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CN119345372A - Application of CD132 in the treatment of AML - Google Patents

Application of CD132 in the treatment of AML Download PDF

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Publication number
CN119345372A
CN119345372A CN202411510259.0A CN202411510259A CN119345372A CN 119345372 A CN119345372 A CN 119345372A CN 202411510259 A CN202411510259 A CN 202411510259A CN 119345372 A CN119345372 A CN 119345372A
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aml
nucleic acid
protein
cell
inhibitor
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赵翔宇
黄晓军
丁亦扬
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Peking University Peoples Hospital
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Peking University Peoples Hospital
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Abstract

The invention discloses the use of CD132 in the treatment of AML. The present invention discovers that CD132 is differentially expressed in AML, that CD132 expression levels correlate with AML malignancy and overall survival of AML patients, that CD132 knockdown can inhibit proliferation capacity of AML cells, affect the cell cycle of AML cells, and promote apoptosis of AML cells.

Description

Use of CD132 in the treatment of AML
Technical Field
The invention belongs to the field of biological medicine, and relates to application of a biomarker in treating AML.
Background
AML (acute myeloid leukemia, AML) is the most frequent adult leukemia, and severely jeopardizes human health and life safety. Treatment of AML typically includes induction chemotherapy, consolidation chemotherapy, hematopoietic stem cell transplantation, and the like. The purpose of chemotherapy is to achieve complete remission in the patient, i.e. <5% of primary cells in bone marrow, and disappearance of clinical symptoms and signs. Common chemotherapy regimens include cytarabine in combination with anthracyclines and the like. Hematopoietic stem cell transplantation may be an effective therapeutic option for high-risk patients or relapsed refractory patients. In recent years, with the development of molecular biology and targeted therapies, some targeted drugs for specific gene mutations or fusion genes are also gradually applied to the treatment of AML, such as FLT3 inhibitors, IDH1/2 inhibitors, and the like.
The prognosis of AML varies with factors such as subtype, age of patient, general condition, cytogenetic and molecular biological characteristics. Overall, M3 leukemia is treated timely and effectively with a relatively good prognosis, while patients with complex chromosomal abnormalities, certain genetic mutations (e.g., FLT3-ITD hypermutation, etc.), have a poor prognosis. Patients older than age and with other diseases often have poor prognosis, with long-term survival rates of 35-40% for AML patients under 60 years old, and only 5-15% for patients under 60 years old and beyond. Acute granulo-mono/monocytic leukemia (including the Fa-Mei-Ying type, i.e., M4 and M5 in FAB type, 29.1% and 8.9% of AML respectively) is an acute leukemia in which both granulocytes and monocytes undergo malignant clonal proliferation at the same time, with a significantly higher recurrence rate than the other AML subtypes, with a recurrence rate of 34% and 50% of bone marrow and extramedullary recurrence after hematopoietic stem cell transplantation, respectively. AML-M4/5 patients have only about 26% of disease-free survival for 3 years, so AML-M4/5 is the major group of refractory to relapse (blood.2018 Jul 26;132 (4): 362-8.; cancer discover.2020 Apr;10 (4): 536-51). Improving long-term survival of AML patients has been a clinical challenge to be addressed.
AML is a complex disease, and despite advances in diagnosis, treatment and prognostic evaluation, there are a number of shortcomings. Further research is needed in the future to develop more accurate diagnostic methods, effective therapeutic means and reliable prognostic evaluation indicators to improve patient survival and quality of life.
Disclosure of Invention
In order to make up for the deficiencies of the prior art, the present invention provides a target that enables the treatment of AML.
In order to achieve the above purpose, the present invention provides the following technical solutions:
In a first aspect the invention provides the use of CD132 as a target in the manufacture of a medicament for the treatment of AML.
Further, the medicament includes an inhibitor of CD 132.
Further, the inhibitor includes any substance that can reduce the expression of a nucleic acid encoding CD132, reduce the level of CD132 protein, or inhibit the activity of CD 132.
Further, the inhibitor includes a nucleic acid inhibitor, a proteolytic enzyme, a protein binding molecule, or a combination thereof.
Further, the nucleic acid inhibitor includes interfering RNA, ribozyme, antisense oligonucleotide, zinc finger, gRNA.
Further, the nucleic acid inhibitor is selected from interfering RNAs.
Further, the nucleic acid inhibitor is shRNA.
Further, the shRNA is shown as SEQ ID NO.1 and SEQ ID NO. 2.
Further, the nucleic acid inhibitor is a gRNA.
Further, the sequence of the gRNA is shown as SEQ ID NO. 3.
Further, the nucleic acid inhibitor includes a gRNA and a Cas9 protein.
Further, the protein binding molecule is selected from the group consisting of antibodies to CD 132.
Further, the anti-CD 132 antibody is REGN7257.
Further, the AML comprises M4 and/or M5 subtypes.
Further, the agents inhibit proliferation of AML cells, affect the cell cycle of AML cells, or promote AML apoptosis.
In a second aspect, the invention provides a medicament for the treatment of AML comprising an inhibitor of CD 132.
Further, the inhibitor includes any substance that can reduce the expression of a nucleic acid encoding CD132, reduce the level of CD132 protein, or inhibit the activity of CD 132.
Further, the inhibitor includes a nucleic acid inhibitor, a proteolytic enzyme, a protein binding molecule, or a combination thereof.
Further, the nucleic acid inhibitor includes interfering RNA, ribozyme, antisense oligonucleotide, zinc finger, gRNA.
Further, the nucleic acid inhibitor is selected from interfering RNAs.
Further, the nucleic acid inhibitor is shRNA.
Further, the shRNA is shown as SEQ ID NO.1 and SEQ ID NO. 2.
Further, the nucleic acid inhibitor is a gRNA.
Further, the sequence of the gRNA is shown as SEQ ID NO. 3.
Further, the nucleic acid inhibitor includes a gRNA and a Cas9 protein.
Further, the protein binding molecule is selected from the group consisting of antibodies to CD 132.
Further, the anti-CD 132 antibody is REGN7257.
Further, the medicament also comprises pharmaceutically acceptable auxiliary agents.
Further, the AML comprises M4 and/or M5 subtypes.
In a third aspect the invention provides the use of CD132 in the screening of candidate agents for the treatment of AML.
Further, a method for screening a candidate drug for treating AML comprises treating a culture system expressing or containing a CD132 gene or a protein encoded thereby with a substance to be screened, and detecting the expression or activity of the CD132 gene or a protein encoded thereby in the system, wherein the substance to be screened is a candidate drug for treating AML when the substance to be screened inhibits the expression level or activity of the CD132 gene or a protein encoded thereby.
Further, the candidate drug inhibits proliferation of AML cells, affects the cell cycle of AML cells, or promotes AML cell apoptosis.
Further, the AML is selected from the group consisting of M4 and/or M5 subtypes.
In a fourth aspect, the invention provides a method of screening for a candidate agent for the treatment of AML, the method comprising treating a culture system expressing or containing a CD132 gene or a protein encoded thereby with a substance to be screened, and detecting the expression or activity of the CD132 gene or a protein encoded thereby in the system, wherein the substance to be screened is a candidate agent for the treatment of AML when the substance to be screened inhibits the expression level or activity of the CD132 gene or a protein encoded thereby.
Further, the method may include a step of functional validation of the candidate drug, such as validating the effect of the candidate drug on AML cell morphology, cell proliferation, cell cycle, apoptosis.
Further, the candidate drug inhibits proliferation of AML cells, affects the cell cycle of AML cells, or promotes AML cell apoptosis.
Further, the AML is selected from the M4 and/or M5 subtypes.
In a fifth aspect, the invention provides a computer-based method for aiding in screening candidate drugs for the treatment of AML, the method comprising:
the domain of CD132 was obtained and,
Screening for agents that modulate CD132 based on the spatial structure of the domain of CD132, the agents that modulate CD132 being candidate agents for the treatment of AML.
Further, the method may include a step of functional validation of the candidate drug, such as validating the effect of the candidate drug on AML cell morphology, cell proliferation, cell cycle, apoptosis.
Further, the AML is selected from the M4 and/or M5 subtypes.
In a sixth aspect, the invention provides the use of a candidate drug screened in accordance with the method of the fourth or fifth aspect of the invention in the manufacture of a medicament for the treatment of AML.
A seventh aspect of the invention provides the use of CD132 in the construction of AML.
Further, the method of constructing an AML model comprises administering an enhancer of CD 132.
Further, the promoter includes any substance that can increase the expression of a nucleic acid encoding CD132, increase the level of CD132 protein, or promote the activity of CD 132.
Further, the promoter includes a CD132 overexpression system.
Further, CD132 overexpression systems include selection of expression vectors including plasmids or viral vectors or transposon vectors, constructed by chemical transfection or physical transfection or viral infection.
Further, the plasmid includes a pCDNA series vector, the viral vector includes a lentiviral vector, an adeno-associated viral (AAV) vector, or an adenovirus vector, and the transposon vector includes a PiggyBac transposon vector or a sleep Beauty transposon vector.
Further, the viral vector is selected from lentiviral vectors.
Further, the chemical transfection method comprises liposome transfection and cationic polymer transfection, and the physical transfection method comprises electroporation method and gene gun method.
Further, the AML comprises M4 and/or M5 subtypes.
In an eighth aspect, the invention provides a method of constructing an AML model, the method comprising administering an agent that modulates CD 132.
Further, the agent includes a CD132 promoter.
Further, the promoter includes any substance that can increase the expression of a nucleic acid encoding CD132, increase the level of CD132 protein, or promote the activity of CD 132.
Further, the promoter includes a CD132 overexpression system.
Further, the AML model is a malignant model.
Further, the AML malignancy model comprises one or more of altered cell morphology, enhanced cell proliferation capacity, cell cycle disorder, cell differentiation disorder, altered cell adhesion and migration capacity, altered metabolism, and altered immunophenotype.
In a ninth aspect, the invention provides a model of AML cells that overexpress CD132.
Further, the AML cell model is constructed by the construction method according to the eighth aspect of the invention.
In a tenth aspect, the invention provides the use of an AML cell model according to the ninth aspect of the invention for screening for a medicament for the treatment of AML or for evaluating the efficacy of a medicament for the treatment of AML.
In an eleventh aspect, the invention provides a method of treating AML comprising administering to a subject an effective amount of an inhibitor of CD 132.
The invention has the advantages and beneficial effects that:
The novel target-CD 132 for diagnosis, typing, prognosis evaluation and treatment of the acute myeloid leukemia is discovered for the first time, the novel target-CD 132 is found to be remarkably high in the AML M4 and M5 subtypes, the CD132 expression level is related to the malignancy degree of the AML M4/5 subtype and the overall survival rate of AML M4/5 subtype patients, CD132 knockdown can inhibit the proliferation capacity of AML cells, influence the cell cycle of the AML cells, promote the apoptosis of the AML cells, and prove the effectiveness of CD132 in preventing, treating and/or diagnosing the AML, and the target is membrane surface protein, thereby being more beneficial to carrying out targeted treatment on the acute myeloid leukemia.
Drawings
FIG. 1 is a graph showing the differential expression of CD132 in AML, wherein 1A is a graph showing the differential expression of CD132 in normal CD34+ cells and AML cells by data set analysis of GEO database, 1B is a graph showing the differential expression of CD132 in AML M4/5 subtype and healthy human in a flow cytometry detection training set, 1C is a graph showing the differential expression of CD132 in AMLM subtype and healthy human in a flow cytometry detection verification set, 1D is a graph showing the differential expression of CD132 in AML M4/5 subtype and AML non-M4/5 subtype in a flow cytometry detection training set, and 1E is a graph showing the differential expression of CD132 in AMLM/5 subtype and AML non-M4/5 subtype in a flow cytometry detection training set;
FIG. 2 is a ROC graph of CD132 for diagnosis of subtype AMLM/4, wherein 2A is a ROC graph of CD132 in training set for diagnosis of subtype AMLM/5 and healthy persons, 2B is a ROC graph of CD132 in validation set for diagnosis of subtype AML M4/5 and healthy persons, 2C is a ROC graph of CD132 in training set for diagnosis of subtype AML M4/5 and subtype AML non-M4/5, and 2D is a ROC graph of CD132 in validation set for diagnosis of subtype AML M4/5 and subtype AML non-M4/5;
FIG. 3 is a graph of correlation analysis of WT1 and CD 132;
FIG. 4 is a graph showing the effect of CD132 on prognosis prediction of the AMLM subtype 4/5, wherein 4A is a graph showing the effect of data set analysis of GEO database on CD132 on differential expression in samples of different dry-strength acute monocytic leukemia (FAB-M4/5) subtypes under the LSC17 (Nature 540,433-437 (2016)) standard, and 4B is a graph showing the effect of data set analysis of OHSU on CD132 and AMLM subtype AMLM/5 on long-term overall survival;
FIG. 5 is a graph of the head of AML cell populations at CD132 high/low;
FIG. 6 is a graph showing the effect of CD132 knockdown on cell function in a human leukemia cell line, wherein 6A is a graph showing the effect on THP-1 cell proliferation, 6B is a graph showing the effect on THP-1 cell colony formation, 6C is a graph showing the effect on THP-1 cell cycle, 6D is a graph showing the effect on THP-1 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;
FIG. 7 is a graph showing the effect on cell function of human leukemia cell line after over-expressing CD132, wherein 7A is the effect on THP-1 cell proliferation, and 7B is the effect on THP-1 cell cycle;
FIG. 8 is a graph showing the effect of CD132 antibodies on leukemia cell cycle.
Note that (p < 0.05), (p < 0.01), (p < 0.001), (p < 0.0001);
Detailed Description
The following provides definitions of some of the terms used in this specification. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The invention provides the use of an agent for detecting CD132 in the preparation of a product for diagnosing AML/predicting the prognosis of AML.
In the present invention, CD132 includes wild-type, mutant-type, or fragments thereof. The term encompasses full length, unprocessed CD132, as well as any form of CD132 derived from processing in a cell. The term encompasses naturally occurring variants (e.g., splice variants or allelic variants) of CD132. The term encompasses, for example, the CD132 gene, human CD132, and CD132 from any other vertebrate source, including mammals, such as primates and rodents (e.g., mice and rats). As a preferred embodiment, in the present invention, CD132 is a human gene, and the gene ID is 3561.
In the present invention, the term "diagnosis" refers to the discovery, judgment, or cognition of an individual's health state or condition based on one or more symptoms, data, or other information associated with the individual. Diagnosis can be generally performed between a patient with a disease and a normal human body, or between different disease subtypes of a patient with a disease. In the present invention, the diagnosis of AML includes diagnosis of an individual not suffering from AML and an individual suffering from AML, or diagnosis of FAB typing of AML. FAB type of Acute Myeloid Leukemia (AML) is mainly classified according to morphological characteristics of leukemia cells and cytochemical staining results, and specifically comprises seven subtypes of M0 (acute myeloblastic leukemia micro-differentiation type), M1 (acute myeloblastic leukemia non-differentiation type), M2 (acute myeloblastic leukemia partial differentiation type), M3 (acute promyelocytic leukemia), M4 (acute myelomonocytic leukemia), M5 (acute monocytic leukemia), M6 (erythroleukemia) and M7 subtype (acute megakaryoblastic leukemia). In some embodiments of the invention, CD132 is used to diagnose different FAB genotypes, such as diagnosing M0/M1/M2 and M4/M5 subtype patients.
In the present invention, the term "prognosis" refers to determining or predicting the course of a disease or disorder. The course of a disease or disorder may be determined, for example, based on life expectancy (survival ) or quality of life. "prognosis" includes the determination of the time course of a disease or condition with or without treatment. Where treatment is considered, prognosis includes determining the efficacy of treatment for a disease or disorder. In the present invention, the prognosis prediction of AML includes predicting the long-term overall survival of AML patients.
The invention provides reagents for detecting CD132 selected from oligonucleotide probes specifically recognizing the CD132 gene, primers specifically amplifying the CD132 gene, or binding agents specifically binding to a protein encoded by the CD132 gene.
In the present invention, the term "probe" refers to a molecule that is capable of binding to a specific sequence or subsequence or other portion of another molecule. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe that is capable of binding to another polynucleotide (often referred to as a "target polynucleotide") by complementary base pairing. Depending on the stringency of the hybridization conditions, the probe is able to bind to a target polynucleotide that lacks complete sequence complementarity with the probe. The probes may be labeled directly or indirectly. Hybridization means, including but not limited to solution phase, solid phase, mixed phase or in situ hybridization assays.
In the present invention, the term "amplification primer" or "primer" refers to an oligonucleotide that is capable of annealing site-specifically to an RNA or DNA region adjacent to a target sequence and serves as an initiator primer for DNA synthesis under suitable conditions under which synthesis of primer extension products is induced, for example, in the presence of nucleotides and polymerization inducers such as DNA-dependent DNA polymerase, as well as suitable temperatures, pH, metal concentrations, and salt concentrations. Typically, PCR reactions use a pair of amplification primers, also referred to as a "primer pair," including an "upstream" or "forward" primer and a "downstream" or "reverse" primer, which define a region of RNA or DNA to be amplified.
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 specifically stated otherwise, "amplification" refers to single copy, or arithmetic, logarithmic, or exponential amplification.
In the present invention, the term "binding agent" refers to all or part of a protein (protein, proteinaceous or protein-containing) molecule that is capable of binding to a membrane protein using a specific intermolecular interaction. The binding agent for a protein is, for example, a receptor for a protein, a lectin that binds a protein, an antibody directed against a protein, a peptide body (peptidebody) directed against a protein, a bispecific dual binding agent, or a bispecific antibody format. 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 that are self-stabilizing and fold 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 up to 500 amino acids and more. Many binding domains can be classified as folded and identifiable, 3-D structures. Some folds are so common in many different proteins that they are given specific names.
Reagents for detecting CD132 provided by the present invention also include a detectable label. A detectable label refers to a composition capable of producing a detectable signal indicative of the presence of a target polynucleotide in an assay sample. Suitable labels include, but are not limited to, radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, bioluminescent moieties. Thus, a label is any composition that can be detected by a device or method, including but not limited to spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical detection means or any other suitable means. In some embodiments, the indicia may be visually detected without the aid of a device.
In the present invention, the radioisotope includes, but is not limited to 3H、14C、35S、125I、131 I. Enzymes include, but are not limited to, horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, acetylcholinesterase. Fluorescent molecules include, but are not limited to, FITC, rhodamine, and lanthanide phosphors (LANTHANIDE PHOSPHORS).
The present invention may use any known method to detect CD132 gene or protein expression levels, including but not limited to RT-PCR, qRT-PCR, biochip detection, southern blotting, in situ hybridization, immunoblotting.
In the present invention, "expression level" refers to the amount, accumulation or rate of biomarker molecules or genomes. The expression level may be expressed, for example, by the amount or rate of synthesis of messenger RNA (mRNA) encoded by the gene, the amount or rate of synthesis of a polypeptide or protein encoded by the gene, or the amount or rate of synthesis of a biomolecule that accumulates in a cell or biological fluid. The term "expression level" refers to the absolute amount of a molecule or the relative amount of the molecule in a sample as determined under steady or non-steady state conditions.
The product of the invention for diagnosing/prognosticating AML may be provided in any format, including but not limited to a chip, a kit, a test paper or a nucleic acid membrane strip.
In the present invention, the term "chip" is also referred to as "array" and refers to a solid support comprising attached 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 locations. These arrays, also known as "microarrays," can generally be produced using mechanical synthesis methods or light-guided synthesis methods that combine a combination of photolithographic methods and solid-phase synthesis methods. The array may comprise a planar surface or may be a bead, gel, polymer surface, fiber such as optical fiber, glass or any other suitable nucleic acid or peptide on a substrate. The array may be packaged in a manner that allows for diagnosis or other manipulation of the fully functional device.
In the present invention, the term "nucleic acid membrane strip" includes a substrate and an oligonucleotide probe immobilized on the substrate, and the substrate may be any substrate suitable for immobilization of an oligonucleotide probe, such as a nylon membrane, a nitrocellulose membrane, a polypropylene membrane, a glass sheet, a silica gel wafer, a micro magnetic bead, or the like.
In the present invention, the term "kit" refers to any delivery system for delivering materials, including kits for research and clinical applications.
The kit comprises reagents for detecting the CD132 gene or protein and one or more substances selected from the group consisting of a container, an instruction for use, a positive control, a negative control, a buffer, an auxiliary agent, a solvent, a preservative and a protein stabilizer. The components of the kit may be packaged in aqueous medium or in lyophilized form. Suitable containers in the kit typically include at least one vial, test tube, flask, baud bottle, syringe, or other container in which one component may be placed, and preferably, an appropriate aliquot may be performed. Where more than one component is present in the kit, the kit will also typically contain a second, third or other additional container in which the additional components are placed separately. However, different combinations of components may be contained in one vial. The kits of the invention will also typically include a container for holding the reagents, sealed for commercial sale. Such containers may include injection molded or blow molded plastic containers in which the desired vials may be retained.
The kit provided by the invention comprises, but is not limited to, a qPCR kit, an ELISA kit, an immunoblotting detection kit, an immunochromatography detection kit, an immunohistochemical detection kit, a flow cytometry analysis kit and an electrochemiluminescence detection kit.
The chips, kits, test strips or nucleic acid membrane strips described herein can be used to detect the expression levels of a plurality of genes or proteins, including the CD132 gene or protein, and their expression products (e.g., AML-related genes or proteins). The detection of multiple markers of AML can greatly improve the accuracy of AML diagnosis or prognosis prediction.
The invention provides the use of CD132 as a target in the manufacture of a medicament for the treatment of AML.
In the present invention, the term "treatment" may refer to a therapeutic treatment or prophylactic measure, wherein the aim is to prevent or slow down (alleviate) an undesired 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, alleviation of symptoms, diminishment of extent of a condition, disorder or disease, stabilization (i.e., not worsening) of the condition, disorder or disease state, delay of the onset or slowing of the progression of the condition, disorder or disease state, amelioration of the condition, disorder or disease state, and remission (whether partial or complete) (whether detectable or undetectable) or amelioration or palliation of the condition, disorder or disease. Treatment may include eliciting a clinically significant response without undue adverse side effects. Treatment also includes an extended survival compared to the expected survival when untreated.
In the present invention, the drug includes an inhibitor of CD 132. An inhibitor is any substance that inhibits the activity of the CD132 protein, inhibits the stability of the CD132 gene or protein, inhibits the expression level of CD132, inhibits the effective duration of the CD132 protein, inhibits the activity of CD132, and as one embodiment of the present invention, is a substance that inhibits the expression level of CD 132.
In the present invention, inhibitors include nucleic acid inhibitors, proteolytic enzymes, protein binding molecules, and combinations thereof.
In some embodiments, the nucleic acid inhibitor is selected from interfering molecules targeting CD132 or transcripts thereof and capable of inhibiting CD132 gene expression or gene transcription, including but not limited to shRNA, siRNA, ribozymes, antisense oligonucleotides, dsRNA, microRNA, zinc fingers, gRNA, or constructs capable of expressing or forming the shRNA, siRNA, ribozymes, antisense oligonucleotides, dsRNA, microRNA, zinc fingers, gRNA. The protein inhibitor is selected from substances capable of inhibiting CD132 protein. The proteolytic enzyme is selected from enzymes capable of catalyzing the proteolysis of CD 132. The protein binding molecule is selected from substances that specifically bind to CD132 protein, such as antibodies or ligands that inhibit the activity of CD132 protein.
In some embodiments, the nucleic acid inhibitor is selected from the group consisting of siRNA. siRNA may include partially purified RNA, substantially pure RNA, synthetic RNA, or recombinantly produced RNA, as well as altered RNA that differs from natural RNA by the addition, deletion, substitution, and/or alteration of one or more nucleotides. The alteration may include the addition of a non-nucleotide material, such as one or more internal nucleotides added to the end of the siRNA or the siRNA, modification of the siRNA to resist nuclease digestion (e.g., modification of the sugar phosphate backbone using 2' -substituted ribonucleotides), or substitution of one or more nucleotides in the siRNA with deoxyribonucleotides.
In some embodiments, the nucleic acid inhibitor is selected from shRNA. shRNA is a non-coding small RNA molecule capable of forming hairpin structures that can inhibit gene expression through RNA interference pathways. When introduced into a cell, the shRNA is recognized by the nuclease Dicer within the cell and cleaved into small interfering RNAs (sirnas) of about 21 nucleotides. The siRNA binds to a protein complex called RNA-induced silencing complex (RNA-induced silencing complex, RISC). The Argonaute protein in RISC recognizes and binds to target mRNA by using the antisense strand of siRNA, and then degrades the target mRNA by means of cleavage or translation inhibition, etc., thereby realizing inhibition of specific gene expression. In a specific embodiment of the invention, the nucleic acid inhibitor is selected from shRNA.
In some embodiments, the nucleic acid inhibitor is selected from gRNA. In some embodiments, the gRNA is a stretch of nucleotides complementary to the target DNA sequence that directs the Cas9 protein to a specific DNA site to effect double-stranded DNA cleavage at the PAM sequence, thus as a preferred embodiment, the nucleic acid inhibitor comprises the gRNA and Cas9 protein.
In some embodiments, the nucleic acid inhibitor is selected from the group consisting of ribozymes, which are 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 their delivery 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. Ter., 4 (11): 1749-1755 (2004), each of which is incorporated in its entirety into the present invention. By cleavage of target RNAs, ribozymes inhibit translation, thus preventing expression of target genes. Ribozymes can be chemically synthesized in the laboratory to increase their stability and catalytic activity by methods known in the art, or ribozyme genes can be introduced into cells by gene delivery mechanisms known in the art.
In some embodiments, the nucleic acid inhibitor is selected from the group consisting of antisense oligonucleotides. Antisense oligonucleotides (antisense nucleic acid sequences) can include nucleotide sequences that are complementary to a sense nucleic acid encoding a protein (e.g., complementary to the coding strand of a double-stranded cDNA molecule 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 herein in their entirety by reference. The antisense oligonucleotide can be complementary to the entire coding strand of the target sequence, or only a portion thereof. The antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or more nucleotides in length.
In some embodiments, the inhibitor is selected from protein binding molecules. Protein binding molecules refer 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 antibodies. Such antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity. An "antibody fragment" comprises a portion of an intact antibody, preferably comprising an antigen binding region thereof. 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 monoclonal antibodies such as REGN7257.
In some embodiments, the inhibitor is selected from the group consisting of proteolytic enzymes. Proteolytic enzymes are a class of enzymes capable of catalyzing the hydrolysis of proteins including, but not limited to, serine proteases, thiol proteases, aspartic proteases, and metalloproteases.
The medicaments described herein may also be used in combination with other AML-treating compounds, which may be administered simultaneously with the primary active ingredient (e.g., an inhibitor of CD 132), even in the same composition, both of which may 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., separately). While being a single formulation in the same formulation, and not a single formulation in different pharmaceutical formulations. Regarding the route of administration, the dosing of other AML-treating compounds with the primary active ingredient (e.g., inhibitors of CD 132) may also vary.
In some embodiments, the medicament comprises pharmaceutically acceptable adjuvants including excipients, disintegrants, sweeteners, binders, coating agents, bulking agents, lubricants, glidants, flavoring agents, solubilizing agents, and the like. For administration, the medicament of the invention may preferably be formulated using at least one pharmaceutically acceptable carrier in addition to the active ingredient. When the composition is formulated as a liquid solution, it may contain at least one pharmaceutically acceptable carrier selected from the group consisting of saline solution, sterile water, ringer's solution, buffered saline, injectable albumin solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures thereof. Other conventional additives including antioxidants, buffers, bacteriostats, etc. may be added if desired. In addition, diluents, dispersants, surfactants, binders and lubricants may be further added to prepare injectable preparations (such as aqueous solutions, suspensions or emulsions, etc.), pills, capsules, granules or tablets.
As one embodiment, diluents such as lactose, sodium chloride, dextrose, urea, starch, water, etc., binders such as starch, pregelatinized starch, dextrin, maltodextrin, sucrose, acacia, gelatin, methylcellulose, carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, alginic acid and alginate, xanthan gum, hydroxypropyl cellulose, hydroxypropyl methylcellulose, etc., surfactants such as polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, monoglyceride of stearic acid, cetyl alcohol, etc., lubricants such as zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolaurosonic acid ester, sodium lauryl sulfate, magnesium lauryl sulfate, etc.
The medicaments of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally or by an implanted reservoir. The medicament of the invention can contain any common nontoxic medicinal carrier, auxiliary material or excipient. In some cases, a pharmaceutically acceptable acid, base or buffer may be used to adjust the pH of the formulation to improve the stability of the formulated compound or dosage form thereof. The term parenteral as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. The agents of the invention may be administered to the subject by any route so long as the target tissue is achieved.
The invention provides the use of CD132 in screening candidate drugs for the treatment of AML. In some embodiments, the drug candidate may modulate any substance of CD132, including, but not limited to, small molecule compounds, protein drugs, nucleic acid drugs. The protein drugs include, but are not limited to, polypeptides, antibodies, and the nucleic acid drugs include, but are not limited to, interfering RNAs, ribozymes, antisense oligonucleotides, zinc fingers, and gRNAs.
The present invention provides a method for screening a candidate drug for treating AML, which comprises treating a culture system expressing or containing CD132 gene or a protein encoded thereby with a substance to be screened, and detecting the expression or activity of CD132 gene or a protein encoded thereby in the system, wherein the substance to be screened is a candidate drug for treating AML when the substance to be screened inhibits the expression level or activity of CD132 gene or a protein encoded thereby.
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 (e.g., an animal model, preferably an animal model of a non-human mammal, such as a mouse, rabbit, sheep, monkey, etc.), and the like.
The invention provides the use of CD132 in the construction of AML models. In some embodiments, the AML model is constructed by administering an enhancer of CD 132. The term "promoter" refers to any substance that can increase the expression of a nucleic acid encoding CD132, increase the level of CD132 protein, or promote the activity of CD132, including CD132 overexpression systems or CD132 agonists.
In some embodiments, the CD132 overexpression system comprises obtaining the CD132 gene by chemical synthesis, organism acquisition methods, or gene library screening methods, and selecting an expression vector comprising a plasmid or viral vector or transposon vector, and constructing by chemical transfection or physical transfection or viral infection methods.
The chemical synthesis method comprises the steps of directly synthesizing a CD132 gene chemically or synthesizing a plurality of short fragments and splicing the short fragments into the CD132 gene, the organism acquisition method comprises the steps of extracting total RNA, carrying out reverse transcription to obtain cDNA, amplifying the CD132 gene by utilizing a PCR technology or directly extracting the CD132 gene from genome DNA, and the gene library screening method comprises the steps of screening from a genome library and a cDNA library.
The term "vector" is a tool capable of carrying a gene of interest into a host cell and replicating and expressing it therein. Common vectors are plasmids, viral vectors, transposon vectors, and the like.
As one embodiment, the vector is selected from the group consisting of 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 vector (Novagen) or pCRTOPO (Invitrogen), λgt11, pJOE, pBBR1-MCS series, pJB861, pBSMuL, pBC2, pUCPKS, pTACT1, pTRE, pCAL-n-EK, pESP-1, pOP13CAT, E-027pCAG Kosak-Cherry (L45 a) 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 vectors 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 expression of proteins in Xenopus (Xenopus) embryos, zebra fish embryos, and a wide variety of mammalian and avian cells is the multipurpose expression vector pCS2+.
As one embodiment, the vector is selected from viral vectors such as retroviral vectors. Such retroviral vectors include, but are not limited to, adenovirus vectors, lentiviral vectors, sendai virus vectors, baculovirus vectors, epstein Barr virus vectors, papova virus vectors, vaccinia virus vectors, herpes simplex virus vectors, heterozygous vectors, and adeno-associated virus (AAV) vectors. In a specific embodiment of the invention, the vector is selected from lentiviral vectors.
The invention provides a construction method of an AML model, which is a malignant model.
In the present invention, the term "malignant model" is an experimental model for studying the mechanisms of malignant transformation of cells and tumorigenesis and development. The malignant model has altered cell morphology, enhanced cell proliferation capacity, cell cycle disorders, cell differentiation disorders, altered cell adhesion and migration capacity, altered metabolism, and immunophenotype as compared to conventional AML models. In particular embodiments of the invention, AML malignancy models are characterized by a cell cycle disorder, enhanced ability to proliferate cells.
The invention will now be described in further detail with reference to the drawings and examples. The following examples are only illustrative of the present invention and are not intended to limit the scope of the invention.
Example 1 expression level of CD132 in AML cells
1. Experimental method
1) Database analysis
Transcriptome differential analysis was performed on AML cells and normal cd34+ cells using the DESeq package in GEO database dataset (GSE 30029) using R language (4.3.2 version).
2) Flow cytometry to detect the expression levels of CD132 and WT1
Collecting bone marrow blood samples of the subjects, and dividing the bone marrow blood samples into a training set and a verification set, wherein the training set comprises 13 healthy donors, 16M 0, M1 and M2 subtypes of AML patients, 21M 4 and M5 subtypes of AML patients, and the verification set comprises 14 healthy donors, 16M 0, M1 and M2 subtypes of AML patients and 22M 4 and M5 subtypes of AML patients.
AML diagnostic criteria are referenced to WHO 2016 hematopoietic and lymphoid tissue tumor classification criteria, 1) peripheral blood or bone marrow primordial cells ≡20%;2) but when patients are confirmed to have clonally reproducible cytogenetic abnormalities t (8; 21) (q 22; q 22), inv (16) (p 13q 22) or t (16; 16) (p 13; q 22) and t (15; 17) (q 22; q 12).
Primary AML patient/healthy donor bone marrow cells were prepared from approximately 3ml of posterior superior iliac spine bone marrow blood, anticoagulated with edta or heparin tubes. The method comprises the steps of extracting bone marrow blood mononuclear cells by using a density gradient centrifugation method, placing the bone marrow mononuclear cells in PBS, labeling CD132-PE and AML surface antigens CD117-FITC/CD34-APC/CD33-BV421 (antibodies are all purchased from BD Biosciences or biolegend), incubating for 15min, washing 2 times by using PBS, analyzing the CD132 expression quantity on the surfaces of CD34+ cells and AML cells in a flow mode, analyzing the expression quantity of WT1 by using RT-qPCR, and performing ROC curve analysis.
3) The correlation of WT1 with CD132 was analyzed for all samples using pearson (pearson) correlation coefficient analysis.
2. Experimental results
1) As a result of analysis of the dataset (GSE 30029) of the GEO database, the transcriptome level of AML cells was significantly higher than that of normal cd34+ cells, and the difference was statistically significant (p=0.0004), as shown in fig. 1A.
2) The flow cytometry measured the expression level of CD132, and as shown in FIGS. 1B-1E, the expression level of CD132 in AML-M4/5 subtype was significantly higher than that in healthy donors and AML patients of non-M4/5 subtype (M0/M1/M2 subtype).
The results of the ROC curve analysis are shown in FIG. 2, CD132 was effective in distinguishing between subtype M4/5 and healthy subjects (training set: AUC 0.8168, sensitivity 100%, specificity 66.67%, cutoff value 42.82; validation set: AUC 0.7727, sensitivity 100%, specificity 59.09%, cutoff value 50.9), and between subtype M4/5 and non-M4/5 subjects (training set: AUC 0.7783, sensitivity 87.5%, specificity 61.9%, cutoff value 48; validation set: AUC 0.7642, sensitivity 81.25%, specificity 59.09%, cutoff value 49.85).
3) Correlation analysis results as shown in fig. 3, WT1 was not correlated with CD 132.
Example 2 use of CD132 in predicting AML cell prognosis
1. Experimental method
1) CD132 expression analysis of the different dry specimens was performed on the acute monocytic leukemia (FAB-M4/5) subtype in the dataset of the GEO database (GSE 14468) according to the LSC17 scoring criteria (Nature.2016Dec 15;540 (7633): 433-437.);
2) Survival analysis was performed on acute myeloid leukemia dataset BeatAML using logrank in OHSU database.
2. Experimental results
1) Analysis of the dataset (GSE 14468) by GEO database showed that CD132 was significantly higher in the first 50 samples with strongest dryness than in the first 50 samples with weakest dryness (p=0.0077), suggesting that CD132 correlates with the degree of dryness of the AMLM/5 subtype.
2) Survival analysis was performed by means of the acute leukemia dataset of OHSU database, taking the diagnosis of 12 months of follow-up as a node, and landmark survival analysis was performed, and the results are shown in fig. 4B, wherein the dotted line represents the condition of total survival before and after 12 months of follow-up, and the survival of patients with high CD132 and low CD132 group after 12 months is significantly different, and the high CD132 expression level is significantly related to the long-term total survival of patients with AML (FAB-M4/5), and the overall survival of patients with high CD132 expression is significantly higher than that of patients with low CD132 expression of AML (FAB-M4/5), so that CD132 can be used for predicting prognosis of patients with AML (FAB-M4/5).
Example 3 effect of CD132 on AML cells
1. Experimental method
1) Bone marrow cell colony formation experiment
(1) Sorting CD132 high and CD132 low cells
Primary leukemia patient/healthy donor bone marrow cells were anticoagulated with approximately 3ml of bone marrow blood from posterior upper iliac spine, edta or heparin tubes. Bone marrow blood mononuclear cells were extracted using density gradient centrifugation, bone marrow mononuclear cells were placed in PBS, CD132-PE and AML surface antigens CD117-FITC/CD34-APC/CD33-BV421 (antibodies were all purchased from BD Biosciences or biolegend), incubated for 15min, washed 2 times with PBS, flow sorter sorted cells for CD117/CD34/CD33+ while CD132high/low, received using a 15ml centrifuge tube, and CFU plated.
(2) Colony formation experiments
Thawing a stem cell culture medium CFU assay (3 mL of stem cell culture medium is arranged in each 15mL centrifuge tube) at room temperature, counting the cells in a resuspension mode, inoculating 5000 cells/hole, adding 1.1mL of stem cell culture solution into a small dish with the thickness of 35mm, and making 1 compound hole. And (5) covering a dish cover. Adding water or PBS into the third dish, placing the three small dishes into a larger dish, covering the dish, culturing for 7-14 days, and observing and counting under a microscope.
2) Functional assay after silencing CD132 in cell lines
(1) Construction of CD132 silencing cell line
Human leukemia cell line ：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 was the negative control, CD132 cloning vector pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO, trueCut TM Cas9 protein v2 (Cefebrifugen TM).
Cell knockdown media containing RPMI1640 with 2% FBS was prepared, cells were resuspended to 5×10 5/mL and plated in 6-well plates. Using the above lentiviral vector, after 16h transfection at 30MOI, 1000rpm 3min condition centrifugation, complete medium changed to 10% FBS was placed in incubator for 48h, drug screening was performed using puromycin for 48h, and the transfection efficiency of cells was verified using RT-qPCR and flow cytometry.
Cell knockdown CD132 gRNA was incubated with TrueCut TM Cas9 protein v2 (zemoer Invitrogen TM) for 15min at normal temperature to construct ribonucleoprotein complex (RNP). The cell line was resuspended to 1X 10 6/mL using the electrotransfer fluid in celetrix cell kit (HY-15559), the incubated RNP complex was mixed well with the resuspended cell line into an electrotransfer cup, the electrotransfer apparatus was opened celetrix to cell line mode, and the electrotransfer cup was placed for electrotransfer. After electrotransformation, the cell line was transferred to complete medium for 72h, and after T7E1 genome verification, functional experiments were performed.
(2) Experiments on the cell proliferation Capacity of the CD 132-silenced human leukemia cell line
Cell plating, namely adjusting the cell concentration to 1X 10 5/mL, 96-well plates to 100 mu L/well and 3 compound wells, paving a plurality of concentration gradients, preparing a standard curve, culturing for 0-4 days according to experimental requirements, adding 10 mu L/well of CCK8, measuring the OD value by an enzyme-labeled instrument at the wavelength of 450nm after 4 hours.
(3) CD132 silencing human leukemia cell line colony formation experiment
Thawing a stem cell culture medium CFU assay (3 mL of stem cell culture medium is arranged in each 15mL centrifuge tube) at room temperature, counting the cell resuspension, inoculating 2000-5000 cells/hole, adding 1.1mL of cell culture solution into a small dish with 35mm, and making 1 multiple holes. And (5) covering a dish cover. Adding water or PBS into the third dish, placing the three small dishes into a larger 100mm dish, covering the dish cover, culturing for 7-14 days, and observing and counting under a microscope.
(4) Cell cycle experiments on CD 132-silenced human leukemia cell lines
A culture medium containing 10. Mu.g/mL Hoechst 33342 was prepared by adding 2% FBS to RPMI 1640. Each 1X10 6 cell was resuspended in 1mL of medium containing Hoechst 33342, incubated at 37℃for 60min, washed once with PBS containing 10. Mu.g/mL of Hoechst 33342, resuspended in 100. Mu.L of PBS, and flow tested in BV421 channels.
(5) Apoptosis experiments on CD 132-silenced human leukemia cell line
Cells were aspirated into the flow tube and washed once with PBS. I.e.1 mL of PBS was added to each tube to resuspend the cells and centrifuged (centrifugation parameters: 1500rpm, 5 min) and washed once with Annexin Vbindingbuffer. Namely, 1mL Annexin Vbinding buffer of resuspended cells were added per tube and centrifuged (centrifugation parameters: 1500rpm, 5 min), 150. Mu.L/tube of AnnexinVbindingbuffer working solution was added to resuspend cells and allow the cells to form a single cell suspension, 2.5. Mu.L of Annexin V-APC and 5. Mu.L of 7-AAD were added per tube, and the mixture was allowed to stand in the dark for 15min and examined by the upper machine.
3) Functional assay after overexpression of cell lines
(1) The over-expression cell line is constructed by human leukemia cell line THP-1 and CD132 over-expression vector pHBLV-CMV-MCS-3FLAG-EF1-ZsGreen-T2A-PURO. Cell overexpression media containing RPMI1640 with 2% FBS was prepared, cells were resuspended to 5X 10 5/mL and plated in 6-well plates. Using the above lentiviral vector, after 16h transfection at 30MOI, 1000rpm 3min condition centrifugation, complete medium changed to 10% FBS was placed in incubator for 48h, drug screening was performed using puromycin for 48h, and the transfection efficiency of cells was verified using RT-qPCR and flow cytometry.
(2) Cell proliferation potency assay of CD132 over-expressed human leukemia cell line
Cell plating, namely adjusting the cell concentration to 1X 10 5/mL, 96-well plates to 100 mu L/well and 3 compound wells, paving a plurality of concentration gradients, preparing a standard curve, culturing for 0-4 days according to experimental requirements, adding 10 mu L/well of CCK8, measuring the OD value by an enzyme-labeled instrument at the wavelength of 450nm after 4 hours.
(3) Cell cycle experiments on CD132 overexpressing human leukemia cell lines
A culture medium containing 10. Mu.g/mL Hoechst 33342 was prepared by adding 2% FBS to RPMI 1640. Each 1X10 6 cell was resuspended in 1mL of medium containing Hoechst 33342, incubated at 37℃for 60min, washed once with PBS containing 10. Mu.g/mL of Hoechst 33342, resuspended in 100. Mu.L of PBS, and flow tested in BV421 channels.
4) Functional assay after blocking CD132 Activity
Primary AML cells were prepared as a1×10 6 cell suspension using IMDM complete medium, CD132 antibody regn725721.1ng/mL, IL-2500IU/mL were added, placed in six well plates and incubated at 37 ℃ for 72h. A culture medium containing IMDM with 2% FBS and 10. Mu.g/mL Hoechst 33342 was prepared. Each 1X 10 6 cell was resuspended in 1mL of medium containing Hoechst 33342, incubated at 37℃for 60min, washed once with PBS containing 10. Mu.g/mL of Hoechst 33342, resuspended in 100. Mu.L of PBS, and flow tested in BV421 channels.
2. Experimental results
1) Bone marrow cell colony formation experimental results as shown in fig. 5, AML cell colony formation by CD132high was significantly elevated compared to CD132low (n=5);
2) The effect of CD132 knockdown on cell function of human leukemia cell line As shown in FIG. 6, after CD132 knockdown of THP-1, molm-13 cells using shRNA, cell proliferation ability (6A, 6E), colony formation ability (6B, 6F) and cell cycle (6C, 6G) of human leukemia cell line were significantly reduced, and 11 days after infection, the knockdown tumor cells showed massive apoptosis (6D), and after CD132 knockdown of OCI-AML3 using gRNA, cell cycle was significantly reduced.
3) Cell cycle experiments and cell proliferation experiments of human leukemia cell lines after CD132 overexpression of cells using lentiviruses are shown in FIGS. 7A and 7B, the cell cycle is remarkably enhanced and the cell proliferation capacity is remarkably enhanced by the human leukemia cell lines with CD132 overexpression.
4) Cell cycle experiments on leukemia cell lines following CD132 blocking of cells using CD132 antibody REGN7257 as shown in figure 8, CD132 antibody REGN7257 significantly reduced the cell cycle of primary AML cells, and was salvaged after simultaneous addition of CD132 ligand IL-2.
The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.

Claims (10)

  1. Use of cd132 as a target in the preparation of a medicament for the treatment of AML;
    preferably, the medicament comprises an inhibitor of CD 132;
    Preferably, the inhibitor comprises any agent that reduces expression of a nucleic acid encoding CD132, reduces CD132 protein levels, or inhibits CD132 activity;
    Preferably, the inhibitor comprises a nucleic acid inhibitor, a proteolytic enzyme, a protein binding molecule, or a combination thereof;
    preferably, the nucleic acid inhibitor comprises interfering RNA, ribozyme, antisense oligonucleotide, zinc finger, gRNA;
    Preferably, the nucleic acid inhibitor is selected from interfering RNAs;
    preferably, the interfering RNA is selected from shRNA, siRNA, microrna;
    Preferably, the nucleic acid inhibitor is shRNA;
    preferably, the sequence of the shRNA is shown as 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 shown as SEQ ID NO. 3;
    preferably, the nucleic acid inhibitor further comprises a Cas protein;
    Preferably, the protein binding molecule comprises an antibody against CD 132;
    preferably, the antibody is REGN7257;
    preferably, the AML is selected from the M4 and/or M5 subtype.
  2. 2. A medicament for treating AML, comprising an inhibitor of CD 132;
    Preferably, the inhibitor comprises any agent that reduces expression of a nucleic acid encoding CD132, reduces CD132 protein levels, or inhibits CD132 activity;
    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 RNAs, ribozymes, antisense oligonucleotides, zinc fingers, grnas;
    preferably, the interfering RNA is selected from shRNA, siRNA, microrna;
    Preferably, the nucleic acid inhibitor is shRNA;
    Preferably, the shRNA is shown as SEQ ID NO.1 and SEQ ID NO. 2;
    optionally, the nucleic acid inhibitor is a gRNA;
    Preferably, the sequence of the gRNA is shown as SEQ ID NO. 3;
    preferably, the nucleic acid inhibitor further comprises a Cas protein;
    Preferably, the protein binding molecule comprises an antibody against CD 132;
    preferably, the antibody is REGN7257;
    Preferably, the medicament further comprises pharmaceutically acceptable adjuvants;
    preferably, the AML comprises the M4 and/or M5 subtype.
  3. Use of cd132 in screening candidate drugs for the treatment of AML;
    Preferably, the method of screening a candidate agent for treating AML comprises treating a culture system expressing or containing a CD132 gene or a protein encoded thereby with a substance to be screened, and detecting the expression or activity of the CD132 gene or a protein encoded thereby in the system, wherein the substance to be screened is a candidate agent for treating AML when the substance to be screened inhibits the expression level or activity of the CD132 gene or a protein encoded thereby;
    preferably, the AML is selected from the M4 and/or M5 subtype.
  4. 4. A method for screening a candidate drug for treating AML, which comprises treating a culture system expressing or containing a CD132 gene or a protein encoded thereby with a substance to be screened, and detecting the expression or activity of the CD132 gene or the protein encoded thereby in the system, wherein the substance to be screened is a candidate drug for treating AML when the substance to be screened inhibits the expression level or activity of the CD132 gene or the protein encoded thereby;
    preferably, the method further comprises a step of functional verification of the candidate drug;
    preferably, the AML is selected from the M4 and/or M5 subtype.
  5. 5. A method of computer-based assisted screening for a candidate drug for the treatment of AML, the method comprising:
    the domain of CD132 was obtained and,
    Screening for agents that modulate CD132 based on the spatial structure of the domain of CD132, the agents that modulate CD132 being candidate agents for the treatment of AML;
    preferably, the method further comprises a step of functional verification of the candidate drug;
    preferably, the AML is selected from the M4 and/or M5 subtype.
  6. 6. Use of a candidate agent screened based on the method of claim 4 or 5 in the manufacture of a medicament for the treatment of AML.
  7. Application of CD132 in construction of AML model;
    Preferably, the method of constructing an AML model comprises administering an enhancer of CD 132;
    Preferably, the promoter comprises any agent that increases expression of a nucleic acid encoding CD132, increases CD132 protein levels, or promotes CD132 activity;
    Preferably, the promoter is selected from the group consisting of CD132 overexpression systems;
    preferably, the CD132 overexpression system comprises selecting an expression vector, including a plasmid or viral vector or transposon vector, constructed by a chemical transfection method or a physical transfection method or a viral infection method;
    Preferably, the plasmid comprises a pCDNA series vector, the viral vector comprising a lentiviral vector, an adeno-associated viral (AAV) vector, or an adenovirus vector, the transposon vector comprising a PiggyBac transposon vector or a sleep Beauty transposon vector;
    Preferably, the viral vector is selected from lentiviral vectors;
    preferably, the chemical transfection method comprises liposome transfection and cationic polymer transfection, and the physical transfection method comprises electroporation method and gene gun method;
    preferably, the AML is selected from the M4 and/or M5 subtype.
  8. 8. A method of constructing an AML model, comprising administering an agent that modulates CD 132;
    preferably, the agent comprises a CD132 promoter;
    Preferably, the promoter comprises any agent that increases expression of a nucleic acid encoding CD132, increases CD132 protein levels, or promotes CD132 activity;
    preferably, the promoter comprises a CD132 overexpression system;
    preferably, the AML model is a malignant model;
    Preferably, the AML malignancy model comprises one or more of altered cell morphology, enhanced cell proliferative capacity, cell cycle disorder, cell differentiation disorder, altered cell adhesion and migration capacity, altered metabolism, and altered immunophenotype.
  9. 9. An AML cell model, wherein said cells overexpress CD132;
    preferably, the AML cell model is constructed by the construction method of claim 8.
  10. 10. Use of the AML cell model of claim 9 for screening a medicament for treating AML or evaluating the efficacy of a medicament for treating AML.
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