Application of fangchinoline in preparation of medicine for treating STAG2 mutation acute myelogenous leukemia

Abstract

The invention provides application of fangchinoline in preparation of a medicine for treating STAG2 mutation acute myelogenous leukemia, and relates to the field of leukemia treatment medicines. According to the application of the fangchinoline in preparation of the medicine for treating the STAG2 mutation acute myelogenous leukemia, aiming at the problems that the STAG2 mutation acute myelogenous leukemia is high in incidence rate, poor in prognosis and lack of effective medicine clinically, proliferation and colony formation of AML cell lines SKM-1, Kasumi-6, THP-1 and U-937 can be inhibited through the fangchinoline, and the STAG2 mutation acute myelogenous leukemia can be treated. The cell apoptosis, cycle arrest and cell differentiation are induced, so that the effect of resisting STAG2 mutation acute myelogenous leukemia is achieved.

Description

Technical Field

[0001] The present invention relates to the field of leukemia therapeutic drugs, and in particular to the use of fangchinoline in the preparation of STAG2 mutation acute myeloid leukemia therapeutic drugs. Background Art

[0002] Leukemia is a malignant clonal disease of hematopoietic stem cells, characterized by abnormal proliferation of bone marrow and damage to normal hematopoietic function. Clinical manifestations include anemia, bleeding, infection and organ infiltration symptoms (such as hepatosplenomegaly). According to the course of the disease, it is mainly divided into acute leukemia and chronic leukemia. According to the cell origin, it is mainly divided into myeloid leukemia and lymphocytic leukemia. It mainly includes: chronic myeloid leukemia, chronic lymphocytic leukemia, acute lymphocytic leukemia, and acute myeloid leukemia.

[0003] Among them, chronic myeloid leukemia is a malignant tumor formed by clonal proliferation of bone marrow hematopoietic stem cells. The presence of Ph chromosome and / or BCR-ABL fusion gene is the main factor leading to the occurrence of this disease. Tyrosine kinase inhibitors such as imatinib are commonly used as first-line treatment options for this disease.

[0004] Acute lymphoblastic leukemia is a leukemia caused by abnormal proliferation of lymphocyte progenitor cells in the bone marrow, which is related to gene mutation or chromosomal aberration. The commonly used treatment plan is based on vincristine or vindesine, anthracycline / anthraquinone drugs (such as daunorubicin, idarubicin, doxorubicin, mitoxantrone, etc.), and glucocorticoids (such as prednisone, dexamethasone, etc.). Among them, the mechanism of action of vincristine is to inhibit microtubule polymerization and block the cell cycle in the M phase.

[0005] Acute myeloid leukemia (AML) is a blood system malignancy caused by abnormal proliferation of hematopoietic stem cells. It is the most common type of adult leukemia, accounting for about 70%, and has a high incidence and mortality rate. Specifically, acute myeloid leukemia is a malignant tumor formed by clonal proliferation of bone marrow hematopoietic stem cells. Existing studies have found that gene mutation is a key factor leading to the occurrence of AML. Multiple genes such as FLT3, NPM1, mLL, STAG2, etc., either alone or in concert, mutations or chromosomal aberrations lead to the occurrence of the disease. Among them, the detection rate of STAG2 (stromal antigen 2) mutation in acute myeloid leukemia and myelodysplastic syndrome is about 5-15%, and is closely related to the poor prognosis of patients [Bachelot A, Bouvier A, Riou J, Thepot S, Giltat A, Nunes Gomes C, Paillassa J, Jouanneau-Courville R, Renard M, Beucher A, Cottin L, Wiber M, Ribourtout B, Geneviève F, Luque Paz D, Tanguy-Schmidt A, Ugo V, Hunault-Berger M, Blanchet O, Orvain C. Relationship between comorbidities, mutational profile, and outcome after intensive chemotherapy in patients older than 60 years with acute myeloid leukemia: Assessment of different risk scores. Am J Hematol. 2023 Jun;98(6):922-931. Tariq H, Barnea Slonim L, Coty Fattal Z, Alikhan MB, SegalJ, Gurbuxani S, Helenowski IB, Zhang H, Sukhanova M, Lu X, Altman JK, Chen QC, BehdadA.Therapy-related myeloid neoplasms with normal karyotype showdistinctgenomic and clinical characteristics compared to their counterpartswith abnormal karyotype. Br J Haematol.2022 Jun;197(6):736-744. Anwar N,MemonFA,Shahid S,Shakeel M,Irfan M,Arshad A,Naz A,Ujjan ID,Shamsi T. The Dawnof next generation DNA sequencing in myelodysplastic syndromes- experiencefromPakistan. BMC Genomics.2021 Dec 16;22(1):903. Eckardt JN,Stasik S,RölligC,Sauer T,Scholl S,Hochhaus A,Crysandt M,BrümmendorfTH,Naumann R,Steffen B etal:Alterations of cohesin complex genes in acute myeloid leukemia:differential co-mutations,clinical presentation and impact onoutcome.BloodCancer J 2023,13(1):18.]。.

[0006] STAG2 is a key subunit of the cohesin complex, located on the X chromosome, responsible for maintaining the tight adhesion of sister chromatids during cell division and ensuring the correct separation of chromosomes in mitosis. Its mutation destroys the differentiation ability of hematopoietic stem cells (HSCs) and enhances the self-renewal potential of HSCs, leading to the occurrence of leukemia [Ochi Y, Kon A, Yoshida K, Kataoka K, Nakagawa MM, Makishima H, Nakayama M, Koseki H, Takaori-Kondo A, Ogawa S: Stag2 regulates Hematopoietic Differentiation and Self-Renewal. Blood 2017, 130(Supplement 1): 790-790.].

[0007] Currently, there is no effective and unified treatment for acute myeloid leukemia with STAG2 mutations. Studies have shown that STAG2 mutations lead to reduced cohesin-DNA binding ability, thereby enhancing sensitivity to PARP inhibitors [Tothova Z, Valton AL, Gorelov RA, Vallurupalli M, Krill-Burger JM, Holmes A, Landers CC, Haydu JE, Malolepsza E, Hartigan C, Donahue M, Popova KD, Koochaki S, Venev SV, Rivera J, Chen E, Lage K, Schenone M, D'Andrea AD, Carr SA, Morgan EA, Dekker J, Ebert BL. Cohesin mutations alter DNA damage repair and chromatin structure and create therapeutic vulnerabilities in MDS / AML. JCI Insight. 2021 Feb 8;6(3):e142149.]. Given the high incidence, poor prognosis and lack of effective drugs in clinical practice of STAG2 mutation AML, it is urgent to find effective drugs for the treatment of STAG2 mutation acute myeloid leukemia.

[0008] Fangchinoline is a dibenzylisoquinoline alkaloid with a molecular formula of Figure 1As shown, it is derived from Fangchinoline which is widely present in nature [Jiang, F., Ren, S., Chen, Y., Zhang, A., Zhu, Y., Zhang, Z., Li, Z., & Piao, D. (2021). Fangchinoline exerts antitumour activity by suppressing the EGFR-PI3K / AKT signalling pathway in colon adenocarcinoma. Oncology reports, 45(1), 139–150.]. Fangchinoline has multiple biological and pharmacological activities, including antioxidant, anti-osteoporosis, anti-inflammatory, immune-enhancing and histamine release inhibition [Winardi, D., Chu, PY, Chen, GY, Wang, K., Hsu, WY, Hsieh, CL, Chen, YH, Wu, YC, & Yang, JC (2022). Novel Aurora A Kinase Inhibitor Fangchinoline Enhances Cisplatin-DNA Adducts and Cisplatin Therapeutic Efficacy in OVCAR-3 Ovarian Cancer Cells-Derived Xenograft Model. International journal of molecular sciences, 23(3), 1868.].Studies have shown that fangchinoline can inhibit the proliferation of tumor cell lines such as gastric cancer, colorectal cancer, liver cancer, gallbladder cancer, kidney cancer, bladder cancer, breast cancer, multiple myeloma, chronic myeloid leukemia, and acute lymphocytic leukemia, induce their apoptosis, and inhibit tumor cell metastasis. In addition, existing in vivo experiments have also confirmed that fangchinoline can inhibit tumor growth [Chen B, Song Y, Zhan Y, Zhou S, Ke J, Ao W, Zhang Y, Liang Q, He M, Li S, Xie F, Huang H, Chan WN, Cheung AHK, Ma BBY, Kang W, To KF, Xiao J. Fangchinoline inhibits non-small cell lung cancer metastasis by reversing epithelial-mesenchymal transition and suppressing the cytosolic ROS-related Akt-mTOR signaling pathway. Cancer Lett. 2022 Sep 1;543:215783. Jung YY, Um JY, Sethi G, Ahn KS. Fangchinolineabrogates growth and survival of hepatocellular carcinoma by negative regulation of c-met / HGF and its associated downstream signaling pathways. Phytother Res. 2022 Dec;36(12):4542-4557.].

[0009] The prior art has disclosed technical means for using stephaniatetrandra to treat chronic myeloid leukemia, multiple myeloma, and acute lymphoblastic leukemia. The specific treatment mechanism is as follows: in the acute lymphoblastic leukemia cell line Jurkat, stephaniatetrandra inhibits the AKT signaling pathway, promotes the production of reactive oxygen species, and oxidative stress induces DNA damage, thereby promoting cell apoptosis [Shao Y, Li C, Miao G, Xu Y. Fangchinoline, an Extract of the Stephaniatetrandra S. Moore Root, Promoted Oxidative Stress-induced DNA Damage and Apoptosis and Inhibited Akt Signaling in Jurkat T Cells. Curr Mol Pharmacol. 2024; 17(1): e100223213590.]. In the chronic myeloid leukemia cell line KBM5 and the multiple myeloma cell line U266, fangchinoline can inhibit NF-κB and AP-1 activation, including attenuating the phosphorylation of IκB kinase (IKK) and p65, and can also significantly enhance TNFα-driven cell apoptosis [Jung YY, Shanmugam MK, Chinnathambi A, Alharbi SA, Shair OHM, Um JY, Sethi G, Ahn KS. Fangchinoline, aBisbenzylisoquinoline Alkaloid can Modulate Cytokine-Impelled Apoptosis via the Dual Regulation of NF-κB and AP-1 Pathways. Molecules. 2019 Aug 28;24(17):3127.]. In addition, in the chronic myelogenous leukemia cell line K562, Fangchinoline can also regulate the expression of CDKN1A and CCND2 and induce cell arrest in the G0 / G1 cycle [Wang Y, Chen J, Wang L, Huang Y, Leng Y, Wang G. Fangchinoline induces G0 / G1 arrest by modulating the expression of CDKN1Aand CCND2 in K562 human chronic myelogenous leukemia cells. Exp Ther Med. 2013Apr;5(4):1105-1112.] Compared with the aforementioned chronic myeloid leukemia, multiple myeloma, and acute lymphoblastic leukemia, STAG2 mutation acute myeloid leukemia is caused by impaired function of the cohesin complex [Fischer A, Hernández-Rodríguez B, Mulet-Lazaro R, Nuetzel M, Hölzl F, van Herk S, Kavelaars FG, Stanewsky H, AckermannU, Niang AH, Diaz N, Reuschel E, Strieder N, Hernández-López I, Valk PJM, Vaquerizas JM, Rehli M, Delwel R, Gebhard C. STAG2 mutations reshape the cohesin-structured spatialchromatin architecture to drive gene regulation in acutemyeloid leukemia. Cell Rep. 2024 Aug 27;43(8):114498.] and dysregulated gene expression [Fischer A,Hernández-Rodríguez B,Mulet-Lazaro R,Nuetzel M,Hölzl F,van Herk S,KavelaarsFG,Stanewsky H,AckermannU,Niang AH,Diaz N,Reuschel E,Strieder N,Hernández-López I,Valk PJM,Vaquerizas JM,Rehli M,Delwel R,Gebhard C.STAG2 mutations reshape the cohesin-structuredspatial chromatin architecture to drive generegulation in acute myeloid leukemia. Cell Rep. 2024 Aug 27;43(8):114498.], hematopoietic stem cell differentiation blockade [Mullenders J, Aranda-Orgilles B, Lhoumaud P, Keller M, Pae J, Wang K, Kayembe C, Rocha PP, RaviramR, Gong Y, Premsrirut PK, Tsirigos A, Bonneau R, Skok JA, Cimmino L, Hoehn D, Aifantis I. Cohesin loss alters adult hematopoieticstem cell homeostasis, leading to myeloproliferative neoplasms. J Exp Med. 2015Oct 19;212(11):1833-50.] and synergistic effects with other gene mutations [Chin CV, Antony J, Ketharnathan S, Labudina A, Gimenez G, Parsons KM, HeJ, George AJ, Pallotta MM, Musio A, Braithwaite A, Guilford P, Hannan RD, Horsfield JA. Cohesin mutations are synthetic lethal with stimulation of WNT signaling. Elife. 2020 Dec 7;9:e61405.], leading to the occurrence of leukemia. .

[0010] Chronic myeloid leukemia is mainly driven by the BCR-ABL fusion gene, which is caused by the overactivation of tyrosine kinase, and its epigenetics is less affected. Therefore, tyrosine kinase inhibitors have a good therapeutic effect on chronic myeloid cells, while demethylation inhibitors alone have no significant effect.

[0011] Multiple myeloma is a malignant blood disease originating from plasma cells in the bone marrow. The disease is caused by mutations in multiple genes such as KRAS, NRAS, DIS3, BRAF, or chromosomal aberrations. Currently, the commonly used treatment for this disease is a combination of proteasome inhibitors (such as bortezomib) and immunomodulators (such as lenalidomide) and dexamethasone. The main effect of proteasome inhibitors is to inhibit the function of 26S proteasomes in myeloma cells, leading to the accumulation of abnormal proteins in the cells, thereby inducing cell apoptosis.

[0012] The main difference between acute lymphoblastic leukemia and acute myeloid leukemia in molecular mechanisms lies in specific chromosomal translocations, fusion genes and gene mutations. BCR-ABL1, ETV6-RUNX1 and TCF3-PBX1 fusion genes, as well as NOTCH1, FBXW7 and KRAS / NRAS mutations are common in acute lymphoblastic leukemia. RUNX1-RUNX1T1, CBFB-MYH11 and PmL-RARA fusion genes, as well as FLT3, NPM1, DNMT3A and IDH1 / IDH2 mutations are common in acute myeloid leukemia. Both involve genomic instability, abnormal signaling pathways and changes in epigenetic regulation, but the genes that mutate are completely different. Demethylating drugs such as azacitidine and decitabine alone have a good therapeutic effect on acute myeloid leukemia carrying mutations such as DNMT3A, IDH1, IDH2, TET2, etc., but the therapeutic effect on chronic myeloid leukemia, acute lymphoblastic leukemia and multiple myeloma is generally poor.

[0013] In short, the molecular mechanisms of STAG2 mutation acute myeloid leukemia are different from those of chronic myeloid leukemia, multiple myeloma, and acute lymphoblastic leukemia, which leads to completely different treatment strategies.

[0014] At the same time, existing studies have shown that the STAG2 mutant cell line SKM-1 is insensitive to commonly used chemotherapy drugs for acute myeloid leukemia, such as cytarabine [Liang SM, Zhou XJ, Cai D, Zhou Q, Wang L. SPARC Overexpression Enhances the Sensitivity of SKM-1 Cells to Ara-C by Regulating CPBP / mL KL. Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2022 Oct; 30(5): 1508-1514. Kimura S, Kuramoto K, Homan J, Naruoka H, ​​Ego T, Nogawa M, Sugahara S, Naito H. Antiproliferative and antitumor effects of azacitidine against the human myelodysplastic syndrome cell line SKM-1. Anticancer Res. 2012 Mar; 32(3): 795-8.]. This directly leads to the fact that there is currently no effective treatment for STAG2 mutation acute myeloid leukemia except hematopoietic stem cell transplantation.

[0015] It can be seen that in view of the high incidence, poor prognosis and lack of effective drugs in clinical practice of STAG2 mutation acute myeloid leukemia, providing effective drugs for the targeted treatment of STAG2 mutation acute myeloid leukemia has important technical significance and research value. Summary of the invention

[0016] In view of the problems of high incidence, poor prognosis and lack of effective drugs in clinical practice of STAG2 mutation acute myeloid leukemia, the present invention provides the use of fangchinoline in the preparation of a drug for treating STAG2 mutation acute myeloid leukemia.

[0017] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows: Application of fangchinoline in the preparation of therapeutic drugs for STAG2 mutation acute myeloid leukemia.

[0018] Furthermore, the drug for treating STAG2 mutant acute myeloid leukemia contains fangchinoline or its salt as an effective ingredient or main effective ingredient.

[0019] A pharmaceutical composition for treating STAG2 mutation acute myeloid leukemia, which uses fangchinoline or its salt as an effective ingredient or a main effective ingredient and is prepared into a pharmaceutically acceptable dosage form with a pharmaceutically acceptable excipient.

[0020] Preferably, the pharmaceutical composition comprises: fangchinoline liposomes.

[0021] Preferably, the fangchinoline liposome comprises the following raw materials in parts by weight: 0.6-0.8 parts of fangchinoline, 1-1.3 parts of cholesterol, and 6-8 parts of phospholipids.

[0022] Furthermore, the fangchinoline liposome is prepared by the following method: S1. adding phospholipids, fanghenoline and cholesterol into an ethanol solution and dispersing them evenly to obtain a mixed organic phase; S2. The mixed organic phase is injected into pure water, and after heating to remove alcohol, pure water is added to the volume before removing alcohol to obtain an initial liposome solution; S3. The liposome initial solution was extruded and filtered using filter membranes with pore sizes of 0.45 μm and 0.22 μm to obtain the fanghemoline liposomes.

[0023] Preferably, in step S1, the weight ratio of fangchinoline, cholesterol and phospholipid is 0.6-0.8:1-1.3:6-8.

[0024] Preferably, in step S1, the volume concentration of the ethanol solution is 70-80%.

[0025] Preferably, in step S2, the mixed organic phase is injected into 4-7 times the volume of pure water.

[0026] Preferably, in step S3, the average particle size of the obtained fangchinoline liposomes is less than 300 nm.

[0027] Compared with the prior art, the present invention has the following beneficial effects: (1) The test of the present invention showed that the use of the fangjinoline can significantly induce apoptosis of STAG2 mutant acute myeloid leukemia cell lines SKM-1, Kasumi-6, THP-1 and U-937, with a 24-hour IC 50 The levels of STAG2 in the acute myeloid leukemia cells were 6.147±0.261μmol / L, 7.070±0.275μmol / L, 13.66±0.584μmol / L and 18.53±0.342μmol / L, respectively. It can be seen that STAG2 mutant acute myeloid leukemia cells are more sensitive to fangchinoline than wild-type AML cells.

[0028] (2) According to the test of the present invention, the use of the described fangchinoline can significantly induce cell cycle arrest of STAG2 mutant acute myeloid leukemia cells; after drug treatment, SKM-1 cells arrested in the G1 phase, and Kasumi-6 cells arrested in the S phase; at the same time, fangchinoline can also induce differentiation of STAG2 mutant acute myeloid leukemia cells and inhibit the formation of AML cell colonies.

[0029] (3) The experiments of the present invention showed that the use of the fangchinoline can significantly increase the levels of lipid peroxides and ROS in SKM-1 and Kasumi-6 cells, regulate SLC7A11 expression through NRF2, and ultimately trigger ferroptosis in STAG2 mutant acute myeloid leukemia cell lines.

[0030] (4) The use of the fangchinoline of the present invention in the preparation of a therapeutic drug for STAG2 mutation acute myeloid leukemia. The fangchinoline or its salt can be used as an effective ingredient or a main effective ingredient in the preparation of the therapeutic drug to inhibit the proliferation and colony formation of AML cell lines SKM-1, Kasumi-6, THP-1 and U-937, induce apoptosis, cell cycle arrest and cell differentiation of these cells, thereby exerting an anti-STAG2 mutation acute myeloid leukemia effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is the chemical formula of fangchinoline.

[0032] Figure 2 These are transmission electron micrographs of fangchinoline liposomes. In the figure, Figure A is a transmission electron micrograph of fangchinoline liposomes (×2000); Figure B is a transmission electron micrograph of fangchinoline liposomes (×20000).

[0033] Figure 3 The expression of STAG2 in AML cell lines SKM-1, Kasumi-6, THP-1, and U-937. In the figure, Figure A shows the expression level of STAG2 mRNA in STAG2 mutant cell lines SKM-1 and Kasmi-6 and STAG2 wild-type cell lines THP-1 and U-937; Figure B shows the expression level of STAG2 protein in STAG2 mutant cell lines SKM-1 and Kasmi-6 and STAG2 wild-type cell lines THP-1 and U-937 (* in each figure) P <0.05;** P <0.01).

[0034] Figure 4 This is a graph showing the proliferation results of STAG2 mutant AML cell lines SKM-1, Kasumi-6 and wild-type AML cell lines THP-1, U-937 after treatment with different concentrations of fangchinoline for 24 hours and 48 hours in the examples of the present invention.

[0035] Figures 5 to 8 The apoptosis results and statistical results of STAG2 mutant and wild-type AML cell lines SKM-1, Kasumi-6, THP-1 and U-937 treated with fangchinoline for 24h and 48h in the embodiments of the present invention (* in each figure) P <0.05;** P <0.01).

[0036] Fig. 9 The results and statistical results of the cell cycle of STAG2 mutant AML cell lines SKM-1 and Kasumi-6 after 24 hours of treatment with fangchinoline in the embodiment of the present invention. In the figure, Figure A shows the effect of fangchinoline on the cell cycle of SKM-1; Figure B shows the effect of fangchinoline on the cell cycle of Kasumi-6 (* in each figure P <0.05;** P <0.01).

[0037] Fig.10 The results and statistical results of the effect of fangchinoline on the differentiation of STAG2 mutant AML cell lines SKM-1 and Kasumi-6 in the embodiments of the present invention are shown in Figure A. Figure A shows the effect of fangchinoline on the morphology of SKM-1 cells; Figure B shows the effect of fangchinoline on the morphology of Kasumi-6 cells.

[0038] Fig.11 The expression of CD15 in SKM-1 and Kasumi-6 cells was changed after fangchinoline was used to treat SKM-1 and Kasumi-6 cells in the embodiment of the present invention (* in the figure) P<0.05;** P <0.01).

[0039] Fig.12 The results and statistical results of STAG2 mutant AML cell lines SKM-1 and Kasumi-6 cell colonies after 15 days of treatment with fangqinoline in the embodiment of the present invention are shown in Figure A. Figure A shows the results of SKM-1 and Kasumi-6 cell colonies; Figure B shows the number of colonies formed by SKM-1 and Kasumi-6 cells (* in each figure P <0.05;** P <0.01).

[0040] Figures 13 to 15 This is a result diagram of the mechanism of action of fangchinoline in the examples of the present invention in inhibiting STAG2 mutant AML cell lines. Fig.13 In the figure, Figure A is a volcano map of differentially expressed genes; Figure B is a heat map of differentially expressed genes; Figure C is the KEGG pathway analysis of differentially expressed genes; Fig.14 Figure D in the middle shows the morphological changes of mitochondria in SKM-1 cells observed by transmission electron microscopy; Fig.15 Figure E in the middle shows the changes in mitochondrial morphology of Kasumi-6 cells observed by transmission electron microscopy.

[0041] Figure 16 to Figure 17 1 is a graph showing the effect of fangqinoline on lipid peroxidation and ROS accumulation in STAG2 mutant AML cell lines in the present invention (* in each figure P <0.05;** P <0.01).

[0042] Fig.18 is a graph showing the results of Fangqinoline down-regulating the expression of NRF2 and SLC7A11 in STAG2 mutant AML cells in the embodiment of the present invention (* in each figure P <0.05;** P <0.01).

[0043] Fig.19 It is a mouse survival curve diagram in an embodiment of the present invention. DETAILED DESCRIPTION

[0044] In order to have a clearer understanding of the technical features, purposes and effects of the present invention, the specific embodiments of the present invention are now described. It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0045] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, "first", "second", etc. are used to distinguish similar objects, and are not used to describe a specific order or sequence. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0046] The embodiment of the present invention provides the use of fangchinoline in preparing a drug for treating STAG2 mutation acute myeloid leukemia.

[0047] Furthermore, the drug for treating STAG2 mutant acute myeloid leukemia contains fangbotoxetine or its salt as an active ingredient or main active ingredient.

[0048] The embodiment of the present invention also provides a pharmaceutical composition for treating STAG2 mutation acute myeloid leukemia, which uses fangchinoline or its salt as an effective ingredient or a main effective ingredient, and is prepared into a pharmaceutically acceptable dosage form with a pharmaceutically acceptable excipient.

[0049] Wherein, the aforementioned compound salt refers to a derivative of the fanghemoline; the parent compound is modified by making an acid salt or an alkaline salt of the parent compound. Examples of compound salts include, but are not limited to: inorganic acid salts or organic acid salts of basic residues (such as amine groups); alkaline salts or organic salts of acidic residues (such as carboxylic acids, etc.). The compound salts of the present invention can be synthesized from a parent compound containing a basic portion or an acidic portion by conventional chemical methods. In addition, the aforementioned compound salts can be prepared in situ during the final separation and purification of the compound.

[0050] The aforementioned excipients refer to any excipients, solvents, dispersion media, absorption retardants, diluents or adjuvants that do not cause secondary reactions such as allergic reactions in humans or animals; such as preservatives or antioxidants, fillers, binders, disintegrants, wetting agents, emulsifiers, suspending agents, solvents, dispersion media, coating agents, antibacterial agents, isotonic agents and absorption delay agents. Examples of typical excipients include, but are not limited to, mannitol, lactose, magnesium stearate, sodium saccharide, talc, cellulose, cross-linked sodium carboxymethyl cellulose, glucose, gelatin, starch, lactose, dicalcium phosphate, sucrose, kaolin, magnesium carbonate, wetting agents, emulsifiers, solubilizers, sterile water, physiological saline, pH buffers, nonionic surfactants, lubricants, stabilizers, binders and edible oils (such as peanut oil, sesame oil, etc.). At the same time, the use of the aforementioned excipients in the pharmaceutical composition or feed additives is foreseeable.

[0051] Furthermore, the pharmaceutical composition for treating STAG2 mutation acute myeloid leukemia is fangchinoline liposomes; the fangchinoline liposomes include the following raw materials in parts by weight: 0.6-0.8 parts of fangchinoline, 1-1.3 parts of cholesterol, and 6-8 parts of phospholipids.

[0052] Furthermore, the preparation method of the fangchinoline liposome comprises the following steps: S1. Dissolve phospholipids in an ethanol solution with a volume concentration of 70-80% to prepare a phospholipid ethanol solution with a mass concentration of 3-5%, and then add fanghemoline and cholesterol to disperse them evenly by ultrasonication to obtain a mixed organic phase.

[0053] Among them, the weight ratio of fanghenoline, cholesterol and phospholipid is 0.6-0.8:1-1.3:6-8.

[0054] S2. Inject the above mixed organic phase into 4-7 times the volume of pure water through a syringe pump, heat it to 55-60°C, stir it to remove ethanol, add pure water to the original volume (i.e. the volume of the material before removing alcohol), and obtain the initial liposome solution.

[0055] S3. The above-mentioned liposome initial solution is extruded and filtered through filter membranes with pore sizes of 0.45 μm and 0.22 μm to obtain fanghemoline liposomes with an average particle size of less than 300 nm.

[0056] The present invention is further described below in conjunction with some specific embodiments.

[0057] Example 1 This example provides the preparation of a pharmaceutical composition (fangchinoline liposomes) for treating STAG2 mutation acute myeloid leukemia, as follows: 1. Materials and reagents: fangchinoline (dissolved to 20 mM in DMSO, purchased from MedChemExpress, Shanghai, China), cholesterol (purchased from MedChemExpress, Shanghai, China), phospholipids, ethanol solution (volume concentration 75%), pure water, 0.45 nm and 0.22 nm filter membranes, 1.5 mL centrifuge tubes, and 10 μL sterile pipette tips.

[0058] 2. Operation steps: Take 25 mg of phospholipids and dissolve them in 0.8 mL of ethanol solution (volume concentration 75%). After stirring and dissolving, take 2.5 mg of effective mass of fangchinoline, weigh 4.1 mg of cholesterol on an electronic balance and put them into it. Ultrasonic dispersion for 10 minutes to fully mix it to obtain a mixed organic phase. Add 5 mL of pure water to a small beaker, and inject the above mixed organic phase into the pure water through a syringe pump while turning on the magnetic stirring. Heat it to 55 ° C and keep it warm. Stir at 20 rpm for 1 hour to remove ethanol, and add pure water to the original volume (i.e. the volume of the material before removing alcohol) to obtain the initial liposome solution. Filter the initial liposome solution three times with filter membranes with pore sizes of 0.45 μm and 0.22 μm to obtain a fangchinoline liposome dispersion.

[0059] 3. Preparation results: After testing, the particle size of the fangchinoline liposome prepared in this example is 244.0 nm, the particle size dispersion coefficient is 0.262, and the encapsulation efficiency is 93.3%; the transmission electron microscopy image of the fangchinoline liposome is as follows: Figure 2 shown.

[0060] Example 2 This example provides real-time fluorescence quantitative PCR and immunoblotting assays, specifically: 1. Materials and reagents: TRIzol, RPMI 1640 medium (GIBCO), fetal bovine serum (GIBCO), penicillin-streptomycin, RIPA, 4× loading buffer, reverse transcription and real-time fluorescence quantitative PCR kit (purchased from Acre Biotechnology Co., Ltd., Changsha, China), GAPDH, HRP-labeled goat anti-rabbit antibody, HRP-labeled goat anti-mouse antibody (purchased from ABclonal, Wuhan, China), STAG2 antibody (purchased from Abways, Shanghai, China), 15 mL sterile centrifuge tubes, 1.5 mL centrifuge tubes, 10 μL sterile pipette tips, 200 μL sterile pipette tips, 1 mL sterile pipette tips, and 6-well cell culture plates.

[0061] 2. Operation steps: Total RNA was extracted from untreated SKM-1, Kasumi-6, THP-1 and U-937 cells using TRIzol. 1 μg of total RNA was reverse transcribed to generate cDNA. STAG2 was quantified by qPCR using the Applied Biosystems 7,500 Fast real-time fluorescence quantitative PCR system based on the SYBR Green method. —△△Ct Method. The primer sequences used are as follows: The forward primer sequence is shown in the sequence listing as SEQ ID NO.1; The reverse primer sequence is shown in the sequence listing as SEQ ID NO.2.

[0062] Equal amounts of cell lysate were loaded onto SDS-PAGE gels and electrophoresed. Proteins were transferred to PVDF membranes. The membranes were blocked with 5% skim milk in TBST and then incubated with GAPDH and STAG2 antibodies overnight. Afterwards, the membranes were incubated in HRP-labeled goat anti-rabbit antibody or HRP-labeled goat anti-mouse antibody solution for 1 h. Finally, enhanced chemiluminescence reagents and Amersham Imager 600 were used to visualize the protein bands.

[0063] 3. Experimental results: Figure 3 As shown, it can be seen that both qPCR and immunoblotting results showed that STAG2 expression in SKM-1 cells and Kasumi-6 cells was significantly reduced, confirming the presence of truncation mutations in the two cell lines.

[0064] Example 3 This example uses fangchinoline to conduct a cell proliferation test (CCK-8 method), specifically: 1. Materials and reagents: fangchinoline (dissolved in DMSO, purchased from MedChemExpress, Shanghai, China), 1640 culture medium (GIBCO), fetal bovine serum (GIBCO), CCK-8 (Solebol), penicillin-streptomycin, 15 mL sterile centrifuge tubes, 1.5 mL centrifuge tubes, 10 μL sterile pipette tips, 200 μL sterile pipette tips, 1 mL sterile pipette tips, and 96-well cell culture plates.

[0065] 2. Operation steps: SKM-1, Kasumi-6, THP-1 and U-937 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum. When they entered the logarithmic growth phase, the cells were collected and counted, and inoculated into 96-well plates with 1×10 cells per well. 4 . After 6 hours, different concentrations of fanghemoline (0, 5, 10, 20, 40, 60, 80, 100 μM) were added, and the DMSO group was used as the control. The cells were cultured in a cell culture incubator at 37°C and 5% CO2 for 24, 48 and 72 hours. 4 hours before the end of incubation, 10 μL CCK-8 was added to each well and incubated in a cell culture incubator for 4 hours. The total volume in each well was 100 μL. Finally, the absorbance at 450 nm was detected using an enzyme reader. The experiment was repeated 3 times, with 3 replicate wells for each experiment.

[0066] 3. Experimental results: Figure 4 As shown in Table 1, it can be seen that fangqinoline significantly inhibits the proliferation of STAG2 mutant AML cells. 50 The results showed that STAG2 mutant AML cell lines were more sensitive to the inhibitory effect of fangchinoline than wild-type AML cell lines, with IC 50 About the latter IC50 In addition, with the increase of incubation time and concentration of fangchinoline, the IC 50 This indicates that the inhibitory effect of fangchinoline on AML cell lines is affected by time and dose.

[0067] Table 1 IC values ​​of fangchinoline against various cell lines 50 Value (μM)

[0068] Note: SKM-1 and Kasumi-6 are 1.5×10 6 / mL were inoculated in 96-well plates, THP-1 and U-937 were inoculated at a density of 1×10 6 / mL was inoculated in a 96-well plate, with 100 μL in each well. The results were expressed as IC 50 ±SD.

[0069] Example 4 This example uses fanghenoline to conduct apoptosis and cell cycle experiments, specifically: 1. Materials and reagents: fangchinoline (dissolved in DMSO, purchased from MedChemExpress, Shanghai, China), 1640 culture medium (GIBCO), fetal bovine serum (GIBCO), penicillin-streptomycin, Annexin-V-FITC / PI apoptosis kit (purchased from BD Bioscience), PI (containing RNase, purchased from BD Bioscience), 15 mL sterile centrifuge tubes, 1.5 mL centrifuge tubes, 10 μL sterile pipette tips, 200 μL sterile pipette tips, 1 mL sterile pipette tips, and 6-well cell culture plates.

[0070] 2. Operation steps: SKM-1, Kasumi-6, THP-1 and U-937 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum. When they entered the logarithmic growth phase, the cells were collected and counted, and incubated with different concentrations of fangchinoline (0, 3, 6, 9 μM). The DMSO group was used as the control. After culturing in a cell culture incubator at 37°C and 5% CO2 for 24h and 48h, the cells were collected, washed twice with binding buffer, and incubated with Annexin V-FITC and PI at room temperature in the dark for 30min. Cell apoptosis was detected and analyzed using flow cytometry.

[0071] SKM-1 and Kasumi-6 cells in the logarithmic growth phase were seeded into 6-well plates, with 2×10 cells per well. 5 , 2mL cell suspension per well. 50Select appropriate concentrations of fangchinoline to treat cells, 3μM, 6μM and 9μM for SKM-1 cells; 2μM, 4μM and 6μM for Kasumi-6 cells. After incubating cells with different concentrations of fangchinoline for 24 hours, collect the cells. Fix the cells with 70% pre-cooled ethanol. Then, add 200μL PI / RNase dye and incubate at room temperature and away from light for 30 minutes. Use flow cytometry for cell cycle analysis.

[0072] 3. Experimental results: Figure 5 As shown in the figure, it can be seen that at 24h and 48h of incubation, SKM-1 showed late apoptosis and increased with the increase of the concentration of fangqinoline. Figure 6 As shown in the figure, it can be seen that when treated with 9μM fangchinoline for 24h and 48h, the apoptosis rate of SKM-1 was 28.56% and 42.19%, respectively. The results of Kasumi-6 incubated for 24h and 48h showed that the late and early apoptosis increased with the increase of fangchinoline concentration. Figure 7-8 As shown in the figure, it can be seen that when treated with 6μM fangchinoline for 24h and 48h, the apoptosis rates of Kasumi-6 were 38.85% and 58.50%, respectively. However, when THP-1 and U-937 cells were treated with 9μM fangchinoline, the apoptosis rates of cells were less than 20% after incubation for 24h and 48h. Fig. 9 As shown, it can be seen that in SKM-1 cells, the proportion of cells in the G1 phase (58.50% vs 62.43% vs 66.90%) increased with the increase of the concentration of fangchinoline; in Kasumi-6 cells, the proportion of cells in the S phase (33.60% vs 40.37% vs 42.67%) increased with the increase of the concentration of fangchinoline.

[0073] The above data can confirm that fanghemoline has a more significant effect on inducing apoptosis in STAG2 mutant AML cell lines. The effect on the cell cycle of STAG2 mutant AML cell lines is related to the cell type. The drug inhibits cell proliferation by inducing cell cycle arrest in the G1 phase or S phase.

[0074] Example 5 This example uses fangchinoline to analyze cell morphology and surface antigens, specifically: 1. Materials and reagents: fangchinoline (dissolved in DMSO, purchased from MedChemExpress, Shanghai, China), Wright-Giemsa stain, APC mouse anti-human CD15 antibody (Cat #551376, purchased from BD Biosciences), PBS, glass slides, 15 mL sterile centrifuge tubes, 1.5 mL centrifuge tubes, 10 μL sterile pipette tips, 200 μL sterile pipette tips, 1 mL sterile pipette tips, and 6-well cell culture plates.

[0075] 2. Operation steps: SKM-1 and Kasumi-6 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum. When they entered the logarithmic growth phase, the cells were collected and counted, and incubated with different concentrations of fangchinoline (SKM-1: 0, 3, 6, 9 μM; Kasumi-6: 0, 2, 4, 6 μM). With the DMSO group as the control, the cells were cultured in a cell culture incubator at 37°C and 5% CO2 for 24 h and 48 h, and then the cells were collected, slides were prepared, and Wright-Giemsa staining was performed. Finally, the morphological characteristics of the cells were observed under an optical microscope. After SKM-1 and Kasumi-6 cells were cultured with the above concentrations of fangchinoline for 24 h, the cells were collected, washed with PBS, and incubated with APC mouse anti-human CD15 antibody. The changes in cell surface antigens were evaluated by flow cytometry.

[0076] 3. Experimental results: Figure 10-11 As shown in the figure, it can be seen that after treatment with fangchinoline, the nuclear / cytoplasmic ratio of SKM-1 and Kasumi-6 cells decreased with the increase of drug concentration, showing a phenotypic change consistent with cell differentiation. Fig.11 As shown, it can be seen that in SKM-1 and Kasumi-6 cells, fangchinoline significantly upregulated the differentiation gene CD15 (a marker of granulocyte differentiation). These results suggest that fangchinoline induces granulocyte differentiation in STAG2 mutant AML cell lines. Therefore, fangchinoline may induce cell cycle arrest by promoting cell differentiation in STAG2 mutant AML cell lines.

[0077] Example 6 In this example, fanghemoline was used to count the number of cell colonies using a fluorescent inverted phase contrast microscope, specifically: 1. Materials and reagents: fangchinoline (dissolved in DMSO, purchased from MedChemExpress, Shanghai, China), 1640 culture medium (GIBCO), fetal bovine serum (GIBCO), penicillin-streptomycin, 15 mL sterile centrifuge tubes, 1.5 mL centrifuge tubes, 10 μL sterile pipette tips, 200 μL sterile pipette tips, 1 mL sterile pipette tips, and 24-well cell culture plates.

[0078] 2. Procedure: SKM-1 and Kasumi-6 cells were mixed with different concentrations of fangchinoline (SKM-1: 0, 6, 9, 12 μM; Kasumi-6: 0, 3, 6, 9 μM) in 500 μL MethoCult TM H4435, which contains 10% fetal bovine serum, 10% cell suspension (cell number 5×10 3) and 1% penicillin / streptomycin, and then inoculated into 24-well plates. The DMSO group was used as the control. After incubation in a cell culture incubator at 37°C and 5% CO2 for 14 days, the number of cell colonies in different groups was counted using a fluorescence inverted phase contrast microscope.

[0079] 3. Experimental results: Fig.12 As shown in Figure AB, it can be seen that with the increase in the concentration of fangchinoline treatment, both SKM-1 and Kasumi-6 showed a significant decrease in the number of colonies. In addition, neither SKM-1 nor Kasumi-6 could form colonies under sufficiently high concentrations of fangchinoline treatment (SKM-1: 12μM; Kasumi-6: 9μM). From this, it seems that the STAG2 mutant AML cell line that has undergone granulocyte differentiation has lost the ability to form colonies. In summary, fangchinoline can effectively inhibit the formation of colonies in STAG2 mutant AML cell lines.

[0080] Example 7 In this example, fangchinoline was used for transcriptome sequencing and transmission electron microscopy observation, specifically: 1. Materials and reagents: fangchinoline (dissolved in DMSO, purchased from MedChemExpress, Shanghai, China), TRIzol, RPMI 1640 medium (GIBCO), fetal bovine serum (GIBCO), penicillin-streptomycin, glutaraldehyde, PBS, osmium acid, double distilled water, ethanol, resin, 15 mL sterile centrifuge tubes, 1.5 mL centrifuge tubes, 10 μL sterile pipette tips, 200 μL sterile pipette tips, 1 mL sterile pipette tips, and 6-well cell culture plates.

[0081] 2. Operation steps: RNA was extracted using TRIzol, and the quality of the extracted RNA samples was assessed. The library was sequenced using the llumina Novasek 6000 sequencing platform. Fastp software was used for data processing. Then, the clean reads obtained were used for subsequent data analysis. HISAT2 software was used for reference genome alignment and FPKM calculation, and the read counts of each gene were obtained by HTSeq-count. KEGG enrichment, GO enrichment and PPI analysis were performed on differentially expressed genes. SKM-1 and Kasumi-6 cells were cultured with the same concentration of fangchinoline for 12h, 24h and 36h. The cells were collected and fixed with glutaraldehyde for 2h. Then, the cells were washed 4 times with 0.1M PBS, fixed with 1% osmium acid for 2h, washed once with 0.1M PBS, and washed 3 times with double distilled water. Subsequently, they were embedded in the resin by ethanol gradient dehydration. Finally, the samples were embedded. Transmission electron microscopy was used to observe the intracellular structure.

[0082] 3. Experimental results: Figure 13-15 As shown, it can be seen that 52 genes were upregulated and 217 genes were downregulated in SKM-1 cells. KEGG analysis showed that differentially expressed genes were enriched in the ferroptosis pathway. Subsequently, this was confirmed by transmission electron microscopy, and the results showed that after drug treatment, SKM-1 and Kasumi-6 cells showed morphological changes consistent with ferroptosis (mitochondrial shrinkage and deformation, increased mitochondrial membrane density, reduced or disappeared mitochondrial cristae, and flocculent amorphous substances appeared in the mitochondrial lumen). The above results indicate that fangchinoline can induce the death of STAG2 mutant AML cells through the ferroptosis pathway.

[0083] Example 8 This example uses fangchinoline to detect lipid peroxides and ROS, specifically: 1. Materials and reagents: fangchinoline (dissolved in DMSO, purchased from MedChemExpress, Shanghai, China), HBSS, Liperfluo solution, 15 mL sterile centrifuge tubes, 1.5 mL centrifuge tubes, 10 μL sterile pipette tips, 200 μL sterile pipette tips, 1 mL sterile pipette tips, and 6-well cell culture plates.

[0084] 2. Operation steps: SKM-1 and Kasumi-6 cells in the logarithmic growth phase were inoculated into 6-well plates; fangchinoline was added, and solvent controls and duplicate wells were set up at the same time. After culturing at 37°C and 5% CO2 for 12 hours, the cells were collected, centrifuged, the supernatant was discarded, and the cells were washed twice with 200μL HBSS, and the HBSS was removed; 200μL of 1μmo1 / L Liperfluo solution diluted with HBSS was added, and the cells were cultured in a 37°C, 5% CO2 incubator for 30 minutes; the solution was removed, and the cells were washed twice with 200μL HBSS, and the intracellular lipid peroxide content and ROS level were determined by flow cytometry.

[0085] 3. Experimental results: Figure 16-17 As shown in the figure, it can be seen that fangchinoline significantly increased the levels of lipid peroxides and ROS in SKM-1 and Kasumi-6 cells. In addition, the level of lipid peroxides increased with the increase of fangchinoline treatment concentration, and the level of ROS increased with the extension of fangchinoline treatment time. These results provide strong evidence that fangchinoline induces ferroptosis in STAG2 mutant AML cell lines.

[0086] Example 9 This example uses fangchinoline to conduct a test to confirm the mechanism of ferroptosis, specifically: 1. Materials and reagents: fangchinoline (dissolved in DMSO, purchased from MedChemExpress, Shanghai, China), TRIzol, RPMI 1640 medium (GIBCO), fetal bovine serum (GIBCO), penicillin-streptomycin, RIPA, 4× loading buffer, SLC7A11 / xCT (purchased from abcam, USA), NRF2 (purchased from Proteintech, Wuhan, China), 15 mL sterile centrifuge tubes, 1.5 mL centrifuge tubes, 10 μL sterile pipette tips, 200 μL sterile pipette tips, 1 mL sterile pipette tips, and 6-well cell culture plates.

[0087] 2. Operation steps: Load equal amounts of cell lysate onto SDS-PAGE gel and perform electrophoresis. Then, transfer the protein to a PVDF membrane. Block the membrane with 5% skim milk in TBST and then incubate with GAPDH, Nrf2, and SLC7A11 antibodies overnight. After that, incubate the membrane in HRP-labeled goat anti-rabbit antibody or HRP-labeled goat anti-mouse antibody solution for 1 hour. Finally, visualize the protein bands using enhanced chemiluminescence reagents and Amersham Imager 600.

[0088] 3. Experimental results: Fig.18 As shown in the figure, it can be seen that compared with the control group, fangchinoline significantly reduced the protein level of SLC7A11 in SKM-1 and Kasumi-6 cells. The protein level of NRF2 was reduced in STAG2 mutant AML cell lines treated with fangchinoline. In general, fangchinoline inhibited NRF2, thereby downregulating the expression of SLC7A11, and ultimately triggering ferroptosis in STAG2 mutant AML cell lines.

[0089] Example 10 In this example, based on the fangchinoline liposomes prepared in Example 1, a preliminary evaluation of the anti-leukemia effect in vivo was conducted, specifically: 1. Materials and reagents: liposomes of fangchinoline, RPMI 1640 medium (GIBCO), fetal bovine serum (GIBCO), penicillin-streptomycin, and cell counting chamber.

[0090] 2. Operation steps: SKM-1 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum. When they entered the logarithmic growth phase, the cells were collected and counted, and inoculated into 6-8 week old NSG mice. 5×10 6 Cells / mouse, 3 days later, fangchinoline liposomes were injected intravenously, and the dosage of fangchinoline was controlled to 3 mg / kg, once every 3 days, and the behavior, body weight, survival status, etc. of the mice were observed every day. At the same time, a normal saline control group was set up for comparison using the same method.

[0091] 3. Experimental results: Fig.19 As shown, it can be seen that the average survival time of NSG mice injected with fangchinoline liposomes was 64.5 days, which was significantly longer than that of the saline control group with an average survival time of 35.5 days (P=0.006). Combined with the test results of the above examples, it is proved that fangchinoline liposomes can inhibit the proliferation and colony formation of AML cell lines SKM-1, Kasumi-6, THP-1 and U-937, induce apoptosis, cell cycle arrest and cell differentiation of these cells, and thus play an anti-AML effect.

[0092] Unless otherwise specified, all percentages used in the present invention are by mass.

[0093] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of fangchinoline in the preparation of therapeutic drugs for STAG2 mutation acute myeloid leukemia.

2. The use of fangchinoline according to claim 1 in the preparation of a drug for treating STAG2 mutation acute myeloid leukemia, characterized in that: The drug for treating STAG2 mutation acute myeloid leukemia uses fangchinoline or its salt as an effective ingredient or a main effective ingredient.

3. A pharmaceutical composition for treating STAG2 mutation acute myeloid leukemia, characterized in that: A pharmaceutically acceptable dosage form is prepared by using fangchinoline or its salt as an effective ingredient or a main effective ingredient and a pharmaceutically acceptable excipient.

4. The pharmaceutical composition for treating STAG2 mutation acute myeloid leukemia according to claim 3, characterized in that: The pharmaceutical composition comprises: fangchinoline liposomes.

5. The pharmaceutical composition for treating STAG2 mutation acute myeloid leukemia according to claim 4, characterized in that: The fangchinoline liposome comprises the following raw materials in parts by weight: 0.6-0.8 parts of fangchinoline, 1-1.3 parts of cholesterol, and 6-8 parts of phospholipids.

6. The pharmaceutical composition for treating STAG2 mutation acute myeloid leukemia according to claim 4, characterized in that: The fangchinoline liposomes are prepared by the following method: S1. adding phospholipids, fanghenoline and cholesterol into an ethanol solution and dispersing them evenly to obtain a mixed organic phase; S2. The mixed organic phase is injected into pure water, and after heating to remove alcohol, pure water is added to the volume before removing alcohol to obtain an initial liposome solution; S3. The liposome initial solution was extruded and filtered using filter membranes with pore sizes of 0.45 μm and 0.22 μm to obtain the fanghemoline liposomes.

7. The pharmaceutical composition for treating STAG2 mutation acute myeloid leukemia according to claim 6, characterized in that: In the step S1, the weight ratio of fangchinoline, cholesterol and phospholipid is 0.6-0.8:1-1.3:6-8.

8. The pharmaceutical composition for treating STAG2 mutation acute myeloid leukemia according to claim 6, characterized in that: In step S1, the volume concentration of the ethanol solution is 70-80%.

9. The pharmaceutical composition for treating STAG2 mutation acute myeloid leukemia according to claim 6, characterized in that: In step S2, the mixed organic phase is injected into 4-7 times the volume of pure water.

10. The pharmaceutical composition for treating STAG2 mutation acute myeloid leukemia according to claim 6, characterized in that: In step S3, the average particle size of the obtained fangchinoline liposomes is less than 300 nm.

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