Biomarkers related to AML diagnosis and prognosis prediction and their applications

Abstract

The present invention discloses biomarkers related to the diagnosis and prognosis prediction of AML and their applications. The present invention discovered that CD132 is differentially expressed in AML, and that CD132 expression levels are correlated with the degree of AML malignancy and the overall survival rate of AML patients. CD132 knockdown can inhibit the proliferation of AML cells, affect the cell cycle of AML cells, and promote apoptosis of AML cells. Therefore, the present invention provides the use of a reagent for detecting CD132 in the preparation of a product for diagnosing AML / predicting AML prognosis, and a product for diagnosing AML / predicting AML prognosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to biomarkers related to AML diagnosis and prognosis prediction and their applications. Background Art

[0002] AML (acute myeloid leukemia, AML), as the most common adult leukemia, seriously endangers human health and life safety. The treatment of AML usually includes induction chemotherapy, consolidation chemotherapy and hematopoietic stem cell transplantation. The goal of induction chemotherapy is to achieve complete remission in patients, 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 gradually been used in the treatment of AML, such as FLT3 inhibitors and IDH1 / 2 inhibitors.

[0003] The prognosis of AML varies depending on factors such as subtype, patient age, general condition, cytogenetic and molecular biological characteristics. Generally speaking, M3 leukemia has a relatively good prognosis with timely and effective treatment; however, patients with complex chromosomal abnormalities and certain gene mutations (such as FLT3-ITD hypermutation) have a poorer prognosis. Older patients and those with other comorbidities also tend to have a poor prognosis. The long-term survival rate for AML patients under 60 years of age is 35-40%, while that for patients 60 years of age and older is only 5-15%. Acute myelomonocytic / monocytic leukemia (including M4 and M5 in the French-American-British classification, or FAB classification, which account for 29.1% and 8.9% of AML, respectively) is an acute leukemia characterized by malignant clonal proliferation of both granulocytes and monocytes. The relapse rate is significantly higher than that of other AML subtypes, with the incidence of bone marrow and extramedullary relapse after hematopoietic stem cell transplantation being 34% and 50%, respectively. The 3-year disease-free survival rate of AML-M4 / 5 patients is only about 26%, making AML-M4 / 5 the main group of patients with relapsed and refractory disease (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. Despite progress in diagnosis, treatment, and prognostic assessment, many deficiencies remain. Further research is needed to develop more accurate diagnostic methods, effective treatments, and reliable prognostic indicators to improve patient survival and quality of life. Summary of the Invention

[0005] In order to overcome the deficiencies of the prior art, the present invention provides a biomarker that can realize the diagnosis and prognosis prediction of AML.

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

[0007] A first aspect of the present invention provides use of a reagent for detecting CD132 in the preparation of a product for diagnosing AML / predicting the prognosis of AML.

[0008] Furthermore, the reagent is 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.

[0009] Furthermore, the reagent also includes a detectable label.

[0010] Furthermore, the detectable label includes radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, and bioluminescent moieties.

[0011] Furthermore, the expression level of CD132 in patients with AML M4 and / or M5 subtypes was negatively correlated with the overall survival of the patients.

[0012] Furthermore, the product includes a chip, a test paper or a nucleic acid membrane strip.

[0013] Furthermore, the chip includes a gene chip and a protein chip. The gene chip includes an oligonucleotide probe targeting the CD132 gene for detecting the transcription level of the CD132 gene, and the protein chip includes a specific binding agent for the CD132 protein.

[0014] Furthermore, the product includes a kit.

[0015] Furthermore, the kit includes a gene detection kit and a protein detection kit. The gene detection kit includes a reagent or chip for detecting the transcription level of CD132 gene, and the protein detection kit includes a reagent or chip for detecting the expression level of CD132 protein.

[0016] Furthermore, the kit further comprises a buffer, a preservative or a protein stabilizer.

[0017] Furthermore, the kit also includes instructions.

[0018] Furthermore, the AML is selected from M4 and / or M5 subtypes.

[0019] A second aspect of the present invention provides a product for diagnosing AML / predicting the prognosis of AML, wherein the product comprises a reagent capable of detecting the expression level of CD132 in a sample.

[0020] Furthermore, the product includes a chip, a test paper or a nucleic acid membrane strip.

[0021] Furthermore, the product includes a kit.

[0022] Furthermore, the kit includes reagents for detecting the expression level of CD132 gene or protein by RT-PCR, qRT-PCR, biochip detection, Southern blotting, in situ hybridization, and immunoblotting.

[0023] Furthermore, the product also includes reagents for processing samples.

[0024] Furthermore, the AML is of M4 and / or M5 subtype.

[0025] A third aspect of the present invention provides a computer-based method for diagnosing AML / predicting AML prognosis, comprising:

[0026] Obtain data: Obtain the expression level data of CD132 in the sample of the test subject,

[0027] Processing data: inputting the CD132 expression level data into a constructed model, wherein the model predicts whether the subject is an AML patient or predicts the AML prognosis based on the CD132 expression data;

[0028] Output the result, output whether the subject to be tested is an AML patient or the result of predicting the AML prognosis of the subject to be tested.

[0029] Furthermore, the AML is of M4 and / or M5 subtype.

[0030] A fourth aspect of the present invention provides a computer-readable storage medium, characterized in that a computer program is stored on the computer-readable storage medium, and when the computer program is executed by a processor, it implements the method for diagnosing AML / predicting AML prognosis described in the third aspect of the present invention.

[0031] In a fifth aspect, the present invention provides a device / apparatus for computer-aided diagnosis of AML / prediction of AML prognosis, the device / apparatus comprising a memory and a processor, the memory being used to store program instructions; the processor being used to call the program instructions, and when the program instructions are executed, implementing the method for diagnosing AML / predicting AML prognosis described in the third aspect of the present invention.

[0032] The present invention has the following advantages and beneficial effects:

[0033] The present invention discovered for the first time a new target for the diagnosis, classification, prognosis assessment and treatment of acute myeloid leukemia - CD132. The present invention found 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, confirming the effectiveness of CD132 in the prevention, treatment and / or diagnosis of AML. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The following are the differential expression effect diagrams of CD132 in AML, among which 1A is the differential expression effect diagram of CD132 in normal CD34+ cells and AML cells analyzed by the GEO database dataset; 1B is the differential expression effect diagram of CD132 in AMLM4 / 5 subtypes and healthy people in the training set detected by flow cytometry; 1C is the differential expression effect diagram of CD132 in AMLM4 / 5 subtypes and healthy people in the validation set detected by flow cytometry; 1D is the differential expression effect diagram of CD132 in AML M4 / 5 subtypes and AML non-M4 / 5 subtypes in the training set detected by flow cytometry; 1E is the differential expression effect diagram of CD132 in AMLM4 / 5 subtypes and AML non-M4 / 5 subtypes in the training set detected by flow cytometry;

[0035] Figure 2 2A is the ROC curve of CD132 for diagnosing AML M4 / 5 subtypes and healthy subjects in the training set; 2B is the ROC curve of CD132 for diagnosing AML M4 / 5 subtypes and healthy subjects in the validation set; 2C is the ROC curve of CD132 for diagnosing AML M4 / 5 subtypes and AML non-M4 / 5 subtypes in the training set; 2D is the ROC curve of CD132 for diagnosing AML M4 / 5 subtypes and AML non-M4 / 5 subtypes in the validation set;

[0036] Figure 3 This is a correlation analysis diagram between WT1 and CD132;

[0037] Figure 4 Figure 4A is a graph showing the effect of CD132 on the prognosis prediction of AMLM4 / 5 subtypes, where 4A is a graph showing the differential expression of CD132 in samples of different stemness-type acute monocytic leukemia (FAB-M4 / 5) subtypes according to the LSC17 (Nature 540, 433–437 (2016)) standard analyzed using the GEO database dataset. Figure 4B is a graph showing the correlation between CD132 and the long-term overall survival rate of AMLM4 / 5 subtypes analyzed using the OHSU database.

[0038] Figure 5 This is a diagram of the differential colony formation of CD132 high / low AML cells;

[0039] Figure 6 Figures 6A and 6B show the effects of CD132 knockdown on cell function in human leukemia cell lines. Figure 6A shows the effects on THP-1 cell proliferation, Figure 6B shows the effects on THP-1 cell colony formation, and Figure 6C shows the effects on THP-1 cell cycle. Figure 6D shows the effects on THP-1 cell apoptosis. Figure 6E shows the effects on Molm-13 cell proliferation, Figure 6F shows the effects on Molm-13 cell colony formation, Figure 6G shows the effects on Molm-13 cell cycle, and Figure 6H shows the effects on OCI-AML3 cell cycle. Note: *(p<0.05), **(p<0.01), ***(p<0.001), ****(p<0.0001).

[0040] Figure 7 Figure 7A shows the effect of overexpression of CD132 on cell function in human leukemia cell lines, wherein Figure 7A shows the effect on THP-1 cell proliferation, and Figure 7B shows the effect on THP-1 cell cycle;

[0041] Figure 8 This is a flowchart of a computer-based method for diagnosing AML / predicting AML prognosis. DETAILED DESCRIPTION

[0042] The following provides definitions of some terms used in this specification. Unless otherwise defined, all technical and scientific terms used in this invention generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0043] The present invention provides use of a reagent for detecting CD132 in preparing a product for diagnosing AML / predicting AML prognosis.

[0044] 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, people's 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 of the people, and gene ID is 3561.

[0045] In the present invention, the term "diagnosis" refers to the discovery, judgment or recognition of an individual's health status or condition based on one or more symptoms, data or other information related to the individual. Diagnosis can usually be performed between a patient with the disease and a normal person, or between patients with different disease subtypes. In the present invention, the diagnosis of AML includes diagnosing individuals who do not have AML and individuals who have AML, or diagnosing AML by FAB typing. The FAB typing of acute myeloid leukemia (AML) is mainly based on the morphological characteristics and cytochemical staining results of leukemia cells. There are seven subtypes: 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), and M7 (acute megakaryocytic leukemia). In some embodiments of the present invention, CD132 is used to diagnose different FAB subtypes, such as diagnosing patients with M0 / M1 / M2 and M4 / M5 subtypes.

[0046] 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 determining the time course of a disease or condition with or without treatment. In the case of considering treatment, prognosis includes determining the therapeutic effect for the disease or condition. In the present invention, the prognosis prediction of AML includes predicting the long-term overall survival rate of AML patients.

[0047] 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.

[0048] In the present invention, the term "probe" refers to a molecule capable of binding to a specific sequence, subsequence, or other portion of another molecule. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe capable of binding 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.

[0049] In the present invention, the term "amplification primer" or "primer" refers to an oligonucleotide that is capable of site-specifically annealing to a region of RNA or DNA adjacent to a target sequence and serving as an initiator of DNA synthesis under suitable conditions in which the synthesis of primer extension products is induced, for example, in the presence of nucleotides and a polymerization inducer such as a DNA-dependent DNA polymerase, and under 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," comprising an "upstream" or "forward" primer and a "downstream" or "reverse" primer that defines the region of RNA or DNA to be amplified.

[0050] As used herein, the term "amplification" refers to a method for 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 copy, or arithmetic, logarithmic, or exponential amplification.

[0051] 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 be bound to a membrane protein using specific intermolecular interactions. The binding agent of an albumen is, for example, a receptor for a protein, a lectin that binds to a protein, an antibody against a protein, a peptide body (peptidebody), 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 overall protein structure that are self-stable 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 to 500 amino acids and more. Many binding domains can be classified as folding and are recognizable, identifiable, 3-D structures. Some foldings are very common in many different proteins, to the extent that they have been given specific names.

[0052] 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 a determination 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 device. In some embodiments, labels can be detected visually without the aid of a device.

[0053] In the present invention, radioactive isotopes include but are not limited to 3 H. 14C. 35 S. 125 I. 131 I. Enzymes include, but are not limited to, horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, and acetylcholinesterase. Fluorescent molecules include, but are not limited to, FITC, rhodamine, and lanthanide phosphors.

[0054] The present invention can 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, and immunoblotting.

[0055] In the present invention, "expression level" refers to the amount, accumulation or rate of a biomarker molecule or gene group. Expression level can be represented, for example, by the amount or synthesis rate of 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.

[0056] 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.

[0057] 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 can comprise a flat surface, or can 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 can be packaged in a manner that allows for diagnostic or other manipulation of a fully functional device.

[0058] 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 oligonucleotide probes, such as nylon membrane, nitrocellulose membrane, polypropylene membrane, glass sheet, silica gel wafer, micro-magnetic beads, etc.

[0059] In the present invention, the term "kit" refers to any delivery system for delivering materials, including kits for research and clinical applications.

[0060] The kit described in the present invention includes a reagent for detecting the CD132 gene or protein, and one or more substances selected from the following group: a container, instructions for use, a positive control, a negative control, a buffer, an adjuvant, a solvent, a preservative, or a 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, plastic bottle, syringe, or other container in which one component can be placed, and preferably, can be appropriately aliquoted. 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 the additional components are placed separately. However, different combinations of components can be contained in a vial. The kit of the present invention will generally also include a container for holding the reactants, sealed for commercial sale. Such a container may include an injection-molded or blow-molded plastic container in which the desired vials can be retained.

[0061] 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.

[0062] 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.

[0063] The present invention provides the use of CD132 in preparing a medicine for treating AML.

[0064] In the present invention, the term "treatment" may refer to therapeutic treatment or preventative 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, relief of symptoms; reduction in the extent of a disorder, disorder or disease; stabilization (i.e., not worsening) of the state of a disorder, disorder or disease; delaying the onset of a disorder, disorder or disease or slowing its progression; improving a disorder, disorder or disease state; and relief (whether partial or complete) (whether detectable or undetectable) or improvement or amelioration of a disorder, disorder or disease. Treatment may include causing a clinically significant response without excessive side effects. Treatment also includes prolonged survival compared to the expected survival if not receiving treatment.

[0065] In the present invention, drugs include CD132 inhibitors. Inhibitors are 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, or inhibit the activity of CD132. As one embodiment of the present invention, the "inhibitor" is a substance that inhibits the expression level of CD132.

[0066] In the present invention, inhibitors include nucleic acid inhibitors, protein inhibitors, proteolytic enzymes, protein binding molecules, and combinations thereof.

[0067] In the present invention, nucleic acid inhibitors are selected from: interfering molecules that target CD132 or its transcript and can inhibit CD132 gene expression or gene transcription, including but not limited to shRNA, siRNA, ribozymes, antisense oligonucleotides, dsRNA, microRNA, zinc fingers, gRNA, or constructs that can express or form the shRNA, siRNA, ribozymes, antisense oligonucleotides, dsRNA, microRNA, zinc fingers, gRNA. Protein inhibitors are selected from substances that can inhibit CD132 protein. Proteolytic enzymes are selected from enzymes that can catalyze the hydrolysis of CD132 protein. Protein binding molecules are selected from substances that specifically bind to CD132 protein, such as antibodies or ligands that can inhibit the activity of CD132 protein.

[0068] 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, as well as RNA that is modified from natural RNA by addition, deletion, substitution, and / or alteration of one or more nucleotides. The alteration may include the addition of non-nucleotide substances, such as addition to the ends of the siRNA or one or more internal nucleotides of the siRNA; modifications that render the siRNA resistant to nuclease digestion (e.g., using 2'-substituted ribonucleotides or modifying the sugar-phosphate backbone); or replacement of one or more nucleotides in the siRNA with deoxyribonucleotides.

[0069] 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, and shRNA can inhibit gene expression through the RNA interference pathway. When shRNA is introduced into a cell, it will be recognized by the nuclease Dicer in the cell and cut into small interfering RNA (siRNA) of about 21 nucleotides. siRNA binds to a protein complex called RNA-induced silencing complex (RISC). The Argonaute protein in RISC uses the antisense chain of siRNA to recognize and bind to the target mRNA, and then degrades the target mRNA by cutting or inhibiting translation, thereby achieving the inhibition of specific gene expression. In a specific embodiment of the present invention, the nucleic acid inhibitor is selected from shRNA.

[0070] In some embodiments, the nucleic acid inhibitor is selected from gRNA. In some embodiments, gRNA is a stretch 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. Therefore, as a preferred embodiment, the nucleic acid inhibitor includes gRNA and Cas9 protein.

[0071] In some embodiments, the nucleic acid inhibitor is selected from ribozymes. Ribozymes 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 for 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. 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.

[0072] In some embodiments, nucleic acid inhibitors are selected from antisense oligonucleotides. Antisense oligonucleotides (antisense nucleic acid sequences) can include nucleotide sequences complementary to the sense nucleic acid of the encoded protein (for example, complementary to the double-stranded cDNA molecule coding strand or complementary to CD132mRNA). 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 can be complementary to the complete coding strand of the target sequence, or only partially complementary to it. The length of antisense oligonucleotides can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or more nucleotides.

[0073] The drugs described in the present invention can also be used in combination with other compounds for treating AML. 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. 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., separately). Simultaneously in the same formulation is as a single formulation, while simultaneously in different pharmaceutical formulations is non-single. 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.

[0074] In the present invention, medicine comprises pharmaceutically acceptable adjuvant, and adjuvant comprises excipient, disintegrating agent, sweetener, adhesive, coating agent, swelling agent, lubricant, glidant, flavoring agent, solubilizing agent etc.For administration, except active component, medicine of the present invention can preferably use at least one pharmaceutical carrier preparation.When composition is formulated as 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 its mixture. If necessary, other conventional additives can be added, comprise antioxidant, buffer, antibacterial agent etc..In addition, diluent, dispersant, surfactant, adhesive and lubricant can be further added to prepare injectable preparation (such as aqueous solution, suspension or emulsion etc.), pill, capsule, granule or tablet.

[0075] 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 alginates, xanthan gum, hydroxypropyl cellulose and hydroxypropyl methylcellulose, etc.; surfactants such as polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, monostearin, 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 ester, sodium lauryl sulfate, magnesium lauryl sulfate, magnesium lauryl sulfate, etc.

[0076] The medicaments of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted drug reservoir. The medicaments of the present invention can contain any conventional non-toxic pharmaceutically acceptable carrier, adjuvant, or vehicle. In some cases, pharmaceutically acceptable acids, bases, or buffers can be used to adjust the pH of the formulation to improve the stability of the formulated compound or its dosage form. The term parenteral as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. The medicaments of the present invention can be administered to the recipient by any route as long as the target tissue is reached.

[0077] The present invention will be further described in detail below with reference to 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.

[0078] Example 1 Expression level of CD132 in AML cells

[0079] 1. Experimental methods

[0080] 1) Database analysis

[0081] 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).

[0082] 2) Flow cytometry detection of CD132 and WT1 expression

[0083] 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.

[0084] 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).

[0085] Preparation of primary bone marrow cells from AML patients / healthy donors: Approximately 3 mL of bone marrow blood was collected from the posterior superior iliac spine and anticoagulated in an EDTA or heparin tube. Mononuclear cells (MNOCs) were isolated by density gradient centrifugation. The MNOCs were placed in PBS and labeled with CD132-PE and the AML surface antigens CD117-FITC / CD34-APC / CD33-BV421 (all antibodies purchased from BD Biosciences or BioLegend). The cells were incubated for 15 minutes and washed twice with PBS. Flow cytometry was performed to analyze the expression of CD132 and WT1 on CD34+ cells and AML cells, and receiver operating characteristic (ROC) curves were used for analysis.

[0086] 3) The Pearson correlation coefficient analysis method was used to analyze the correlation between WT1 and CD132 in all samples.

[0087] 2. Experimental results

[0088] 1) The results of the GEO database (GSE30029) analysis 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).

[0089] 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).

[0090] The ROC curve analysis results are as follows Figure 2As shown, CD132 can effectively distinguish M4 / 5 subtypes 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 M4 / 5 subtypes 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).

[0091] 3) Correlation analysis results are as follows Figure 3 As shown, WT1 does not associate with CD132.

[0092] Example 2 Application of CD132 in Predicting the Prognosis of AML Cells

[0093] 1. Experimental methods

[0094] 1) In the GEO database dataset (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).

[0095] 2) Use logrank to perform survival analysis on the acute myeloid leukemia dataset BeatAML in the OHSU database.

[0096] 2. Experimental results

[0097] 1) The results of the GEO database (GSE14468) analysis 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), suggesting that CD132 is associated with the malignancy of AMLM4 / 5 subtypes.

[0098] 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 4As 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 in the CD132 high and CD132 low 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 longer than that of patients with AML (FAB-M4 / 5) with low CD132 expression. Therefore, CD132 can be used to predict the prognosis of AML (FAB-M4 / 5) patients.

[0099] Example 3 Effect of CD132 on AML cells

[0100] 1. Experimental methods

[0101] 1) Bone marrow cell colony formation assay

[0102] (1) Sorting CD132 high and CD132 low cells

[0103] Primary bone marrow cells from leukemia patients / healthy donors: Obtain approximately 3 mL of bone marrow blood from the posterior superior iliac spine and anticoagulate with EDTA or heparin. Isolate bone marrow mononuclear cells (MNNCs) using density gradient centrifugation. Place the MNNCs in PBS and label with CD132-PE and the AML surface antigens CD117-FITC / CD34-APC / CD33-BV421 (all antibodies purchased from BD Biosciences or BioLegend). Incubate for 15 minutes, wash twice with PBS, and separate cells with CD117 / CD34 / CD33+ and CD132 high / low levels using a flow cytometer. Collect the cells in 15 mL centrifuge tubes and plate CFU.

[0104] (2) Colony formation assay

[0105] Thaw stem cell culture medium for the CFU assay at room temperature (3 mL of stem cell culture medium per 15 mL centrifuge tube); resuspend and count cells, seeding 5,000 cells per well; add 1.1 mL of stem cell culture medium to each 35 mm dish, performing a replicate well. Cover the dish. Add water or PBS to a third dish; place the three dishes in a larger dish, cover, and culture for 7-14 days; then observe and count the cells under a microscope.

[0106] 2) Functional experiments after CD132 silencing in cell lines

[0107] (1) Construction of CD132-silenced cell line

[0108] 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 as a negative control; CD132 cloning vector: pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO; TrueCut TM Cas9 protein v2 (Thermo Fisher Scientific Invitrogen TM ).

[0109] Cell knockdown: Prepare a medium containing RPMI1640 supplemented with 2% FBS and resuspend the cells to 5×10 5 / mL, and placed in a 6-well plate. Using the above-mentioned lentiviral vector, at an MOI of 30, 16 hours after transfection, the cells were centrifuged at 1000 rpm for 3 minutes, replaced with complete medium containing 10% FBS, and cultured in an incubator for 48 hours. Drug selection was performed using puromycin for 48 hours, and transfection efficiency was verified by RT-qPCR and flow cytometry.

[0110] Cell knockout: CD132 gRNA and TrueCut TM Cas9 protein v2 (Thermo Fisher Scientific Invitrogen TM ) were incubated at room temperature for 15 minutes 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 complexes and resuspended cell lines evenly into an electroporation cuvette, switch the celetrix electroporator to cell line mode, and place the cuvette for electroporation. After electroporation, transfer the cell lines to complete medium and culture for 72 hours. After T7E1 genome verification, functional experiments were performed.

[0111] (2) Cell proliferation ability experiment of CD132-silenced human leukemia cell lines

[0112] Cell plating: adjust the cell concentration to 1×10 5 / mL, 100 μL / well in a 96-well plate, 3 replicates; lay out multiple concentration gradients to create a standard curve; after culturing for 0-4 days according to experimental requirements, add 10 μL / well of CCK8; after 4 hours, measure the OD value at a wavelength of 450 nm using a microplate reader.

[0113] (3) Colony formation experiment of CD132-silenced human leukemia cell lines

[0114] Thaw stem cell culture medium for the CFU assay at room temperature (3 mL of stem cell culture medium per 15 mL centrifuge tube); resuspend and count cells, seeding 2,000-5,000 cells per well; add 1.1 mL of cell culture medium to a 35 mm dish, performing a replicate. Cover the dish. Add water or PBS to a third dish; place all three dishes in a larger 100 mm dish, cover, and culture for 7-14 days; observe and count the cells under a microscope.

[0115] (4) Cell cycle experiment of CD132-silenced human leukemia cell lines

[0116] Prepare a culture medium containing RPMI1640 supplemented with 2% FBS and 10 μg / mL Hoechst 33342. 6 The cells were resuspended in 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 in 100 μL of PBS; and detected by flow cytometry on the BV421 channel.

[0117] (5) Apoptosis experiment of CD132-silenced human leukemia cell lines

[0118] Pipette cells into flow cytometry tubes; wash once with PBS. Add 1 mL of PBS to each tube to resuspend the cells and centrifuge at 1500 rpm for 5 minutes. Wash once with Annexin V binding buffer. Add 1 mL of Annexin V binding buffer to each tube to resuspend the cells and centrifuge at 1500 rpm for 5 minutes. Add 150 μL / tube of Annexin V binding buffer to resuspend the cells to form a single-cell suspension. Add 2.5 μL of Annexin V-APC and 5 μL of 7-AAD to each tube. Incubate in the dark for 15 minutes before analysis.

[0119] 3) Functional experiments after overexpression in cell lines

[0120] (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 supplemented with 2% FBS, resuspend the cells to 5×10 5 / mL, and placed in a 6-well plate. Using the above-mentioned lentiviral vector, at an MOI of 30, 16 hours after transfection, the cells were centrifuged at 1000 rpm for 3 minutes, replaced with complete medium containing 10% FBS, and cultured in an incubator for 48 hours. Drug selection was performed using puromycin for 48 hours, and transfection efficiency was verified by RT-qPCR and flow cytometry.

[0121] (2) Cell proliferation ability experiment of CD132 overexpressing human leukemia cell lines

[0122] Cell plating: adjust the cell concentration to 1×10 5 / mL, 100 μL / well in a 96-well plate, 3 replicates; lay out multiple concentration gradients to create a standard curve; after culturing for 0-4 days according to experimental requirements, add 10 μL / well of CCK8; after 4 hours, measure the OD value at a wavelength of 450 nm using a microplate reader.

[0123] (3) Cell cycle experiment of CD132 overexpressing human leukemia cell lines

[0124] Prepare a culture medium containing RPMI1640 supplemented with 2% FBS and 10 μg / mL Hoechst 33342. 6 The cells were resuspended in 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 in 100 μL of PBS; and detected by flow cytometry on the BV421 channel.

[0125] 2. Experimental results

[0126] 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);

[0127] 2) Effects of CD132 knockdown on human leukemia cell lines on cell function Figure 6 As shown, after CD132 in THP-1 and Molm-13 cells was knocked down 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 was significantly affected (6C, 6G). 11 days after infection, a large number of knocked-down tumor cells showed apoptosis (6D); after CD132 in OCI-AML3 was knocked down using gRNA, the cell cycle was significantly weakened.

[0128] 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 7As shown in A and 7B, the human leukemia cell line overexpressing CD132 significantly enhanced the cell cycle and cell proliferation ability.

[0129] Example 4 Computer-based method for diagnosing AML or predicting AML prognosis

[0130] Figure 8 1 is a flow chart of a method for computer-aided diagnosis of AML provided by an embodiment of the present invention. Specifically, the method includes the following steps:

[0131] S101: Acquire data: Acquire the expression level data of CD132 in the sample of the test subject;

[0132] In one embodiment, the expression level of CD132 includes the protein expression level and nucleic acid expression level of CD132.

[0133] In one embodiment, methods for determining the protein expression level of CD132 include but are not limited to Western blotting, immunohistochemistry, immunofluorescence, enzyme-linked immunosorbent assay, mass spectrometry, and flow cytometry.

[0134] In one embodiment, methods for determining the nucleic acid expression level of CD132 include but are not limited to RT-PCR, qRT-PCR, biochip detection, Southern blotting, Northern blotting, and gene chip methods.

[0135] In one embodiment, the AML is of M4 and / or M5 subtype.

[0136] S102: Processing data: Inputting the CD132 expression level data into the constructed model, wherein the model predicts whether the subject is an AML patient or predicts the AML prognosis based on the CD132 expression data.

[0137] In one embodiment, a machine learning method is used to construct a model, and the machine learning method includes but is not limited to a support vector machine learning model, a linear discriminant analysis model, a recursive feature removal model, a predictive analysis of a microarray model, a logistic regression model, a CART algorithm, a flextree algorithm, a LART algorithm, a random forest algorithm, a MART algorithm, a machine learning algorithm, a penalized regression method, and a combination thereof.

[0138] S103: Output result: output whether the subject to be tested is an AML patient or predict the AML prognosis of the subject to be tested.

[0139] In one embodiment, when the expression level of CD132 is higher than the normal reference value in the model, the subject to be tested is an AML patient.

[0140] In one embodiment, when the expression level of CD132 is higher than the reference value for good prognosis of AML in the model, the prognosis of the AML subject to be tested is poor.

[0141] Those skilled in the art will understand that all or part of the steps in the various methods of the above embodiments can be completed by instructing related hardware through a program, and the program can be stored in a computer-readable storage medium, which may include: read-only memory (ROM), random access memory (RAM), disk or optical disk, etc.

[0142] The above embodiments are only provided for understanding the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by a person skilled in the art, and such improvements and modifications shall fall within the scope of protection of the claims of the present invention.

Claims

1. Use of a reagent for detecting CD132 in the preparation of a product for diagnosing AML / predicting the prognosis of AML, wherein the AML is selected from the M4 and / or M5 subtypes.

2. The use according to claim 1, characterized in that The reagent is 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.

3. The use according to claim 1, characterized in that The reagent also includes a detectable label.

4. The use according to claim 3, characterized in that The detectable labels include radioisotopes, nucleotide chromophores, enzymes, fluorescent molecules, and magnetic particles.

5. The use according to claim 1, characterized in that The products include chips, test papers or nucleic acid membrane strips.

6. The use according to claim 5, characterized in that The chip includes a gene chip and a protein chip. The gene chip includes an oligonucleotide probe targeting the CD132 gene for detecting the transcription level of the CD132 gene, and the protein chip includes a specific binding agent for the CD132 protein.

7. The use according to claim 1, characterized in that The product includes a kit.

8. The use according to claim 7, characterized in that The kit includes a gene detection kit and a protein detection kit. The gene detection kit includes a reagent or chip for detecting the transcription level of CD132 gene, and the protein detection kit includes a reagent or chip for detecting the expression level of CD132 protein.

9. The use according to claim 8, characterized in that The kit may also include a buffer, a preservative, or a protein stabilizer.

10. The use according to claim 1, characterized in that The product also includes reagents for processing the sample.

11. A computer-based method for diagnosing AML / predicting AML prognosis, characterized in that: include: Acquiring data: acquiring CD132 expression level data in samples of test subjects; Processing data: inputting the CD132 expression level data into a constructed model, wherein the model predicts whether the subject is an AML patient or predicts the AML prognosis based on the CD132 expression data; Output result: output whether the subject to be tested is an AML patient or predict the AML prognosis of the subject to be tested; Wherein, the AML is of M4 and / or M5 subtype.

12. A computer-readable storage medium, characterized in that The computer-readable storage medium stores a computer program, and when the computer program is executed by a processor, the method for diagnosing AML / predicting the prognosis of AML according to claim 11 is implemented.

13. A device / apparatus for computer-aided diagnosis of AML / prediction of AML prognosis, characterized in that: The device / apparatus includes a memory and a processor, the memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, the method for diagnosing AML / predicting AML prognosis according to claim 11 is implemented.

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