Application of combination of RUNX1, SPI1 and CEBPE in induced differentiation treatment of acute myelogenous leukemia

Abstract

The invention relates to the field of biological medicines, and provides application of combination of RUNX1, SPI1 and CEBPE in induced differentiation treatment of acute myelogenous leukemia. The invention particularly relates to a method and application for inducing differentiation of differentiated and blocked leukemia cells into mature blood cells by using three transcription factors of RUNX1, SPI1 and CEBPE so as to be applied to hematologic tumor treatment. Researches show that by introducing exogenous RUNX1, SPI1 and CEBPE genes into acute myelogenous leukemia cells, the acute myelogenous leukemia cells can be effectively induced to be differentiated into mature blood cells, so that a new method for tumor induced differentiation treatment is provided.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and relates to a new medical application of RUNX1, SPI1 and CEBPE, and specifically to an application of RUNX1, SPI1 and CEBPE in inducing differentiation to treat acute myeloid leukemia. Background Art

[0002] Acute myeloid leukemia (AML) is a malignant blood tumor disease caused by abnormal proliferation of myeloid hematopoietic stem cells. Clinical manifestations include anemia, bleeding, infection and fever, organ infiltration, metabolic abnormalities, etc. Most cases are acute and severe, with a poor prognosis, posing a serious threat to the health level of Chinese citizens. Currently, the first-line treatments for leukemia are mainly chemotherapy and bone marrow transplantation, but the high heterogeneity, high relapse rate, and lack of donors still limit the effectiveness of leukemia treatment. Therefore, the treatment research of leukemia urgently needs more effective and more universal new treatment methods.

[0003] Cell differentiation blockage caused by gene mutation is an important feature of leukemia, so differentiation therapy has always been an important research direction for the development of leukemia treatment methods. Taking acute promyelocytic leukemia, a common leukemia, as an example, the combined use of all-trans retinoic acid and arsenic trioxide can make tumor cells that are blocked in the promyelocytic stage redifferentiate into mature granulocytes, and then achieve clinical remission through programmed cell death of mature granulocytes. The 5-year survival rate of patients treated with trans-retinoic acid and arsenic trioxide combination therapy reached over 95% with no obvious side effects, and has become a model for drug-induced differentiation therapy for leukemia (de The H, Differentiation therapy revisited. Nature reviews, Cancer, Feb 2018; 18(2): 117-127.)(Chen L, Arsenic trioxide replacing or reducing chemotherapy in consolidation therapy for acute promyelocytic leukemia (APL 2012 trial), Proceedings of the National Academy of Sciences of the United States of America, Feb 92021; 118(6).). However, except for acute promyelocytic leukemia, there is still a lack of effective differentiation induction schemes for other types of leukemia.

[0004] Key transcription factors for hematopoietic development are a class of transcription factors that regulate the differentiation of hematopoietic cells during normal development, including multiple transcription factor families such as ETS, CCAAT / enhancer binding protein (C / EBP), and HOX. These transcription factors promote the fate decision mechanism of multiple hematopoietic cell lineages such as lymphoid, myeloid, and erythroid during the differentiation of hematopoietic stem and progenitor cells through specific gene regulatory networks (Krumsiek, Hierarchical Differentiation of Myeloid Progenitors Is Encoded in the Transcription Factor Network, PLoS One, 2011; 6(8): e22649.). Mutations or abnormal expressions of key transcription factors for hematopoietic development are highly correlated with the occurrence, typing, and prognosis of leukemia. Taking the drug-induced differentiation therapy of promyelocytic leukemia as a template, the research team of the inventor has previously identified and reported the key role of two transcription factors, SPI1 and CEBPE, in inducing terminal differentiation of leukemia cells (Tang Y, Induced lineage promiscuity undermines the efficiency of all-trans-retinoid-acid-induced differentiation of acute myeloid leukemia, iScience, 2021Apr 11; 24(5): 102410.). However, subsequent studies have found that the combination of these two transcription factors only partially promotes differentiation of leukemia cells of a specific type, suggesting that further optimization is still needed to obtain a more effective and wider range of induced differentiation therapy. Therefore, this field aims to develop a combination of more key transcription factors in different hematopoietic development stages to induce the differentiation of leukemia cells, providing a more effective mitigation method for leukemia treatment. Summary of the invention

[0005] The present invention is based on the above research and studies the application of RUNX1, SPI1 and CEBPE in inducing differentiation to treat acute myeloid leukemia.

[0006] Through further transcription factor function screening experiments, it was identified that RUNX1 can effectively enhance the effect of SPI1 and CEBPE in inducing terminal differentiation of leukemia cells. Referring to the hematopoietic development map, RUNX1, as one of the most critical transcription factors in the early stage of hematopoietic development, is likely to have relieved the restrictive conditions of leukemia cell differentiation at the chromosomal level, creating conditions for SPI1 and CEBPE to induce terminal differentiation of leukemia cells.

[0007] The research process of the present invention is as follows: first, a DOX-induced expression of an RSE three-factor combination lentiviral plasmid is constructed, and the sequence correctness is verified by Sanger sequencing; then, the RSE three-factor combination lentiviral plasmid is transfected into AML-related cells, showing that it can significantly inhibit the proliferation of leukemia cells and induce their transformation. Under a microscope, it can be seen that the suspended leukemia cells begin to adhere to the wall, and undergo morphological and cell nucleus type changes, while promoting AML cell apoptosis.

[0008] Based on the above research, the technical solution to be protected by the present invention is as follows:

[0009] One of the main purposes of the present invention is to provide new medical uses of the genes of RUNX1, SPI1, CEBPE and their product proteins; another purpose is to provide a combination drug with RUNX1, SPI1, and CEBPE as active components, and the combination drug can be used as a leukemia differentiation treatment drug; the third purpose is to provide a new AML treatment method, which combines the use of multiple key hematopoietic development transcription factors to induce leukemia cell differentiation.

[0010] The first aspect disclosed in the present invention provides the use of RUNX1, SPI1 and CEBPE in combination for preparing a drug for treating acute myeloid leukemia.

[0011] Preferably, the drug for treating acute myeloid leukemia is a drug that induces differentiation-blocked leukemia cells to differentiate into mature blood cells, inhibits leukemia cell proliferation, promotes their apoptosis, and improves the effect when used in combination with other treatment methods, thereby achieving the purpose of treating leukemia.

[0012] The RUNX1, SPI1, and CEBPE combination is selected from the combination of exogenous RUNX1 protein, SPI1 protein, and CEBPE protein, the combination of exogenous RUNX1 gene, SPI1 gene, and CEBPE gene, a recombinant expression vector simultaneously loaded with RUNX1 gene, SPI1 gene, and CEBPE gene, a combination of substances that promote the expression of RUNX1 gene, SPI1 gene, and CEBPE gene, and a combination of mRNA, cDNA, or precursors of RUNX1, SPI1, and CEBPE.

[0013] In a preferred embodiment of the present invention, the RUNX1 gene is selected from any of the following situations:

[0014] (i) a nucleic acid molecule having a nucleotide sequence as shown in GENBANK ID: NM_001754.5;

[0015] (ii) a molecule that hybridizes to the nucleotide sequence defined in (i) under stringent conditions;

[0016] (iii) a nucleic acid molecule homologous to or having sequence identity with the nucleotide sequence shown in GENBANK ID: NM_001754.5;

[0017] (iv) a nucleic acid molecule in which one or more nucleotides are substituted, deleted or added in the nucleotide sequence of (i), (ii) or (iii);

[0018] The SPI1 gene is selected from any of the following situations:

[0019] (i) a nucleic acid molecule having a nucleotide sequence as shown in GENBANK ID: NM_003120.3;

[0020] (ii) a molecule that hybridizes to the nucleotide sequence defined in (i) under stringent conditions;

[0021] (iii) a nucleic acid molecule homologous to or having sequence identity with the nucleotide sequence shown in GENBANK ID: NM_003120.3;

[0022] (iv) a nucleic acid molecule in which one or more nucleotides are substituted, deleted or added in the nucleotide sequence of (i), (ii) or (iii);

[0023] The CEBPE gene is selected from any of the following situations:

[0024] (i) a nucleic acid molecule having a nucleotide sequence as shown in GENBANK ID: NM_001805.4;

[0025] (ii) a molecule that hybridizes to the nucleotide sequence defined in (i) under stringent conditions;

[0026] (iii) a nucleic acid molecule homologous to or having sequence identity with the nucleotide sequence shown in GENBANK ID: NM_001805.4;

[0027] (iv) A nucleic acid molecule in which one or more nucleotides are substituted, deleted or added in the nucleotide sequence of (i), (ii) or (iii).

[0028] In some embodiments, RUNX1 protein, SPI1 protein, CEBPE protein are selected from: naturally purified proteins, chemically synthesized products, or produced from prokaryotic or eukaryotic hosts using recombinant technology. The host is selected from: bacteria, yeast, higher animals and mammalian cells, preferably human RUNX1 protein, SPI1 protein, CEBPE protein.

[0029] Furthermore, the present invention also provides an RSE three-factor recombinant expression vector, comprising an expression vector and nucleic acid molecules of RUNX1 gene, SPI1 gene and CEBPE gene inserted into the expression vector, wherein the nucleic acid molecules of RUNX1 gene, SPI1 gene and CEBPE gene are as shown above.

[0030] Wherein, the expression vector is a conventional vector such as a plasmid vector, a cosmid vector, a phage vector or a viral vector, and the specific type is selected from the prior art according to the actual situation. The "viral vector" includes adenovirus, lentivirus, retrovirus and adeno-associated virus. Suitable viral vectors are well known to those of ordinary skill in the art. The remaining "non-viral vectors" include liposomes or lipid complexes, cationic polymers, chitosan polymers and nanoparticle vectors. Suitable non-viral vectors are well known to those of ordinary skill in the art.

[0031] Of course, constructing three recombinant expression vectors based on three genes respectively is also applicable to the present invention and also falls within the protection scope of the present invention.

[0032] In the second aspect disclosed in the present invention, a combination drug or combined drug for treating acute myeloid leukemia is provided, wherein the active ingredients include any of the following:

[0033] (a) RUNX1 protein, SPI1 protein and CEBPE protein;

[0034] (b) the coding sequence of RUNX1 protein, the coding sequence of SPI1 protein and the coding sequence of CEBPE protein;

[0035] (c) A recombinant expression vector containing the coding sequence of RUNX1 protein, the coding sequence of SPI1 protein and the coding sequence of CEBPE protein.

[0036] In addition, the combination drug also includes a pharmaceutically acceptable carrier or excipient. The pharmaceutically acceptable carrier or excipient of the present invention refers to additives commonly used in the pharmaceutical field except for active ingredients, such as diluents (starch, sugar, cellulose and inorganic salts), excipients, etc., fillers such as starch sucrose, adhesives such as water, ethanol, cellulose derivatives, gelatin and polyvinyl pyrrolidone, disintegrants such as dry starch, sodium carboxymethyl starch, solubilizers such as polysorbates and polyoxyethylene fatty acid esters, etc., absorption promoters, surfactants such as Tween, Span, adsorption carriers, lubricants such as magnesium stearate, micropowder silica gel, etc. In addition, other adjuvants such as flavoring agents, sweeteners, etc. can also be added to the composition.

[0037] In the third aspect disclosed in the present invention, a method for treating AML is provided, the method comprising simultaneously or sequentially administering an effective amount of proteins, coding sequences or expression vectors containing the coding sequences of the key transcription factors for hematopoietic development, RUNX1, SPI1 and CEBPE.

[0038] In the fourth aspect disclosed in the present invention, a method for inducing or promoting leukemia differentiation in mammals is provided, that is, a new method for treating leukemia. The method comprises the steps of: jointly applying the key transcription factors of hematopoietic development RUNX1, SPI1, CEBPE protein, their coding sequences or expression vectors of the coding sequences into leukemia cells, inhibiting the proliferation of leukemia cells and promoting their apoptosis.

[0039] In terms of administration, the drug can be administered to patients who need such treatment in the form of a pharmaceutical composition by oral administration, nasal inhalation, rectal administration, parenteral administration or transdermal administration. When used for oral administration, it can be made into conventional solid preparations such as tablets, powders, granules, capsules, pills, sustained-release pellets, solid dispersions, inclusion compounds, etc., and liquid preparations such as suspensions, emulsions, melts, syrups, mixtures, solutions, etc. When used for parenteral administration, it can be made into solutions for injection, water or oily suspensions, emulsions, liposomes, microcapsules, microspheres, nanoparticles, etc., and can also be made into various sustained-release and controlled-release preparations. The preferred form is injection, and injections with targeted release at specific sites are particularly preferred.

[0040] The pharmaceutical composition and the combination drug of the present invention are also used to inhibit the formation of leukemia in vivo. Preferably, the pharmaceutical combination can also be used in combination with other AML chemotherapy drugs.

[0041] When the drug combination of the present invention is administered to animals including humans, the dosage varies depending on the patient's age and weight, disease characteristics and severity, and administration route. The results of animal experiments and various circumstances can be referenced, and the total dosage cannot exceed a certain range.

[0042] Beneficial protection and effects of the present invention:

[0043] The present invention provides the application of RUNX1, SPI1, and CEBPE in the induction differentiation treatment of acute myeloid leukemia. By introducing exogenous RUNX1, SPI1, and CEBPE genes into acute myeloid leukemia cells, acute myeloid leukemia cells can be effectively induced to differentiate into mature blood cells. Under a microscope, it can be seen that the suspended leukemia cells begin to adhere to the wall, and morphological and karyotype changes occur, while promoting AML cell apoptosis. Therefore, the present invention provides a new medical use for key transcription factors for hematopoietic development, and also provides a new method for introducing multiple key transcription factors for hematopoietic development into leukemia cells to work synergistically, induce leukemia differentiation, and then treat leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The present disclosure will be further described below in conjunction with the accompanying drawings, wherein these drawings are only for illustrating the embodiments of the present disclosure rather than for limiting the scope of the present disclosure.

[0045] Figure 1 The TetON-RUNX1-SPI1-CEBPE three-factor inducible expression lentiviral plasmid structure is shown.

[0046] Figure 2 The inhibition rate of RSE three-factor combination induced expression on leukemia cells such as K562, KASUMI-1, OCI-AML3, MOLM13, and MV-4-11 was calculated by CCK8 experiment.

[0047] Figure 3 The quantitative analysis of related mRNAs after 2 and 4 days of induction of the RSE three-factor combination in OCI-AML3 cells is shown. A is the expression of RUNX1, SPI1, and CEBPE, and B is the expression of a group of mature neutrophil marker genes.

[0048] Figure 4 The images show the cell morphology observations after 0 and 4 days of RSE triple factor combination induction expression in OCI-AML3 cells.

[0049] Figure 5 The results of observing the karyotype changes of leukemia cell lines by Wright-Giemsa staining 8 days after the induction of the expression of the RSE three-factor combination are shown. A is the OCI-AML3 cell line, B is the MOLM13 cell line, and C is the THP-1 cell line.

[0050] Figure 6 The expression of mature neutrophil surface markers in OCI-AML3 cells after 0, 2, 4 and 6 days of induction of RSE three-factor combination by flow cytometry is shown. A is the proportion of CD11b, CD66b, and CD62L positive cells, and B is the statistical analysis of cells in different groups.

[0051] Figure 7 The figure shows the quantitative analysis of apoptosis in OCI-AML3 cells by flow cytometry after 0-6 days of induction of expression of the RSE triple factor combination. DETAILED DESCRIPTION

[0052] The following examples and experimental examples further illustrate the present invention and should not be construed as limiting the present invention. The examples do not include a detailed description of conventional methods, such as methods for constructing vectors and plasmids, methods for inserting protein-encoding genes into vectors and plasmids, or methods for introducing plasmids into host cells. Such methods are well known to those skilled in the art and are described in many publications.

[0053] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the present invention, and the preferred implementation methods and materials described in the specific implementation methods are for demonstration purposes only.

[0054] Example 1: Construction of a DOX-induced expression RSE three-factor combination lentiviral plasmid and verification of sequence correctness by Sanger sequencing

[0055] (1) RNA was extracted from human peripheral blood mononuclear cells using an RNA extraction kit according to the instructions, and the RNA content was determined using an ultraviolet spectrophotometer, followed by immediate next step or storage at -80°C; (2) reverse transcription was performed to obtain cDNA;

[0056] Table 1 RNA extraction and reverse transcription reaction system and process

[0057]

[0058]

[0059] (3) RT-PCR was performed using RSE three-factor combination coding sequence specific primers. The reaction system is shown in Table 2 below, and the primer sequences are shown in Table 3:

[0060] Table 2 RT-PCR reaction system

[0061]

[0062] The reaction procedure was as follows: pre-denaturation at 98°C for 30 seconds; denaturation at 98°C for 10 seconds; annealing for 15 seconds, with the temperature adjusted according to the primers; extension at 72°C for 20 seconds / kb, with a total of 30 cycles of the above three steps; and complete extension at 72°C for 2 minutes.

[0063] Table 3 RSE three-factor combination coding sequence specific primer sequences

[0064]

[0065] (4) 1.5% (w / v) agarose gel electrophoresis, 120V, 40min. Cut the target fragment under blue light, purify the target gene coding sequence DNA using a commercial recovery kit (purchased from QIAGEN), and determine the DNA content using an ultraviolet spectrophotometer.

[0066] (5) The target gene coding sequence DNA fragment is cloned into the vector by the Gibson Assembly method. Specifically, the vector plasmid is linearized by PCR. A 15-20bp long homologous sequence is added to both ends of the target gene coding sequence DNA fragment by the PCR method. Prepare the Gibson Assembly Master Mix reaction system and incubate at 50°C for 1h. The product is transformed into DH5α competent cells, and then spread on LB solid culture medium supplemented with Ampicilin and cultured at 37°C overnight. Pick a single clone colony in LB liquid culture medium supplemented with Ampicilin, shake the bacteria at 37°C and 250rpm for 12-16h, then use a plasmid extraction kit to extract the plasmid, and use a UV spectrophotometer to determine the plasmid content. A small amount of plasmid is taken and sent for Sanger sequencing and compared with the reference sequence. The qualified plasmid can be used immediately in the next step or stored at -20°C. The constructed plasmid map is shown as follows. Figure 1 shown.

[0067] Example 2 Inducing the expression of the three-factor combination of RSE can significantly inhibit the proliferation of leukemia cells

[0068] In a 96-well plate, K562, KASUMI-1, OCI-AML3, MOLM13, and MV-4-11 leukemia cells containing RSE three-factor combination expression elements were added at a density of 10,000 cells per well. Doxycycline was added to each well of the experimental group to induce the expression of the RSE three-factor combination. After 4 days, 10 μl of CCK8 reagent was added to each well, and after incubation at 37°C for 3 hours, the absorbance value was measured at a wavelength of 450 nm in an ELISA reader to calculate the inhibitory ability of leukemia cell proliferation after expressing the RSE three-factor combination. The inhibition rate calculation formula is:

[0069] [(Ac-As) / (Ac-Ab)]×100%

[0070] As: absorbance of the experimental group; Ac: absorbance of the control group; Ab: absorbance of the blank well.

[0071] The results are as follows Figure 2The results showed that the RSE three-factor combination had the lowest inhibition rate on human chronic myeloid leukemia cells K562, about 50%, and the highest inhibition rate on human acute myeloblastic leukemia cells Kasumi-1, close to 90%, and the inhibition rate on human acute myeloid leukemia cells OCI-AML3, human acute myeloid leukemia cells MOLM13, and human myelomonocytic leukemia cells MV-4-11 was between 70% and 80%, indicating that inducing the expression of the RSE three-factor combination can significantly inhibit the proliferation of leukemia cells.

[0072] Example 3 RSE three factors promote OCI-AML3 leukemia cell differentiation

[0073] The virus packaging method is as follows: inoculate about 1×10 7 293T / 17 cells, and the confluence reached more than 80% after 16 hours. Prepare lentiviral packaging plasmids psPAX2, pMD2.G and RSE three-factor combination gene coding sequence expression plasmids. Use 10ng of plasmid for each 10cm dish, and the ratio of RSE three-factor combination gene coding sequence expression plasmid, psPAX2, and pMD2.G is 4:3:1, using 5ng, 3.75ng, and 1.25ng respectively. Add 10ng of plasmid to 500μl jetPrimebuffer, vortex mix for 10sec, and centrifuge briefly. Then add 20μl jetPrime reagent, mix well, and let stand at room temperature for 10min. Then slowly add the mixture dropwise to the culture dish. After 6h, the cell culture medium was replaced with complete culture medium (90% high glucose DMEM + 10% FBS) without transfection reagent. Collect the supernatant 36h, 48h, and 60h after transfection and replace with fresh culture medium. The supernatant containing lentiviral particles was collected and concentrated using PEG8000 or used immediately in the next cell infection experiment, and the rest was stored at -80°C.

[0074] OCI-AML3 leukemia cells were infected with RSE triple factor combination lentivirus, and the medium was changed after 24 hours. RFP was sorted using a flow cytometer produced by BD. + Cells. After obtaining cells with the RSE three-factor combination expression sequence inserted, 2 μg / ml of Doxycycline was used to induce the expression of the RSE three-factor combination gene, and the cells were collected after 2 days and 4 days of induction expression of the RSE three-factor combination. RNA was extracted and reverse transcribed to obtain cDNA according to the method in Example 1. Neutrophil function-related genes such as ITGAM, S100A8, S100A12, ARG1, and CEACAM1 were selected as cell differentiation detection indicators, and real-time quantitative PCR was performed. The reaction system is shown in Table 4, and the primer sequences of neutrophil characteristic genes are shown in Table 5:

[0075] Table 4 Real-time quantitative PCR reaction system

[0076]

[0077] Pre-denaturation at 95°C for 30 sec, amplification at 95°C for 10 sec, and 60°C for 30 sec were performed for 40 cycles, and then the melting curve was collected.

[0078] Table 5 Primer sequences of neutrophil characteristic genes

[0079]

[0080] Note: Integrin Subunit Alpha M (ITGAM); S100 Calcium Binding Protein A8 (S100A8); S100 Calcium Binding Protein A12 (S100A12); Arginase 1 (ARG1); CEA cell adhesion molecule 1 (CEA Cell Adhesion Molecule 1, CEACAM1).

[0081] The results are as follows Figure 3 The results showed that when Doxycycline was used to induce the expression of the RSE three-factor combination for 2 days, the expression levels of RUNX1, SPI1, and CEBPE increased significantly, but the expression levels on the 4th day were almost the same as those on the 2nd day, indicating that the RSE three-factor transfection cells can rapidly increase in the cells ( Figure 3 A); The high expression of RUNX1, SPI1, and CEBPE also led to a significant increase in the expression of multiple neutrophil marker genes, including ITGAM, S100A8, S100A12, ARG1, and CEACAM1 ( Figure 3 B).

[0082] Example 4 RSE three-factor combination induces leukemia cell morphological changes

[0083] After 4 days of inducing the expression of the RSE trio in OCI-AML3 cells using Doxycycline, the leukemia cells that were originally growing in suspension were observed under the microscope ( Figure 4 A) Begins to adhere to the wall and undergoes morphological changes ( Figure 4 B).

[0084] After inducing the expression of the RSE three-factor combination in OCI-AML3, MOLM13, and THP-1 cells for 1, 4, and 7 days, the cells were collected and leukemia cell slides were prepared. After drying, Wright-Giemsa stain A was used for staining for 1 minute, followed by stain B for staining for 8 minutes, and the stain was rinsed with running water and dried. Changes in cell karyotype were observed under a microscope.

[0085] The results showed that after 4 days of Doxycycline-induced RSE triple factor expression, a large number of neutrophil-like segmented nuclear cells appeared in OCI-AML3, MOLM13, and THP-1 cells ( Figure 5 ).

[0086] Example 5 RSE three-factor combination promotes leukemia cells to express neutrophil marker protein

[0087] After 1 to 6 days of inducing the expression of the RSE three-factor combination in OCI-AML3 cells using Doxycycline, the cells were collected, washed with PBS, resuspended in flow cytometry buffer (containing BSA and EDTA), treated with human Fc receptor blocking solution for 20 minutes, and added with mature neutrophil markers CD11b (FITC), CD66b (APC) and CD62L (PE-CY7) flow cytometry antibodies, and incubated on ice for 30 minutes. The expression of each marker was detected by flow cytometry ( Figure 6 A).

[0088] The results showed that after the induction of the RSE three-factor combination, OCI-AML3 cells gradually expressed neutrophil-specific cell membrane surface proteins ( Figure 6 B).

[0089] Example 6 The combined expression of the three RSE factors promotes apoptosis of leukemia cells

[0090] After 1 to 6 days of inducing the expression of the RSE three-factor combination in OCI-AML3 cells using Doxycycline, about 300,000 cells were collected by centrifugation at 300g for 5 minutes and washed twice with DPBS. The cells were resuspended in 100μl 1×Binding Buffer, and 5μl AnnexinV-FITC and 5μl 7-AAD were added. After incubation at room temperature for 10 minutes in the dark, 400μl 1×Binding Buffer was added, and cell apoptosis was analyzed by flow cytometry ( Figure 7 A).

[0091] The results showed that the induction of expression of the three RSE factors promoted the apoptosis of leukemia cells, early apoptosis (Annexin V + / 7-AAD - ) and late apoptosis and death (AnnexinV+ / 7-AAD + ) increased the proportion of OCI-AML3 cells ( Figure 7 B).

[0092] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. Use of RUNX1, SPI1 and CEBPE in combination for the preparation of drugs for the treatment of acute myeloid leukemia.

2. The use according to claim 1, characterized in that The drug for treating acute myeloid leukemia is a drug for inducing differentiation-blocked leukemia cells to differentiate into mature blood cells.

3. The use according to claim 1, characterized in that The RUNX1, SPI1, and CEBPE combination is selected from the combination of exogenous RUNX1 protein, SPI1 protein, and CEBPE protein, the combination of exogenous RUNX1 gene, SPI1 gene, and CEBPE gene, a recombinant expression vector simultaneously loaded with RUNX1 gene, SPI1 gene, and CEBPE gene, a combination of substances that promote the expression of RUNX1 gene, SPI1 gene, and CEBPE gene, and a combination of mRNA, cDNA, or precursors of RUNX1, SPI1, and CEBPE.

4. The use according to claim 3, Features: Wherein, the RUNX1 gene is selected from any of the following situations: (i) a nucleic acid molecule having a nucleotide sequence as shown in GENBANK ID: NM_001754.5; (ii) a molecule that hybridizes to the nucleotide sequence defined in (i) under stringent conditions; (iii) a nucleic acid molecule homologous to or having sequence identity with the nucleotide sequence shown in GENBANK ID: NM_001754.5; (iv) a nucleic acid molecule in which one or more nucleotides are substituted, deleted or added in the nucleotide sequence of (i), (ii) or (iii); The SPI1 gene is selected from any of the following situations: (i) a nucleic acid molecule having a nucleotide sequence as shown in GENBANK ID: NM_003120.3; (ii) a molecule that hybridizes to the nucleotide sequence defined in (i) under stringent conditions; (iii) a nucleic acid molecule homologous to or having sequence identity with the nucleotide sequence shown in GENBANK ID: NM_003120.3; (iv) a nucleic acid molecule in which one or more nucleotides are substituted, deleted or added in the nucleotide sequence of (i), (ii) or (iii); The CEBPE gene is selected from any of the following situations: (i) a nucleic acid molecule having a nucleotide sequence as shown in GENBANK ID: NM_001805.4; (ii) a molecule that hybridizes to the nucleotide sequence defined in (i) under stringent conditions; (iii) a nucleic acid molecule homologous to or having sequence identity with the nucleotide sequence shown in GENBANK ID: NM_001805.4; (iv) A nucleic acid molecule in which one or more nucleotides are substituted, deleted or added in the nucleotide sequence of (i), (ii) or (iii).

5. A RSE three-factor recombinant expression vector, characterized in that: The recombinant vector includes an expression vector and nucleic acid molecules of RUNX1 gene, SPI1 gene and CEBPE gene inserted into the expression vector. The nucleic acid molecules of RUNX1 gene, SPI1 gene and CEBPE gene are as shown in claim 4.

6. The RSE three-factor recombinant expression vector according to claim 5, characterized in that: in, The expression vector is a plasmid vector, a cosmid vector, a phage vector or a virus vector.

7. Use of the RSE three-factor recombinant expression vector according to claim 5 or 6 in the preparation of a drug for treating acute myeloid leukemia.

8. A combination drug for treating acute myeloid leukemia, characterized in that: Its active ingredients include any of the following: (a) RUNX1 protein, SPI1 protein and CEBPE protein; (b) the coding sequence of RUNX1 protein, the coding sequence of SPI1 protein and the coding sequence of CEBPE protein; (c) a recombinant expression vector containing a coding sequence of RUNX1 protein, a coding sequence of SPI1 protein and a coding sequence of CEBPE protein, The coding sequence of the protein is shown in claim 4.

9. The combination drug for treating acute myeloid leukemia according to claim 8, characterized in that: Used in combination with other AML chemotherapy drugs.

Images