Medicine for treating AML (acute myelogenous leukemia) and / or prognosis of AML patient and application of ubiquitin specific protease 20 serving as target spot in treatment of acute myelogenous leukemia

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a medicine for treating AML (acute myelogenous leukemia) and / or prognosis of AML patients and application of ubiquitin specific protease 20 serving as a target spot in treating acute myelogenous leukemia. The invention provides a medicine for treating AML (acute myelogenous leukemia) and / or prognosis of an AML patient and application of ubiquitin specific protease 20 as a target spot in treating acute myelogenous leukemia, USP20 is used as a super enhancer regulation gene, and the progress of the AML is promoted by combining with CTNNB1, ERG, ELF1 and RUNX1. The knock-down of the USP20 can significantly inhibit AML proliferation in vivo and in vitro. The wnt-beta-catenin pathway can be influenced by interfering the expression of the USP20 so as to influence the progress of AML (acute myeloid leukemia). And the inhibition effect of the inhibitor AS1517499 subjected to virtual screening on the growth of the AML cells is superior to that of a commercial inhibitor GSK2643943A of USP20.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a drug for treating the prognosis of AML and / or AML patients and the application of ubiquitin-specific protease 20 as a target in the treatment of acute myeloid leukemia. Background Technology

[0002] Acute myeloid leukemia (AML) is a cancer characterized by the infiltration of proliferative, clonal, abnormally differentiated, and occasionally poorly differentiated cells from the hematopoietic system into the bone marrow, blood, and other tissues. AML is highly heterogeneous, and its pathogenesis involves multiple genetic and epigenetic abnormalities, such as mutations in genes like FLT3, IDH1 / 2, and NPM1, as well as RNA methylation modifications (e.g., METTL13-mediated m6A) and signaling pathway dysregulation (e.g., aberrant MYC expression). Current treatments for AML include conventional chemotherapy, targeted therapy, immunotherapy, and stem cell transplantation. Although these treatments have improved the clinical prognosis of some AML subtypes, the overall prognosis for AML patients remains poor, with a 5-year survival rate of only 29%. Furthermore, due to the high heterogeneity of AML, drug resistance, and the effectiveness of specific targets, the treatment of AML still faces many challenges. Therefore, the exploration of new targets for AML remains crucial for improving the survival environment and prognosis of AML patients.

[0003] Isopeptidases that cleave ubiquitin at its carboxyl terminus are called deubiquitinating enzymes (DUBs). DUBs are divided into five distinct groups, with the USP family being the largest, most versatile, and most diverse family of deubiquitinating enzymes. USP20 was first identified as a deubiquitinating enzyme in 2002, belonging to the USP subfamily of DUBs. Ubiquitin-specific protease 20 (USP20) belongs to the ubiquitin-specific peptidase (USP) subfamily of DUBs and was first identified as a substrate for pVHL in 2002. The human USP20 gene is located at q34.11 on chromosome 9. This gene is transcribed into three variants: variant 1, variant 2, and variant 3. USP20 has been shown to induce deubiquitination of D2, HIF-1α, β2-AR, TRAF6, Tax, and PKM2, triggering the activation of signaling pathways such as Wnt, HIF1A, and NF-κB, thereby regulating vital processes such as thyroid hormone activation, cell cycle regulation, inflammatory responses, tumorigenesis, and tumor progression. There are no reports on the relationship between inhibiting the activity of specific inhibitors of USP20 and tumor treatment. Summary of the Invention

[0004] The purpose of this invention is to provide a drug for treating AML and / or improving the prognosis of AML patients and the application of ubiquitin-specific protease 20 as a target in the treatment of acute myeloid leukemia. The compound AS1517499 can reduce the expression of ubiquitin-specific protease 20, inhibit the growth of AML cells, and its effect is superior to that of the commercially available inhibitor of USP20, GSK2643943A.

[0005] To address the aforementioned technical problems, the present invention proposes the following technical solution: This invention provides the use of a substance for detecting ubiquitin-specific protease 20 or its encoding gene in at least one of the following: 1) Preparation of diagnostic products for acute myeloid leukemia; 2) Prepare products for evaluating the therapeutic effects of acute myeloid leukemia; 3) Develop products for prognostic evaluation of acute myeloid leukemia.

[0006] The amino acid sequence of ubiquitin-specific protease 20 is shown in SEQ ID NO.1 (NCBI accession number NP_001103773.2).

[0007] SEQIDNO.1:MGDSRDLCPHLDSIGEVTKEDLLLKSKGTCQSCGVTGPNLWACLQVACPYVGCGESFADHSTIHAQAKKHNLTVNLTTFRLWCYACEKEVFLEQRLAAPLLGSSSKFSEQDSPPPSHPLKAVPIAVADEGESESEDDDLKPRGLTGMKNLGNSCYMNAALQALSNCPPLTQFFLECGGLVRTDKKPALCKSYQKLVSEVWHKKRPSYVVPTSLSHGIKLVNPMFRGYAQQDTQEFLRCLMDQLHEELKEPVVATVALTEARDSDSSDTDEKREGDRSPSEDEFLSCDSSSDRGEGDGQGRGGGSSQAETELLIPDEAGRAISEKERMKDRKFSWGQQRTNSEQVDEDADVDTAMAALDDQPAEAQPPSPRSSSPCRTPEPDNDAHLRSSSRPCSPVHHHEGHAKLSSSPPRASPVRMAPSYVLKKAQVLSAGSRRRKEQRYRSVISDIFDGSILSLVQCLTCDRVSTTVETFQDLSLPIPGKEDLAKLHSAIYQNVPAKPGACGDSYAAQGWLAFIVEYIRRFVVSCTPSWFWGPVVTLEDCLAAFFAADELKGDNMYSCERCKKLRNGVKYCKVLRLPEILCIHLKRFRHEVMYSFKINSHVSFPLEGLDLRPFLAKECTSQITTYDLLSVICHHGTAGSGHYIAYCQNVINGQWYEFDDQYVTEVHETVVQNAEGYVLFYRKSSEEAMRERQQVVSLAAMREPSLLRFYVSREWLNKFNTFAEPGPITNQTFLCSHGGIPPHKYHYIDDLVVILPQNVWEHLYNRFGGGPAVNHLYVCSICQVEIEALAKRRRIEIDTFIKLNKAFQAEESPGVIYCISMQWFREWEAFVKGKDNEPPGPIDNSRIAQVKGSGHVQLKQGADYGQISEETWTYLNSLYGGGPEIAIRQSVAQPLGPENLHGEQKIEAETRAV。

[0008]

[0009] This invention discovers the function and mechanism of superenhancers (SE) regulating the gene USP20 in AML, which holds promise for developing more effective targeted therapy strategies and bringing better clinical outcomes to AML patients. Specifically, the superenhancer-regulated gene USP20 was first discovered in AML, where it is specifically highly expressed and plays an important role. USP20 promotes the proliferation and survival of AML cells by stabilizing the CTNNB1 protein through deubiquitination. The USP20-specific inhibitor AS1517499, screened using a virtual screening method, provides a new approach to AML treatment, and USP20 shows promise as a potential therapeutic target for AML.

[0010] USP20, a super-enhancer regulatory gene, promotes the progression of AML by binding to factors such as CTNNB1, ERG, ELF1, and RUNX1. In both in vivo and in vitro experiments, interference with USP20 expression significantly inhibits AML cell proliferation, an effect related to its influence on the Wnt / β-catenin signaling pathway. Furthermore, the inhibitor AS1517499, obtained through virtual screening, showed superior inhibitory effects on AML cell growth compared to the commercially available USP20 inhibitor GSK2643943A.

[0011] Preferably, the acute myeloid leukemia includes one or more of the following: acute myeloid leukemia microdifferentiation, acute myeloid leukemia undifferentiated, acute myeloid leukemia partially differentiated, acute promyelocytic leukemia, acute myelomonocytic leukemia, erythroleukemia, and megakaryocytic leukemia.

[0012] Preferably, the prognosis includes one or more of the following: the clinical outcome of the prognostic subject, the treatment effect of the prognostic subject, and the survival of the prognostic subject.

[0013] Preferably, the product includes a drug and / or a reagent kit.

[0014] This invention provides the use of an inhibitor of ubiquitin-specific protease 20 or its encoding gene in the preparation of a medicament for treating acute myeloid leukemia and / or improving the prognosis of patients with acute myeloid leukemia.

[0015] Preferably, the inhibitor is selected from the following substances: (1) Substances that inhibit the enzymatic activity of ubiquitin-specific protease 20; or / and, (2) Substances that reduce or block the expression of the ubiquitin-specific protease 20 encoding gene.

[0016] Preferably, the substance that inhibits the enzymatic activity of ubiquitin-specific protease 20 includes an antibody or inhibitor of ubiquitin-specific protease 20 protein, AS1517499.

[0017] Preferably, the substance that reduces or blocks the expression of the ubiquitin-specific protease 20 encoding gene includes siRNA targeting the ubiquitin-specific protease 20 encoding gene, and / or antisense nucleic acid or a CRISPR / Cas gene editing system that targets and knocks out the ubiquitin-specific protease 20 encoding gene.

[0018] Preferably, the treatment of acute myeloid leukemia and / or improvement of the prognosis of patients with acute myeloid leukemia includes one or more of inhibiting AML cell line proliferation, inhibiting AML cell line migration, and inhibiting AML cell line invasion.

[0019] The present invention provides a medicament for treating AML and / or improving the prognosis of AML patients, comprising substances including substances that inhibit the enzymatic activity of ubiquitin-specific protease 20; and / or substances that reduce or block the expression of the gene encoding ubiquitin-specific protease 20.

[0020] Preferably, the dosage form of the drug includes one or more of the following: oral dosage form, injection dosage form, inhalation dosage form, and suppository.

[0021] The beneficial effects of this invention: This invention is the first to experimentally discover that the expression level of ubiquitin-specific protease 20 (USP20) is significantly increased in AML, and it shows a negative correlation with the survival of AML patients. It also reveals that USP20 can serve as a novel regulatory factor for AML, providing not only new theoretical basis for the pathogenesis of AML, but more importantly, a new approach for AML drug development. Therefore, the deubiquitinating enzyme USP20 can be used as a marker molecule for AML diagnosis, as a target molecule for screening new AML therapeutic drugs, and as a detection indicator for evaluating AML therapeutic drugs. The results of the examples show that compound AS1517499 can reduce the expression of ubiquitin-specific protease 20, inhibit AML cell growth, and its effect is superior to that of the commercially available USP20 inhibitor GSK2643943A. Attached Figure Description

[0022] Figure 1To identify USP20 as a superenhancer regulatory gene in AML and to demonstrate that its high expression is associated with poor AML prognosis, the study included the following steps: A) Screening for potential superenhancers in the USP family by analyzing H3K27acChIP-seq data from seven AML cell lines and 13 AML patients; B) Using IGV visualization tools to compare and analyze H3K27acChIP-seq results from seven AML cell lines and 13 AML patients, an enriched region was identified near the USP20 gene, indicating a high probability of the presence of a superenhancer; however, no enrichment near USP20 was observed in H3K27acChIP-seq data from CD34-positive hematopoietic stem cells (HSCs) from the GEO database; C) Knockdown of BRD4 in AML cells led to decreased USP20 mRNA expression; D) According to public databases, AML samples showed significantly higher USP20 mRNA expression levels compared to healthy bone marrow samples; E) According to the TARGET database, AML patients with higher USP20 expression levels had lower survival rates. Figure 2 The graph shows the expression level of USP20 protein. In A, the use of BRD4 inhibitors and knockdown of BRD4 in AML cells can lead to a decrease in the expression level of USP20 protein; B shows the expression of USP20 in AML cells detected by Western blot technology; and C shows that high expression of USP20 is associated with poor prognosis in AML patients in the TCGA database. Figure 3 This paper describes the regulatory role of relevant super-enhancers on USP20. A shows a ChIP-seq gene mapping map displaying the H3K27ac signal at the USP20 gene locus, illustrating Hi-C interactions in AML and labeling three candidate enhancer regions of USP20 (E1, E2, and E3). B evaluates the efficiency of transfecting MV4-11 and kasumi-1 cells with dual-luciferase reporter vectors containing negative controls or USP20 enhancer elements. C is a schematic diagram illustrating the inactivation of Cas9 by the fusion transcriptional repressor domain (KRAB) to inhibit U… Methods for SP20 constitutive enhancer; D shows the Western blot results of MV4-11 and kasumi-1 cells transfected with the sgRNA-Cas9 vector; E shows the mRNA expression level of USP20 in MV4-11 and kasumi-1 cells transfected with the sgRNA-Cas9 vector; F shows the cell viability (viability) of MV4-11-Cas9, MV4-11, kasumi-1-Cas9, and kasumi-1 cells after transfection with non-targeted negative control sgRNA and E3-targeting sgRNA, as assessed by CCK8 assay. Figure 4This study highlights the crucial role of key transcription factors in regulating USP20 expression driven by super-enhancers. A shows the signaling of transcription factors (TFs) in the super-enhancer region of the USP20 gene locus in the AML cell line (CMK) using the CUT&Tag IGV map; B shows the signaling of transcription factors (TFs) in the super-enhancer region of the USP20 gene locus in the AML cell line (MV4-11); C assesses the activity of the E3 luciferase reporter gene after ETV6 knockdown in MV4-11 and CMK cells; D shows the USP20 mRNA level after ETV6 knockdown in MV4-11 and CMK cells using qRT-PCR analysis; and E shows a scatter plot indicating a strong positive correlation between the transcriptional levels of USP20, ERG, ETV6, RUNX1, and LDB1 in AML cases (GSE114868n=194). Figure 5 To investigate the activity of the E3 luciferase reporter gene and the mRNA expression level of USP20 after knocking down ELF1 and ZNF217 in MV4-11 and CMK cells, respectively, A shows the activity of the E3 luciferase reporter gene after knocking down ELF1 and ZNF217 in MV4-11 and CMK cells, respectively; B shows the mRNA expression level of USP20 after knocking down transcription factors ELF1 and ZNF217 in MV4-11 and CMK cells, respectively, by qRT-PCR analysis. Figure 6 To demonstrate that USP20 knockdown inhibits the proliferation and progression of AML both in vitro and in vivo; A) Western blotting validated the USP20 knockdown level in MV4-11, kasumi-1, and CMK cells; B) qRT-PCR was used to detect the USP20 mRNA level in MV4-11, kasumi-1, and CMK cells infected with sh-NC, sh-USP20#2, and sh-USP20#3; C) CCK-8 assay was used to calculate the proliferation of MV4-11, kasumi-1, and CMK cells after USP20 knockdown; D) Flow cytometry was used to determine the knockdown level of USP20. The apoptosis levels of MV4-11, kasumi-1, and CMK cells after SP20 treatment; E shows the upregulation of PARP and cleaved caspase-8 expression in MV4-11, kasumi-1, and CMK cells after USP20 knockdown, as shown by Western blot analysis; F is the in vivo experimental design flowchart; G shows the relevant bioluminescence images of USP20 knockdown and control mice obtained at days 15, 20, 25, and 30; H is a bar chart showing the bioluminescence signal values ​​of the two mouse groups at different time points; I shows that the sh-USP20 treatment group had a longer survival time than the control group. Figure 7 To demonstrate the effect of USP20 knockdown on inhibiting the proliferation and progression of AML in vitro, A shows the colony formation assay of MV4-11, kasumi-1, and CMK cells transfected with sh-USP20#2, sh-USP20#3, and sh-NC; B shows the microscopic morphological changes of MV4-11, kasumi-1, and CMK cells after USP20 knockdown. Figure 8 Cell cycle distribution and apoptosis levels of MV4-11, kasumi-1, and CMK cells are shown in Figure A, where A represents the cell cycle distribution measured by propidium iodide (PI) staining after USP20 knockdown in MV4-11, kasumi-1, and CMK cells; and B represents the apoptosis levels of infected MV4-11, kasumi-1, and CMK cells as determined by flow cytometry. Figure 9 To demonstrate the effect of USP20 knockdown on inhibiting the proliferation and progression of AML in vivo; A shows in vivo bioimaging images of the average radiouptake of liver, spleen, and bone tissue in both groups of mice; bar charts show the bioluminescence signal values ​​of liver, spleen, and bone; B shows a significant decrease in the number of human CD45-positive cells in liver, spleen, and bone marrow after USP20 knockdown compared to the control group; C shows no significant weight loss observed between the two groups of mice; D shows representative immunohistochemical Ki67 staining images of mouse liver, spleen, and bone marrow. Figure 10 The image shows the results of hematoxylin-eosin (H&E) staining analysis of mouse liver, spleen and bone marrow. Compared with the control group, the number of tumor cells in the USP20 knockdown group was significantly reduced. Figure 11The diagram shows the process of virtual screening for USP20 inhibitors; A is a diagram of the predicted structure of USP20 and its small molecule binding sites; B is a flowchart of the virtual screening process; C is a molecular weight-score distribution diagram, showing the molecular weight-score distribution corresponding to the docking scores of the top 50% of small molecules in the T001 compound library with USP20; D is a protein-ligand interaction fingerprint (PLIF) analysis diagram of 262 screened compounds; the same amino acids are marked with the same color, and the higher the vertical density, the higher the interaction frequency between the site and the compound; E is a binding conformation diagram of compound AS1517499 with the USP20 structure; E (1) in E is a panoramic view of the binding site of AS1517499 in the USP20 structure. The carbon atoms of the compound are green, the oxygen atoms are red, and the nitrogen atoms are blue. The protein is shown in gray cartoon form. (2) in E is a 3D close-up view of the binding of AS1517499 and USP20. The carbon atoms of the compound are green, the oxygen atoms are red, and the nitrogen atoms are blue. The surrounding amino acids are marked with black numbers in white sticks. Yellow dashed lines represent hydrogen bonds, and cyan dashed lines represent aromatic ring-hydrogen interactions. (3) in E is a 2D close-up view of the binding of AS1517499 and USP20. Purple arrows represent hydrogen bond interactions. Figure 12 The diagram shows the inhibition of AML proliferation in vivo and in vitro by the virtual screening inhibitor AS1517499. A shows the detailed interaction pattern between compound AS1517499 and USP20, with hydrogen bonds and aromatic ring-hydrogen interactions indicated by yellow and cyan dashed lines, respectively. B shows the effects of nine inhibitors from the virtual compound library and the commercially available USP20 inhibitor GSK2643943A in MV4-11 cells, detected using a CCK-8 assay. The results showed that inhibitor AS1517499 had the strongest inhibitory effect on MV4-11 cell growth. C shows the effect of Western blot analysis on protein immunoblotting, indicating that after A... The graph shows the changes in USP20, PARP, and cleaved caspase-8 protein levels in MV4-11 cells after S1517499 treatment; D is the experimental design flowchart; E is the bioluminescence imaging at days 7, 10, 14, and 17 revealing the differences between the AS1517499 group and the solvent control group; F is a bar chart showing the bioluminescence signal values ​​of the two mouse groups at different time points; G shows that the AS1517499 treatment group showed a longer survival time compared to the untreated group; H is an HE-stained section image of mice in the untreated group and the AS1517499 treatment group. Figure 13The images show the effects of AS1517499 on MV4-11 cells. A shows the microscopic morphological changes of MV4-11 cells after treatment with AS1517499; B shows that flow cytometry analysis revealed that AS1517499 treatment induced more apoptotic cells in a dose-dependent manner; C shows the cell cycle distribution of MV4-11 cells after treatment with AS1517499, measured by propidium iodide staining; and D shows the colony formation assay of MV4-11 cells treated with AS1517499. Figure 14 This diagram illustrates the inhibition of AML proliferation and progression by the USP20 inhibitor AS1517499 in mice. A shows the average radioactive uptake in the liver, spleen, and bone tissue of both groups of mice, with bar charts displaying the bioluminescence signal values ​​of the liver, spleen, and bone. B shows a significant decrease in the number of human CD45-positive cells in the bone marrow of the AS1517499 treatment group compared to the control group. C shows the hematoxylin-eosin (H&E) staining analysis of the liver, spleen, and bone marrow of mice in the AS1517499 group and the solvent control group. D shows a comparison of the size of the liver, spleen, and bone marrow of mice in the AS1517499 group and the solvent control group under white light. E shows the hematoxylin-eosin (H&E) staining analysis of the intestines and kidneys of mice in the AS1517499 group and the solvent control group, indicating that no intestinal or kidney damage was observed after AS1517499 treatment. Figure 15 Representative immunohistochemical Ki67 staining images of liver, spleen, and bone marrow from mice in the AS1517499 treatment group and solvent control group; Figure 16 The effect of GSK2643943A inhibitor is shown in Figure A. Figure A represents the effect of GSK2643943A inhibitor in AML cells, and its IC50 value, as determined by the CCK-8 assay. 50 B shows that GSK2643943A treatment induced apoptosis in MV4-11 cells in a dose-dependent manner, as demonstrated by flow cytometry. C shows that GSK2643943A treatment altered the PARP protein level in MV4-11 cells. Figure 17The diagram shows the molecular mechanism by which USP20 inhibits proliferation and progression in AML cells. A represents differentially expressed genes between the USP20 knockdown group and the untreated group, analyzed using a volcano plot in RNA-seq data; upregulated and downregulated genes are represented in red and blue, respectively. B is a KEGG pathway enrichment analysis diagram. C shows a Gene Set Enrichment Analysis (GSEA) map, indicating that genes in the WNT-BETA-CATENIN, cholesterol, and MYC signaling pathways are enriched in USP20 knockdown-treated AML cells. D is a heatmap showing the expression levels of WNT-BETA-CATENIN, cholesterol, and MYC pathway genes in USP20 knockdown-treated CMK cells. E shows qPCR analysis indicating decreased expression levels of related genes. F shows PCR results after USP20 knockdown in AML cells, indicating downregulation of WNT-BETA-CATENIN, cholesterol, and MYC pathway genes (such as CCND2, HMGCR, and AIMP2). Figure 18 CTNNB1 is highly expressed in AML cells and is associated with poor prognosis. A shows that, according to public databases, the mRNA expression level of CTNNB1 in AML samples is significantly higher than that in healthy bone marrow samples; B shows that AML patients with high CTNNB1 expression in the TCGA and TARGET databases have lower survival rates. Figure 19To illustrate how USP20 promotes AML progression through deubiquitination of CTNNB1 and SQSTM1, the following diagram is presented: A is a schematic diagram of mass spectrometry analysis; B shows the detection of USP20, CTNNB1, and SQSTM1 proteins by Western blotting in MV4-11 and kasumi-1 cells overexpressing USP20 after immunoprecipitation; C shows the levels of USP20, CTNNB1, SQSTM1, and β-actin proteins in MV4-11 and kasumi-1 cells stably expressing sh-NC, sh-USP20#2, and sh-USP20#3 by Western blotting (WB); D shows the mRNA levels of USP20 and CTNNB1 in MV4-11 and kasumi-1 cells stably expressing sh-NC, sh-USP20#2, and sh-USP20#3 by qRT-PCR; E shows the reaction of 20 μM MMG-132 with stably expressing sh-NC, sh-... After 6 hours of incubation with USP20#2 and sh-USP20#3 MV4-11 and kasumi-1 cells, protein levels of USP20, CTNNB1, SQSTM1, and β-actin were detected by Western blotting. F represents the protein levels of sh-NC, sh-USP20#2, and sh-USP20#3 MV4-11 and kasumi-1 cells after treatment with 100 μg / ml actinomycin (CHX) for a specified time, followed by Western blotting. Western blot was used to assess the protein levels of USP20, CTNNB1, SQSTM1, and β-actin; G represents the addition of MG132 (20 μM) to AML cells transfected with Flag-USP20, GFP-CTNNB1, wild-type ubiquitin, a ubiquitin mutant with only 48 lysine residues (Ub-K48), and a ubiquitin mutant with only 63 lysine residues (Ub-K63) 6 hours before harvest, followed by detection of the ubiquitination status of CTNNB1.

[0023] Figure 20The diagram illustrates the progression of USP20 through deubiquitination of CTNNB1 and its role in promoting AML. A shows the mRNA levels of USP20 and SQSTM1 in MV4-11, kasumi-1, and CMK cells stably expressing sh-NC, sh-USP20#2, and sh-USP20#3, detected by qRT-PCR. B shows the protein levels of USP20, CTNNB1, SQSTM1, and β-actin in MV4-11 cells treated with AS1517499, detected by Western blotting. C shows the levels of USP20, CTNNB1, SQSTM1, and β-actin in CMK cells stably expressing sh-NC, sh-USP20#2, and sh-USP20#3 after incubating 20M mMG-132 for 6 hours, as determined by Western blotting. The protein levels of n are shown in Figure 1. D represents the protein levels of USP20, CTNNB1, SQSTM1, and β-actin detected by Western blotting after incubating CMK cells treated with AS1517499 for 6 hours with 20 μM MMG-132. Figure E shows the changes in CTNNB1 protein in MV4-11 and kasumi-1 cells of sh-NC, sh-USP20#2, and sh-USP20#3 after treatment with 100 μg / ml actinomycin (CHX) for a specified time. Figure F shows the changes in SQSTM1 protein in MV4-11 and kasumi-1 cells of sh-NC, sh-USP20#2, and sh-USP20#3 after treatment with 100 μg / ml actinomycin (CHX) for a specified time.

[0024] Figure 21 This diagram illustrates how USP20 regulates gene transcription via CTNNB1. A, a heatmap generated by CUT&Tag experiments, reveals that USP20 and CTNNB1 occupy common genomic loci in the CMK cell line. B shows the distribution of CTNNB1 and USP20 binding peaks within ±1000 bp of the USP20 binding site. C, CUT&Tag analysis shows that USP20 shares highly similar binding motifs with ELF1, ERG, and RUNX1. D, an IGV map showing the signals of ELF1, ERG, RUNX1, USP20, CTNNB1, and H3K27ac at the CCND2 gene locus. E, PCR data showing the mRNA expression level of CCND2 after knocking down ELF1, ERG, RUNX1, USP20, and CTNNB1. F, a schematic diagram of the mechanism. Figure 22The pie chart shows the distribution of DNA binding sites of different antibodies in CMK cells using the CUT&Tag assay; A represents USP20 antibody / CMK cells; B represents CTNNB1 antibody / CMK cells; C represents ELF1 antibody / CMK cells; D represents ERG antibody / CMK cells; and E represents RUNX1 antibody / CMK cells. Figure 23 The results of CUT&Tag are shown in Figure A. A heatmap generated by CUT&Tag reveals that ELF1, ERG, RUNX1, USP20, and CTNNB1 occupy common genomic loci in the CMK cell line. Figure B shows the distribution of binding peaks for ELF1, ERG, RUNX1, USP20, and CTNNB1 within ±1000 bp of the USP20 binding site. Figure C shows the IGV map of CUT&Tag, displaying the binding peaks of ELF1, ERG, RUNX1, and USP20 at the JAG1 gene locus. D shows the signals of USP20, CTNNB1, and H3K27ac; E shows the mRNA expression level of JAG1 after knockdown of ELF1, ERG, RUNX1, USP20, and CTNNB1 by PCR; E shows the IGV map of CUT&Tag showing the signals of ELF1, ERG, RUNX1, USP20, CTNNB1, and H3K27ac at the ADAM17 gene locus; F shows the mRNA expression level of ADAM17 after knockdown of ELF1, ERG, RUNX1, USP20, and CTNNB1 by PCR. Figure 24 To use the Chronos algorithm to compute the cancer dependency map of the CCND2 gene in the DepMap project. Detailed Implementation

[0025] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from commercial sources.

[0027] This invention discloses the regulatory mechanism of the super-enhancer-regulated deubiquitinating enzyme USP20 in AML and its application as a drug target.

[0028] The specific experimental methods and procedures are given below.

[0029] 1. Cell Culture Human primitive megakaryocyte cell line (CMK), human acute monocytic resuscitation cell line (MV4-11), human acute myeloid leukemia cells (kasumi-1), human acute promyelocytic leukemia cell line (NB4), human monocytic leukemia (THP-1), human acute myeloid leukemia cell line (MOLM-13), human acute promyelocytic leukemia cells (HL-60), and human embryonic kidney cells (HEK293FT) were all obtained from Guangzhou Genio Biotechnology Co., Ltd. All cells except HEK293FT were acute myeloid leukemia (AML) cells. AML cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (P / S), while HEK293FT cells were cultured in high-glucose DMEM medium containing 10% FBS and 1% P / S.

[0030] The RPMI1640 medium was sourced from VivaCell, China; all cells were correctly identified by STR and regularly tested with a mycoplasma detection kit to ensure they were free of mycoplasma contamination.

[0031] 2. Dual-luciferase reporter assay The USP20 superenhancer was divided into three fragments: E1, E2, and E3. Using an M5 HiperLipo2000 Transfection Reagent (mei5bio, China), 2 μg of each enhancer plasmid fragment and the Relina internal control were transfected into CMK cells. After 48 hours, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, USA). The ratio of firefly luciferase activity to Renilla luciferase activity was calculated and normalized.

[0032] 3. Cell apoptosis and cell cycle experiments Apoptosis assay: (1~5)×10⁵ cells were collected, washed with PBS (1×) (Servicebio, China), and detected on a Beckman Gallios™ Flow Cytometer (Beckman, Germany) using Annexin V-FITC / PIApoptosis Detection Kit (Vazyme, China). Cell cycle assay: (5~10)×10⁵ cells were collected, washed with PBS, and then incubated overnight at 4°C in 70% ethanol. The cells were then analyzed using the Cell Cycle and Apoptosis Analysis Kit (PIstaining) (MCE, USA) on a Beckman Gallios™ Flow Cytometer (Beckman, Germany). The data were analyzed using the fine flowjo software.

[0033] 4. Cell proliferation and cytotoxicity After being screened with puromycin following viral infection, cells were seeded in 96-well plates at a density of (2-3) × 10³ cells per well and cultured in 100 μL of RPMI 1640 medium. Cell counting kit-8 (CCK-8) (APExBIO, USA) was added on days 1, 3, 5, and 7 of culture, and the cells were incubated in a cell culture incubator for 3 hours. The absorbance at 450 nm was then measured using a spectrometer (Thermo, USA).

[0034] Add (1~2)×10⁴ cells to each well of a 96-well plate and add AS1517499 and GSK2643943A inhibitors in a 2-fold serial dilution starting from 6 μM to each well. After incubating in a cell culture incubator at 37°C and 5% CO₂ for 24 h, 48 h, and 72 h, add CCK8 (APExBIO, USA) to each well and incubate. Measure the absorbance at 450 nm using a spectrometer (Thermo, USA).

[0035] 5. Western Bolting Cells were collected, washed with PBS (1×) (Servicebio, China), and lysed using RIPAlysis buffer (Beyotime, China). Samples were added to SDS-PAGE (gelGenScript, China) at a protein concentration of 20–30 μg per well for electrophoresis, followed by transfer using a PVDF (Merck Millipore, USA) membrane. After blocking with 5% skim milk prepared by TBST at room temperature for one hour, the cells were incubated overnight at 4°C with primary antibody. Finally, chemiluminescence imaging was performed using an AI600 image gellimaging analyzer (GE, USA).

[0036] 6. Immunoprecipitation The Co-ip experiment used the Protein A / G Magnetic IP / Co-IP Kit (Vazyme, China). 1×10⁶ cells were used to spin-bind with USP20 antibody at room temperature for 30 min, followed by binding with NP-40 washed beads at room temperature for another 30 min. Finally, denaturation elution was performed using an SDS-PAGE Sample Loading Buffer, and the resulting supernatant was used for Western bolting assays.

[0037] 7. Cut & Tag Cut & tag experiments were performed using CMK cells according to the Hyperactive Universal Cut & Tag Assay Kit (Vazyme, China). All cut & tag libraries were sequenced end-to-end in PE150 mode using the Illumina NovaSeq 6000 sequencing platform (SHBIO, China). Raw data from the CUT & Tag experiments were aligned to the UCSChg38 reference genome using Bowtie2 (v2.4.1), and target enriched regions (peaks) were identified using MACS3 (v3.0.2).

[0038] 8. In vivo experiments Four to six-week-old NSG mice (SHANGHAI MODELORGANISMS, China) were randomly divided into two groups. One × 10⁶ firefly luciferin-labeled CMK cells transfected with sh-NC and sh-USP20 were injected via the tail vein. D-luciferin sodium salt (GoldBio, USA) was injected intraperitoneally into the mice at days 15, 20, 25, and 30. Five to 15 minutes after injection, the mice were anesthetized and in vivo imaging was performed using a Berthold imager (Berthold, Germany).

[0039] The mice were euthanized after being anesthetized by introducing CO2 gas. The liver, spleen, bones and other tissues of the mice were then removed for subsequent experiments, including hematoxylin-eosin staining (HE staining), detection of CD45-positive cells and immunohistochemical analysis.

[0040] In in vivo experiments with AS1517499 (targetmol, China), female NSG mice were randomly divided into two groups. Each mouse received an intravenous injection of 1 × 10⁶ MV4-11 cells expressing firefly luciferase via the tail vein. One week later, the experimental group received an intraperitoneal injection of 10 mg / kg AS1517499, while the control group received an equal volume of saline. In vivo imaging was performed every seven days using a Bertholdimager (Berthold, Germany), and liver, spleen, and bone tissue were harvested for subsequent experiments.

[0041] 9. Cloning experiment The lower gel was prepared using 1.2% agarose gel (SIGMA, USA) with 20% FBS, 2×RPMI 1640 (Pricella, China) and antibiotics. After solidification, cells transfected with sh-NC and sh-USP20 were mixed with 0.7% agarose gel and cultured in a cell culture incubator at 37°C and 5% CO2. The culture medium was changed every two days. After three weeks, colony formation was stained using Wright-Giemsa Staining Solution (Beyotime, China), and the number of colonies was counted.

[0042] 10. RNA-seq Total RNA was extracted and a transcriptome library was constructed by Novogene Technology Co., Ltd. (Beijing, China). Sequencing was performed using the Illumina NovaSeq 6000 platform. Novogene was responsible for data processing, including quality control and sequence alignment.

[0043] 11. Quantitative Real-Time PCR RNA was extracted using the FastPureCell / TissueTotalRNAIsolationKitV2 (Vazyme, China), and then reverse transcribed into cDNA using the Hifair® III 1st Strand cDNA Synthesis Kit (YEASEN, China). qRT-PCR analysis was performed using the Hifair® III OneStep RT-qPCR SYBR Green Kit (YEASEN, China) on a LightCycler 480 real-time quantitative PCR system (Roche, Germany). All genes were analyzed using three biological replicates, and the average value was calculated.

[0044] 12. Packaging and transfection of lentiviruses USP20 shRNA was synthesized by Aigi Biotechnology Co., Ltd. (China) and constructed into the pLKO.1-puro vector. HEK293FT cells at a density of 70%–80% were inoculated with a pre-mixed packaging plasmid containing 8 μg psPAX2, 6 μg pMD2.G (Cambridge, USA), and 2 μg USP20 knockdown plasmid (pre-mixed with PEI, Sigma-Aldrich, USA). After culturing at 37°C and 5% CO2 for 48 h, the cells were concentrated using 4×PEG8000 and prepared as a pre-packaged lentivirus for later use.

[0045] For lentiviral transfection, lentivirus was mixed with AML cells resuspended in 1640 and 10 μg / mL polybrene (Sigma-Aldrich) and placed in a 12-well plate. After 12 h, the medium was replaced with fresh 1640 medium, and selection was performed using 1 μg / mL puromycin (Beyotime, China) 48 h after transfection.

[0046] 13. CRISPR-mediated silencing of USP20 First, CRISPR / Cas9 was established: lentiviruses containing Cas9 were transfected into MV4-11 and kasumi-1 cells, and selected using 6 μg / mL Blastidin S. Next, sgRNA targeting USP20 was cloned into the lenti-crispr plasmid synthesized by IGE Biotechnology LTD (Guangzhou, China). Finally, sg-RNA-USP20 and a control sequence were transfected into cells stably expressing Cas9 using lentiviral packaging and transfection.

[0047] The results are as follows: 1. USP20 is a SE-regulated gene in AML, and its high expression is associated with poor prognosis. To systematically identify USP family genes regulated by superenhancers in AML, H3K27acChIP-seq data from 13 AML patients and 7 AML cell lines (MV4-11, kasumi-1, MOLM-13, HL-60, THP-1, NB4, and CMK) were analyzed. Visualization analysis was performed using ROSE (Rank Ordering of Super-Enhancers) software, resulting in superenhancer maps for these 13 AML patients. It was found that the USP4, USP20, and USP36 loci intersected with superenhancer locations defined by ROSE software in multiple samples. Figure 1(A) This suggests that USP20 may play a role in the progression of AML through regulation by a superenhancer. Next, further H3K27acChIP-seq analysis using IGV visualization on samples from 7 AML cell lines and 13 AML patients revealed consistent SE characteristics at the USP20 locus. However, no superenhancer was found near the USP20 gene in CD34+ hematopoietic stem cells, suggesting that USP20 may be a SE-regulated gene specifically present in AML. Figure 1 (B) In AML cells, knockdown of the super-enhancer core regulator BRD4 resulted in a decrease in both the protein and mRNA levels of USP20, and the BRD4 inhibitor GNE987 exhibited the same effect in AML cells. Figure 1 C in the middle Figure 2 (A). This indicates that USP20 is a super-enhancer-regulated gene in AML cells. Analysis of USP20 in AML cells and normal tissues using public databases shows that USP20 gene expression is significantly higher in AML than in normal tissues. Figure 1 (D). Furthermore, USP20 is generally highly expressed in AML cell lines ( Figure 2 (B)

[0048] According to the TARGET and TCGA databases, patients with high expression of the USP20 gene in AML have significantly lower survival rates than those with low expression of the USP20 gene. Figure 1 E, Figure 2 (C). The above data all suggest that USP20 is a super-enhancer regulatory gene in AML, and its high expression is associated with poor prognosis in AML patients.

[0049] 2. The central role of master transcription factors in the regulation of USP20 expression driven by super-enhancers To confirm the extensive interactions between the enhancer and promoter regions of the USP20 gene, previous Hi-C data from AML patients and cell lines were reanalyzed. The results showed significant chromatin-long-range interactions enriched at the USP20 locus. Subsequently, connect lines superimposed on the Chip-seq peak plots were used to reveal key enhancer-promoter interaction connections. Figure 3(A). Finally, within the identified super-enhancer region (USP20-SE), three potential enhancer components with core functional activity were located: E1, E2, and E3. A region without enhancer activity was selected as a negative control. The three candidate enhancers were cloned into the PGL3 promoter luciferase reporter vector and transfected into MV4-11 and kasumi-1 cell lines. Both E2 and E3 showed reporter gene activity, with E3 exhibiting higher activity than E2, consistent with its high H3K27ac signaling characteristic. Figure 3 Based on this, E3 was confirmed as the core functional unit of the USP20 superenhancer. To verify the enhancer's transcriptional regulatory function on USP20, specific inhibition was performed using sgRNA targeting the USP20 E3 element. Figure 3 (C), Western blot and PCR results showed that sgRNA2, sgRNA4+5 and sgRNA6+7 could significantly inhibit the protein and mRNA levels of USP20 (C). Figure 3 China D and Figure 3 In MV4-11-Cas9 and kasumi-1-Cas9 cells, transfection with sgRNA that knocked out the USP20 superenhancer significantly inhibited cell proliferation, while transfection with sgRNA alone had no significant effect in MV4-11 and kasumi-1 cells. Figure 3 (Middle F).

[0050] Based on literature reports and previous research results, ELF1, ERG, IKZF1, RUNX1, TAL1, and ZNF217 play important roles in promoting the proliferation and development of AML, and are crucial transcription factors in AML. Therefore, CUT & Tag analysis of these major AML transcription factors revealed that ETV6, ELF1, and ZNF217 have multiple enrichment sites in the super-enhancer region of USP20 in MV4-11 and CMK cells, with the most significant enrichment in the E3 enhancer region. Figure 4 China A and Figure 4 (B). To verify whether these major transcription factors directly regulate USP20 expression through SE, ETV6, ELF1, and ZNF217 were knocked down, and the PGL3 promoter luciferase reporter vector of the USP20-E3 enhancer was transfected into MV4-11 cells and CMK cells. The results showed a significant decrease in the reporter activity of USP20-E3. Figure 4 C and Figure 5 In MV4-11 and CMK cells, the absence of any transcription factor (ETV6, ELF1, ZNF217) at the mRNA level significantly downregulated USP20 mRNA expression. Figure 4 China D and Figure 5 In the expression data analysis of AML patient samples from the GEO database, USP20 was positively correlated with the expression of ERG, ETV6, RUNX1, and LDB1. Figure 4 (E). The above results indicate that the above transcription factors coordinately regulate transcription by directly occupying the super-enhancer region of USP20.

[0051] 3. Knocking down USP20 can inhibit the proliferation of AML cells in vivo and in vitro. according to Figure 1 To investigate the expression of USP20 in AML cell lines, MV4-11, kasumi-1, and CMK were selected to validate the role of USP20 in AML. In vitro experiments were performed by transfecting three AML cell lines (MV4-11, kasumi-1, and CMK) with USP20-targeting sh-RNAs (sh-USP20#1, sh-USP20#2, and sh-USP20#3) and control sh-RNA (sh-NC). Western blot and qRT-PCR results showed that, compared with the control group, the expression levels of USP20 protein and mRNA were significantly reduced in cells transfected with USP20 sh-RNAs (#2 and #3). Figure 6 China A and Figure 6 (B). In plate cloning experiments, the absence of USP20 significantly reduced the ability of cells to form clones on soft agar. Figure 7 (A). Compared with the control group (sh-NC), the reduction of USP20 significantly inhibited the proliferation of CMK, MV4-11 and kasumi-1 cells. Figure 7 (C) and flow cytometry results showed that knocking down USP20 increased the proportion of apoptotic cells. Figure 6 D, Figure 8 In PI staining, compared to the control group, USP20 knockdown resulted in more cells being arrested in the G2 / M phase, thereby inhibiting cell proliferation. Figure 8 (A). Simultaneously, white light spectroscopy showed that, compared to the control group (sh-NC), a decrease in USP20 induced cell death. Figure 7 The results from experiment B also corroborate the above findings. Furthermore, Western blot analysis showed that downregulation of USP20 expression significantly cleaved the apoptosis proteins PARP and caspase-8. Figure 6 (E). Further in vivo experiments were conducted to verify the function of USP20 in AML development. Kasimi-1 cells labeled with luciferase and transfected with sh-USP20 and sh-NC were injected into NSG mice via tail vein injection. Figure 6In vivo imaging of mice at days 15, 20, 25, and 30 revealed a significant decrease in fluorescence flux in mice injected with sh-USP20 compared to the control group (sh-NC). Figure 6 China G and Figure 6 (H). Subsequently, fluorescence imaging was performed on the liver, spleen, and bone of both groups of mice. The results showed that USP20 knockdown significantly reduced the tumor burden in mice compared with the control group. Figure 9 (A). Meanwhile, CD45+ detection of the above tissues showed that the CD45+ ratio in the sh-USP20 group mice was significantly lower than that in the control group (A). Figure 9 (B) and there was no significant difference in body weight between the two groups of mice. Figure 9 (C). Survival analysis showed that sh-USP20 mice had a significant median survival advantage (C). Figure 6 (I). Based on H&E staining and Ki67 immunohistochemical staining of mouse liver, spleen, and bone marrow, it was observed that the USP20 knockdown group had a significantly reduced number of tumor cells compared to the control group. Figure 9 D, Figure 10 In summary, USP20 knockdown significantly inhibits AML proliferation both in vivo and in vitro.

[0052] 4. Protein preparation and binding site prediction Since the full-length crystal structure of USP20 is not included in the Protein Database (PDB), the protein structure used in this invention is derived from the AlphaFold2 prediction results. Given that the catalytic activity of the deubiquitinase USP20 depends on Cys154 and His643 residues, the binding site of the AlphaFold2 predicted USP20 structure was analyzed using MOE software. Site1, located near His643, was ultimately selected as the virtual screening target. This site contains the following amino acids: Gln229, Gln230, Asp231, Glu234, Phe589, His591, His635, Thr638, Ala639, Gly640, Ser641, Gly642, His643, and Tyr644. Figure 11 (A)

[0053] The USP20 structure was processed using the protein preparation wizard module of the Maestro platform (Schrödinger, NY, 2021). This included bond order assignment, hydrogen atom addition, disulfide bond designation, and protonation at pH 7.0 using the PROPKA method. Energy optimization was performed using OPLS4 force fields to apply positional constraints, eliminating atomic conflicts and converging the heavy atom RMSD to 0.3 Å. The protein structure was further refined through side-chain conformation optimization, ultimately yielding a pre-processed receptor structure suitable for subsequent screening.

[0054] The T001 compound library (TargetMol, containing 26,836 known active compounds) was used for screening. The library was processed using Maestro's LigPrep module: protonation, desalting, and tautomerization were performed at pH 7.0 ± 2.0 using the Epik method while preserving the original atomic chirality. To account for global conformational flexibility, a maximum of 32 conformations were generated for each molecule. The processed compound library was used as ligand input for virtual screening. Figure 11 (B)

[0055] like Figure 11 As shown in Figure B, after protein preparation, binding site prediction, and small molecule compound library preprocessing, virtual screening based on molecular docking was conducted to identify novel small molecules targeting the USP20 protein. The preprocessed receptor and ligand files were imported into Maestro, and molecular docking was performed using the GlideSP docking mode. Energy optimization and Glide scoring were then performed after docking. The top 50% of molecules by score (a total of 13,418 compounds) were retained, with Glide scores ranging from -9.8233 to -4.0739 kcal / mol.

[0056] Based on binding affinity distribution, 262 compounds with Glide scores below -7 kcal / mol were screened. Protein-ligand interaction fingerprinting (PLIF) analysis revealed the interaction frequencies of these compounds with USP20 residues (including hydrogen bonds, ionic bonds, π-π stacking, π-cation stacking, and π-hydrogen stacking, excluding van der Waals interactions). Notably, Asp231 and Glu234 residues exhibited higher interaction frequencies compared to other sites.

[0057] Structural diversity analysis of 262 compounds was performed using MOE software via Jarvis-Patrick clustering (70% similarity threshold), resulting in 167 clusters. The compound with the highest affinity in each cluster was retained. After combining conformational visual inspection, adhering to Lipinski's five rules, and excluding sugars, peptides, and compounds with inappropriate structures, nine candidate compounds were selected for bioactivity testing. The 2D and 3D diagrams of the interaction between the finally selected candidate compound AS1517499 and USP20 are shown below. Figure 11 C.

[0058] 5. Screening inhibitors and commercially available inhibitors of USP20 can inhibit the growth of AML cells in vivo and in vitro. The bioactivity of nine retained compounds—BC-1215, NN'-Bis(salicylidene)ethylenitamine, Dobutamine hydrochloride, Lys01, Dihydromyricetin, Mirabegron, Progabide, AS1517499, CRT0066101dihydrochloride, and GSK2643943A—was determined. Compound AS1517499 exhibited the best activity. To investigate the structural basis of this optimal activity, molecular docking analysis was performed between compound AS1517499 and the USP20 protein using the ligand docking module in Maestro. The results are as follows: Figure 12 As shown in Figure A, this compound interacts with multiple amino acid residues within the Site1 site: the hydrogen atom in its amide group forms hydrogen bonds with Gln230 and Glu234, while the hydrogen atom bound to the nitrogen atom of the phenethylamine side chain forms a hydrogen bond with Ala639. Furthermore, the hydrogen atom on the aromatic ring of the benzylamine side chain forms an aromatic ring-hydrogen interaction with Thr638. Notably, the docking results also revealed additional interactions beyond the Site1 range: the oxygen atom on the amide group of AS1517499 forms a hydrogen bond with Arg319. It is evident that compound AS1517499 may inhibit the deubiquitination activity of USP20 by simultaneously forming multiple hydrogen bonds with Site1 residues and surrounding amino acids—this interaction hinders the binding of the substrate to USP20, thereby affecting downstream signaling pathways and ultimately manifesting as enhanced biological activity.

[0059] In summary, compound AS1517499 may inhibit the deubiquitination activity of USP20 by simultaneously forming multiple hydrogen bonds with Site1 residues and surrounding amino acids, thereby hindering the binding of the substrate to USP20. Figure 12 (A). This interference further affects downstream pathways, ultimately resulting in enhanced biological activity. Subsequently, several screened inhibitors were added to MV4-11 cells at specific concentration gradients. CCK8 results showed that, compared to other small molecule inhibitors and the currently known commercially available USP20 inhibitor GSK2643943A, the IC50 of the screened small molecule inhibitor AS1517499 in MV4-11 cells was significantly lower than that of other inhibitors (A). Figure 12 (B). Next, MV4-11 cells were treated with a specific concentration gradient. While USP20 protein levels decreased in a concentration-dependent manner, the cleavage of PARP and caspase 8 proteins increased. Figure 12 (C). Simultaneously, flow cytometry results showed that treatment with 0, 150, and 200 nM compound AS1517499 increased the apoptosis rate of MV4-11 cells in a concentration-dependent manner. Figure 13 (B) The corresponding PI staining results showed that the cell G2 phase increased, causing cell cycle arrest in MV4-11 cells. Figure 13 (C). In plate cloning experiments, AS1517499 significantly reduced the ability of MV4-11 cells to form clones on plates in a concentration-dependent manner. Figure 13 (D), and the white light spectrum also demonstrated that AS1517499 can promote AML cell death in a concentration-dependent manner ( Figure 13 Next, the antitumor effect of AS1517499 in mice was investigated. First, MV4-11 cells labeled with luciferase were injected into mice via the tail vein. Five days after modeling, the experimental group mice were injected daily with 5 mg / kg of AS1517499, while the control group mice were injected intraperitoneally with the same volume of saline. In vivo imaging of the mice was performed on days 6, 10, 14, and 17. Figure 12 (D, E). The results showed that the fluorescence flux in the drug-treated group of mice was significantly lower than that in the control group (D, E). Figure 12 The results (F) indicate that AS1517499 can significantly delay the proliferation of MV411 in vivo. Survival curves show that the survival time of mice in the drug treatment group is prolonged (F). Figure 12 (G). At the experimental endpoint, the signal intensity of leukemia cells in the liver, spleen, and bone marrow of the treatment group mice was significantly lower than that in the control group (G). Figure 14 (A); White light showed no significant differences in the appearance of the liver, spleen, and bones between the NC group and the treatment group. Figure 14 (D). Furthermore, the CD45+ ratio in the liver, spleen, and bone marrow was significantly lower than that in the control group ( Figure 14 (B). HE staining and immunohistochemical results both showed that, compared with the control group, the drug treatment group had reduced tumor cell infiltration in the liver, spleen, and bone marrow. Figure 12 H in the middle Figure 14 C in the middle Figure 15 Furthermore, no obvious lesions were observed in the kidneys and intestines. Figure 14 (E).

[0060] Treatment of AML cells with the currently known commercially available USP20 inhibitor GSK2643943A at different concentrations significantly inhibited AML cell proliferation. The half-maximal inhibitory concentration (IC50) curves and IC50 values ​​are shown in the figure. Figure 16 In the study, treatment of MV4-11 cells with different concentration gradients of GSK2643943A resulted in a concentration-dependent increase in PARP protein splice levels. Based on previous findings, GSK2643943A did not reduce USP20 protein levels in cells, unlike AS1517499. Figure 16(B). Flow cytometry results showed that the apoptosis level of cells in the GSK2643943A treatment group increased ( Figure 16 (C). The above results indicate that both the virtual selection inhibitor and the commercial inhibitor of USP20 can promote AML cell death, and the inhibitor AS1517499 selected by virtual selection has a better inhibitory effect on AML cell growth than the commercial inhibitor GSK2643943A of USP20.

[0061] 6. Analyze the molecular mechanism by which USP20 inhibits the progression of AML. To investigate the transcriptional regulatory mechanism of USP20 in AML, RNA-seq transcriptome sequencing was performed on a control group based on CMK and a stable knockdown cell line with USP20 knockdown. Differential expression analysis showed that 772 genes were significantly downregulated and 997 genes were significantly upregulated in the USP20 knockdown group. Figure 17 In the USP20 knockdown group, gene enrichment analysis (GSEA) of downregulated genes showed that WNT-BETA-CATENIN, cholesterol, and the MYC pathway were significantly downregulated. Figure 17 China B and Figure 17 (C). A heatmap further visualizes the changes in expression patterns of core genes in the WNT-BETA-CATENIN, cholesterol, and MYC pathways after USP20 knockdown. Figure 17 Dysregulation of the WNT pathway has been confirmed in AML, and according to the TARGET and TCGA databases, CTNNB1 expression is significantly upregulated in AML cells compared to normal tissues, and high CTNNB1 expression is associated with poor prognosis. Figure 18 China A and Figure 18 (Middle B). MYC represents a typical oncogene that dysregulates and reprograms many aspects of cellular metabolism in AML, including glucose uptake and glycolysis, glutamine breakdown, serine / glycine metabolism, and lipid biosynthesis. RTqPCR results showed that USP20 knockdown significantly reduced the mRNA levels of WNT-BETA-CATENIN, cholesterol, and key MYC pathway genes NOTCH1, HMGCR, and LDHA. Figure 17 These findings suggest that USP20 is a key regulator of oncogenic signaling in AML, particularly through its regulation of the WNT / β-catenin pathway.

[0062] 7. USP20 promotes the progression of AML by exerting a deubiquitination effect on CTNNB1 and / or P62. To further explore the mechanism by which USP20 regulates the aforementioned pathways, mass spectrometry combined with software analysis identified 36 target proteins of USP20, including H2AC14, SQSTM1, H2BC13, CTNNB1, and HIF1A. Based on unique peptides, coverage, and intensity, CTNNB1 and SQSTM1 were selected as the primary targets for this invention. Figure 19 (A). Next, USP20 was overexpressed in MV4-11 and kasumi-1 cells, and immunoprecipitation experiments confirmed that USP20 interacts with CTNNB1 and SQSTM1 proteins. Figure 19 (Middle B). Furthermore, in MV4-11, kasumi-1, and CMK cells, knockdown of USP20 led to a decrease in CTNNB1 and SQSTM1 protein levels, but their mRNA levels remained unchanged. Figure 19 C, Figure 19 D, Figure 20 (A) indicates that USP20 does not regulate the decrease of CTNNB1 and SQSTM1 by regulating their RNA levels, and that different concentration gradients of AS1517499 can also cause a concentration-dependent decrease in the protein levels of CTNNB1 and SQSTM1. Figure 20 (B). Given that USP20 is a deubiquitinating enzyme, it is speculated that USP20 may stabilize these two proteins by regulating the ubiquitination levels of CTNNB1 and SQSTM1. In MV4-11, kasumi-1, and CMK cells, MG132 can reverse the downregulation of CTNNB1 and SQSTM1 protein levels caused by USP20 knockdown. Figure 19 E, Figure 20 (C). Following the inhibition of protein synthesis with actinomycin (CHX), USP20 knockdown accelerated the degradation of CTNNB1 and SQSTM1 protein levels in MV4-11 and kasumi-1 cells. Figure 19 China F, Figure 20 E, Figure 20 The above demonstrates that USP20 stabilizes CTNNB1 and SQSTM1 by stabilizing their ubiquitination. Given USP20's function as a deubiquitination enzyme, it is hypothesized that it maintains CTNNB1 stability by inhibiting K48-linked polyubiquitination. To verify this hypothesis, the following were co-expressed in AML cells: Flag-empty vector / Flag-USP20, GFP-CTNNB1, wild-type ubiquitin (Ub-WT), K48-specific ubiquitin (Ub-K48), and K63-specific ubiquitin (Ub-K63). Western blot results showed that USP20 overexpression significantly reduced the total ubiquitination level of CTNNB1. Figure 19 In the middle G lane, lanes 1 vs 2 (from left to right, lanes are 1, 2, 3, and 4) specifically inhibited polyubiquitination of K48 linkages, but had no significant effect on K63 linkages. Figure 19 (Middle G lane 3 vs 4). Then, cut & tag experiments were performed to analyze the regulatory role of USP20 on CTNNB1 in AML. The results showed that the binding sites of USP20 and CTNNB1 in CMK cells significantly overlapped. Figure 21 China A and Figure 21 (B). Based on the cut & tag results of USP20, it was found that USP20 has a highly similar motif to ELF1, ERG1, and RUNX1. Figure 21 C in the middle Figure 22 Furthermore, the binding sites of ELF1, ERG, RUNX1, USP20, and CTNNB1 in CMK cells highly overlap. Figure 23 China A and Figure 23 According to IGV visualization analysis, USP20 can co-regulate the transcription of the Wnt-bate-catenin pathway genes CCND2, JAG1, and ADAM17 by co-localizing with CTNNB1-1, CTNNB1-2, ELF1, ERG, and RUNX1. Figure 21 D, Figure 23 C and Figure 23 In MV4-11 cells, knocking down any one of the following genes—USP20, CTNNB1, ELF1, ERG, and RUNX1—leads to a decrease in the mRNA levels of the CCND2, JAG1, and ADAM17 genes. Figure 21 Chinese E, Figure 23 Chinese E, Figure 23 According to the Cancer Dependency Map, knocking out CCND2 in MV4-11 and kasumi-1 cells significantly inhibited cancer cell proliferation. Figure 24 In summary, as shown in () Figure 21 As shown in Figure F), USP20, as a super enhancer regulatory gene, promotes the progression of AML by binding to CTNNB1, ERG, ELF1, and RUNX1.

[0063] In summary, this invention is the first to discover that USP20 expression is significantly elevated in AML and negatively correlated with AML patient survival. It also reveals that USP20 can serve as a novel regulatory factor for AML, providing not only new theoretical evidence for the pathogenesis of AML but, more importantly, a new approach for AML drug development. Therefore, the deubiquitinase USP20 can be used as a biomarker for AML diagnosis, a target molecule for screening new AML treatments, and a detection indicator for evaluating AML treatments.

[0064] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of substances that detect ubiquitin-specific protease 20 or its encoding gene in at least one of the following: 1) Preparation of diagnostic products for acute myeloid leukemia; 2) Prepare products for evaluating the therapeutic effects of acute myeloid leukemia; 3) Preparation of prognostic evaluation product for acute myeloid leukemia; the amino acid sequence of the ubiquitin-specific protease 20 is shown in SEQ ID NO. 1; the nucleotide sequence encoding the ubiquitin-specific protease 20 is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The prognosis includes one or more of the following: clinical outcome of the prognostic subject, treatment effect of the prognostic subject, and survival of the prognostic subject; the product includes a drug and / or a reagent kit.

3. The application according to claim 1, characterized in that, The acute myeloid leukemia includes one or more of the following: acute myeloid leukemia undifferentiated, acute myeloid leukemia undifferentiated, acute myeloid leukemia partially differentiated, acute promyelocytic leukemia, acute myelomonocytic leukemia, erythroleukemia, and megakaryocytic leukemia.

4. The use of inhibitors of ubiquitin-specific protease 20 or its encoding gene in the preparation of drugs for the treatment of acute myeloid leukemia and / or to improve the prognosis of patients with acute myeloid leukemia.

5. The application according to claim 4, characterized in that, The inhibitor is selected from the following substances: (1) Substances that inhibit the enzymatic activity of ubiquitin-specific protease 20; or / and, (2) Substances that reduce or block the expression of the ubiquitin-specific protease 20 encoding gene.

6. The application according to claim 5, characterized in that, The substances that inhibit the enzymatic activity of ubiquitin-specific protease 20 include antibodies or inhibitors of ubiquitin-specific protease 20, such as AS1517499.

7. The application according to claim 5, characterized in that, The substances that reduce or block the expression of the ubiquitin-specific protease 20 encoding gene include siRNA targeting the ubiquitin-specific protease 20 encoding gene, and / or antisense nucleic acid or a CRISPR / Cas gene editing system that targets and knocks out the ubiquitin-specific protease 20 encoding gene.

8. The application according to claim 4, characterized in that, The treatment of acute myeloid leukemia and / or improvement of prognosis in patients with acute myeloid leukemia includes one or more of the following: inhibiting AML cell line proliferation, inhibiting AML cell line migration, and inhibiting AML cell line invasion.

9. A drug for treating acute myeloid leukemia and / or improving the prognosis of patients with acute myeloid leukemia, characterized in that, This includes substances that inhibit the enzymatic activity of ubiquitin-specific protease 20; and / or substances that reduce or block the expression of the gene encoding ubiquitin-specific protease 20.

10. The medicament according to claim 9, characterized in that, The dosage form of the drug includes one or more of the following: oral dosage form, injection dosage form, inhalation dosage form, and suppository.

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