATTGGAGTGAATGGAGCCACCCA A-3' (SEQ ID NO.2); gRNA 5'-CAAAACACTGAACCTCTGGG-3' (SEQ ID NO.3), shScr is a negative control; CD132 cloning vector: pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO; TrueCut TM Cas9 protein v2 (Thermo Fisher Scientific Invitrogen TM ).

[0160] Cell knockdown: Prepare a medium containing RPMI1640 supplemented with 2% FBS and resuspend the cells to 5×10 5 / mL, placed in a 6-well plate. Using the above lentiviral vector, under 30 MOI conditions, after 16 hours of transfection, centrifuge at 1000 rpm for 3 minutes, change to 10% FBS complete medium and place in an incubator for 48 hours, use puromycin for drug screening for 48 hours, and use RT-qPCR and flow cytometry to verify the transfection efficiency of the cells.

[0161] Cell knockout: CD132 gRNA and TrueCut TM Cas9 protein v2 (Thermo Fisher Scientific Invitrogen TM ) were incubated at room temperature for 15 min to construct the ribonucleoprotein complex (RNP). The cell line was resuspended in the electroporation solution in the celetrix cell kit (HY-15559) to 1×10 6 / mL, mix the incubated RNP complex and the resuspended cell line evenly into the electroporation cup, turn on the celetrix electroporator to the cell line mode, and place the electroporation cup for electroporation. After electroporation, transfer the cell line to complete medium and culture for 72 hours. After T7E1 genome verification, perform functional experiments.

[0162] (2) Experiment on cell proliferation ability of CD132-silenced human leukemia cell lines

[0163] Cell plating: adjust the cell concentration to 1×10 5 / mL, 100μL / well of 96-well plate, 3 replicates; lay multiple concentration gradients to make a standard curve; after culturing for 0-4 days according to experimental requirements, add 10μL / well of CCK8; after 4 hours, measure the OD value at a wavelength of 450nm using an enzyme reader.

[0164] (3) Colony formation experiment of CD132 silencing human leukemia cell line

[0165] Thaw stem cell culture medium for CFU assay at room temperature (3 mL of stem cell culture medium in each 15 mL centrifuge tube); resuspend and count cells, inoculate 2000-5000 cells / well; take 1.1 mL of cell culture medium and add it to a 35 mm small dish, and make 1 duplicate well. Cover the dish lid. Add water or PBS to the third dish; put the three small dishes into a larger 100 mm dish, cover the dish lid, and culture for 7-14 days; observe and count under a microscope.

[0166] (4) Cell cycle experiment of CD132 silencing human leukemia cell line

[0167] Prepare a culture medium containing RPMI1640 supplemented with 2% FBS and 10 μg / mL Hoechst 33342. 6The cells were resuspended with 1 mL of culture medium containing Hoechst 33342; incubated at 37°C for 60 min; washed once with PBS containing 10 μg / mL Hoechst 33342; resuspended with 100 μL of PBS; and flow cytometry was performed on the BV421 channel.

[0168] (5) Apoptosis experiment of CD132 silencing human leukemia cell line

[0169] Aspirate the cells into the flow tube; wash once with PBS. That is, add 1mL of PBS to each tube to resuspend the cells and centrifuge (centrifugation parameters: 1500rpm, 5min); wash once with Annexin Vbinding buffer. That is, add 1mL of Annexin Vbinding buffer to each tube to resuspend the cells and centrifuge (centrifugation parameters: 1500rpm, 5min); add 150μL / tube of Annexin Vbinding buffer working solution to resuspend the cells and make the cells form a single cell suspension; add 2.5μL of Annexin V-APC and 5μL of 7-AAD to each tube; keep it away from light for 15min; detect on the machine.

[0170] 3) Functional experiments after overexpression in cell lines

[0171] (1) Construction of overexpression cell line: Human leukemia cell line: THP-1, CD132 overexpression vector: pHBLV-CMV-MCS-3FLAG-EF1-ZsGreen-T2A-PURO. Cell overexpression: Prepare medium containing RPMI1640 with 2% FBS, resuspend the cells to 5×10 5 / mL, placed in a 6-well plate. Using the above lentiviral vector, under 30 MOI conditions, after 16 hours of transfection, centrifuge at 1000 rpm for 3 minutes, change to 10% FBS complete medium and place in an incubator for 48 hours, use puromycin for drug screening for 48 hours, and use RT-qPCR and flow cytometry to verify the transfection efficiency of the cells.

[0172] (2) Cell proliferation ability experiment of CD132 overexpressing human leukemia cell line

[0173] Cell plating: adjust the cell concentration to 1×10 5 / mL, 100μL / well of 96-well plate, 3 replicates; lay multiple concentration gradients to make a standard curve; after culturing for 0-4 days according to experimental requirements, add 10μL / well of CCK8; after 4 hours, measure the OD value at a wavelength of 450nm using an enzyme reader.

[0174] (3) Cell cycle experiment of human leukemia cell lines with overexpression of CD132

[0175] Prepare a culture medium containing RPMI1640 supplemented with 2% FBS and 10 μg / mL Hoechst 33342. 6 The cells were resuspended with 1 mL of culture medium containing Hoechst 33342; incubated at 37°C for 60 min; washed once with PBS containing 10 μg / mL Hoechst 33342; resuspended with 100 μL of PBS; and flow cytometry was performed on the BV421 channel.

[0176] 4) Functional experiments after blocking CD132 activity

[0177] Primary AML cells were cultured at 1 × 10 cells / mL using IMDM complete medium. 6 The cell suspension was added with CD132 antibody REGN725721.1ng / mL and IL-2500IU / mL, placed in a six-well plate, and cultured at 37°C for 72h. The culture medium containing IMDM with 2% FBS and 10μg / mL Hoechst 33342 was prepared. 6 The cells were resuspended with 1 mL of culture medium containing Hoechst 33342; incubated at 37°C for 60 min; washed once with PBS containing 10 μg / mL Hoechst 33342; resuspended with 100 μL of PBS; and flow cytometry was performed on the BV421 channel.

[0178] 2. Experimental results

[0179] 1) Bone marrow cell colony formation assay results Figure 5 As shown, the colony formation of AML cells with CD132high was significantly higher than that with CD132low (n=5);

[0180] 2) Effects of CD132 knockdown on human leukemia cell lines on cell function Figure 6 As shown, after knocking down CD132 in THP-1 and Molm-13 cells using shRNA, the cell proliferation ability (6A, 6E) and colony formation ability (6B, 6F) of the human leukemia cell lines were significantly reduced, and the cell cycle (6C, 6G) was significantly affected. Eleven days after infection, a large number of knocked-down tumor cells showed apoptosis (6D). After knocking down CD132 in OCI-AML3 using gRNA, the cell cycle was significantly weakened.

[0181] 3) The results of cell cycle and cell proliferation experiments on human leukemia cell lines after overexpression of CD132 using lentivirus are as follows Figure 7 As shown in A and 7B, the cell cycle and cell proliferation ability of human leukemia cell lines overexpressing CD132 were significantly enhanced.

[0182] 4) The results of the cell cycle experiment of leukemia cell lines after CD132 blocking using CD132 antibody REGN7257 are as follows Figure 8 As shown, the CD132 antibody REGN7257 significantly reduced the cell cycle of primary AML cells, which could be rescued after the simultaneous addition of the CD132 ligand IL-2.

[0183] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. Application of CD132 as a target in the preparation of drugs for the treatment of AML; Preferably, the drug comprises an inhibitor of CD132; Preferably, the inhibitor includes any substance that can reduce the expression of nucleic acid encoding CD132, reduce the level of CD132 protein, or inhibit the activity of CD132; Preferably, the inhibitor comprises a nucleic acid inhibitor, a proteolytic enzyme, a protein binding molecule or a combination thereof; Preferably, the nucleic acid inhibitor includes interfering RNA, ribozyme, antisense oligonucleotide, zinc finger, gRNA; Preferably, the nucleic acid inhibitor is selected from interfering RNA; Preferably, the interfering RNA is selected from shRNA, siRNA, and microRNA; Preferably, the nucleic acid inhibitor is shRNA; Preferably, the sequence of the shRNA is as shown in SEQ ID NO.1 and SEQ ID NO.2; Preferably, the nucleic acid inhibitor is selected from gRNA; Preferably, the sequence of the gRNA is as shown in SEQ ID NO.3; Preferably, the nucleic acid inhibitor further comprises a Cas protein; Preferably, the protein binding molecule comprises an anti-CD132 antibody; Preferably, the antibody is REGN7257; Preferably, the AML is selected from M4 and / or M5 subtypes.

2. A drug for treating AML, characterized in that: The drug includes an inhibitor of CD132; Preferably, the inhibitor includes any substance that can reduce the expression of nucleic acid encoding CD132, reduce the level of CD132 protein, or inhibit the activity of CD132; Preferably, the inhibitor comprises a nucleic acid inhibitor, a proteolytic enzyme, a protein binding molecule or a combination thereof; Preferably, the nucleic acid inhibitor is selected from interfering RNA, ribozyme, antisense oligonucleotide, zinc finger, gRNA; Preferably, the interfering RNA is selected from shRNA, siRNA, and microRNA; Preferably, the nucleic acid inhibitor is shRNA; Preferably, the shRNA is as shown in SEQ ID NO.1 and SEQ ID NO.2; Optionally, the nucleic acid inhibitor is gRNA; Preferably, the sequence of the gRNA is as shown in SEQ ID NO.3; Preferably, the nucleic acid inhibitor further comprises a Cas protein; Preferably, the protein binding molecule comprises an anti-CD132 antibody; Preferably, the antibody is REGN7257; Preferably, the drug further comprises a pharmaceutically acceptable adjuvant; Preferably, the AML comprises M4 and / or M5 subtypes.

3. Application of CD132 in screening candidate drugs for the treatment of AML; Preferably, the method for screening candidate drugs for treating AML is as follows: treating a culture system expressing or containing the CD132 gene or a protein encoded by it with a substance to be screened; and detecting the expression or activity of the CD132 gene or a protein encoded by it in the system; wherein, When the substance to be screened inhibits the expression level or activity of the CD132 gene or the protein encoded by it, the substance to be screened is a candidate drug for treating AML; Preferably, the AML is selected from M4 and / or M5 subtypes.

4. A method for screening candidate drugs for treating AML, characterized in that: The method comprises treating a culture system expressing or containing the CD132 gene or a protein encoded by it with a substance to be screened; and detecting the expression or activity of the CD132 gene or a protein encoded by it in the system; wherein, when the substance to be screened inhibits the expression level or activity of the CD132 gene or a protein encoded by it, the substance to be screened is a candidate drug for treating AML; Preferably, the method further comprises a step of functional verification of the candidate drug; Preferably, the AML is selected from M4 and / or M5 subtypes.

5. A method for computer-assisted screening of candidate drugs for treating AML, characterized in that: The method comprises: Obtain the domain structure of CD132, Screening a substance that regulates CD132 based on the spatial structure of the domain of CD132, wherein the substance that regulates CD132 is a candidate drug for treating AML; Preferably, the method further comprises a step of functional verification of the candidate drug; Preferably, the AML is selected from M4 and / or M5 subtypes.

6. Use of the candidate drug screened by the method according to claim 4 or 5 in the preparation of a drug for treating AML.

7. Application of CD132 in constructing AML models; Preferably, the method for constructing an AML model comprises administering a promoter of CD132; Preferably, the promoter includes any substance that can increase the expression of nucleic acid encoding CD132, increase the level of CD132 protein, or promote the activity of CD132; Preferably, the promoter is selected from a CD132 overexpression system; Preferably, the CD132 overexpression system comprises selecting an expression vector including a plasmid, a viral vector or a transposon vector, and constructing the expression vector by chemical transfection, physical transfection or viral infection; Preferably, the plasmid includes a pCDNA series vector, the viral vector includes a lentiviral vector, an adeno-associated virus (AAV) vector or an adenoviral vector, and the transposon vector includes a PiggyBac transposon vector or a Sleeping Beauty transposon vector; Preferably, the viral vector is selected from a lentiviral vector; Preferably, the chemical transfection method includes liposome transfection and cationic polymer transfection, and the physical transfection method includes electroporation and gene gun method; Preferably, the AML is selected from M4 and / or M5 subtypes.

8. A method for constructing an AML model, characterized in that: The method comprises administering an agent that modulates CD132; Preferably, the reagent includes a CD132 promoter; Preferably, the promoter includes any substance that can increase the expression of nucleic acid encoding CD132, increase the level of CD132 protein, or promote the activity of CD132; Preferably, the promoter comprises a CD132 overexpression system; Preferably, the AML model is a malignant model; Preferably, the AML malignant model includes one or more of changes in cell morphology, enhanced cell proliferation ability, cell cycle disorder, cell differentiation disorder, changes in cell adhesion and migration ability, metabolic changes and immune phenotype changes.

9. An AML cell model, characterized in that The cells overexpress CD132; Preferably, the AML cell model is constructed by the construction method described in claim 8.

10. Use of the AML cell model according to claim 9 in screening drugs for treating AML or evaluating the efficacy of drugs for treating AML.

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