Application of deubiquitinating enzyme USP20 as target spot in treatment of T-ALL

Abstract

The invention discloses application of a deubiquitinating enzyme USP20 as a target spot in treatment of T-ALL, belongs to the field of genetic engineering, discovers the function and mechanism of a super enhancer (SE) regulation gene USP20 in T-ALL, is expected to develop a more effective targeted therapy strategy, and brings a better clinical effect for T-ALL patients. Specifically, firstly, a gene USP20 regulated by a super enhancer is found in T-ALL, and the gene USP20 is specifically and highly expressed in the T-ALL and plays an important role. The USP20 stabilizes the HIF1A protein through deubiquitination, and promotes the proliferation and survival of T-ALL cells. The USP20 specific inhibitor GSK2643943A provides a new thought for the treatment of the T-ALL, and the USP20 is expected to become a potential target spot for the treatment of the T-ALL.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and specifically relates to the application of super enhancer regulatory gene USP20 as a target in the treatment of T-ALL. Background Art

[0002] T-ALL is a highly aggressive hematological malignancy, an acute lymphoblastic leukemia caused by abnormal clonal proliferation of T lymphocytes, accounting for about 15% of childhood ALL cases and 25% of adult ALL cases. In recent years, with the continuous improvement of treatment options, the overall survival rate (OS) of T-ALL has improved, but it is still less than 50%, so the treatment of T-ALL needs to be improved. Because T-ALL is highly invasive and has stronger resistance to chemotherapy, relapsed and / or refractory T-ALL is particularly difficult to treat, with an overall survival rate of less than 10%, which is one of the clinical problems. Therefore, in-depth research on the pathogenesis of T-ALL can provide important clues for the development of new targeted treatment strategies, which is of great significance for improving the clinical treatment effect of T-ALL.

[0003] An increasing number of studies have shown that deubiquitinating enzymes (DUBs) can regulate various physiological or pathological processes by stabilizing tumor or anti-tumor proteins, and ubiquitin-specific peptidases (USPs) are the largest subfamily of DUBs involved in the progression of multiple tumors. USP20 was first identified as a deubiquitinating enzyme in 2002 and belongs to the USP subfamily of DUBs. It has been confirmed that USP20 can cause deubiquitination of D2, HIF-1α, β2-AR, TRAF6, Tax, and PKM2, triggering the activation of signaling pathways such as Wnt, HIF 1A, and NF-κB, thereby regulating life processes such as thyroid hormone activation, cell cycle regulation, inflammatory response, tumorigenesis, and tumor progression. In addition, USP20 often plays an adverse role in cancer treatment and has been found to be overexpressed in a variety of tumor cells and cancer patients. New studies have revealed the key role of USP 20 in tumorigenesis in various cancer types, such as breast cancer, colon cancer, lung cancer, gastric cancer, and adult T-cell leukemia. Therefore, specific inhibition of USP20 activity is a new tumor treatment method. The small molecule inhibitor GSK2643943A has been shown to inhibit the deubiquitination activity of USP20.

[0004] However, the specific mechanism of action of USP20 gene in T-ALL remains unclear. Summary of the invention

[0005] The technical problem to be solved by the present invention is: application of USP20 as a target in the treatment of T-ALL.

[0006] The technical scheme of the present invention is: use of a deubiquitinating enzyme USP20 protein or an inhibitor of its encoding gene in the preparation of a drug for treating acute T lymphocytic leukemia.

[0007] Furthermore, the inhibitor is selected from the following substances:

[0008] a. a substance that inhibits the enzymatic activity of the deubiquitinating enzyme USP20; or / and

[0009] b. A substance that reduces or blocks the expression of a gene encoding the deubiquitinating enzyme USP20.

[0010] Furthermore, the substance that inhibits the enzymatic activity of the deubiquitinating enzyme USP20 is an antibody or inhibitor GSK2643943A of the deubiquitinating enzyme USP20 protein.

[0011] Furthermore, the substance that reduces or blocks the expression of the gene encoding the deubiquitinating enzyme USP20 is siRNA, antisense nucleic acid or CRISPR / Cas gene editing system for knocking out the gene encoding the deubiquitinating enzyme USP20.

[0012] Use of a substance for detecting the expression amount of deubiquitinating enzyme USP20 in preparing a kit for diagnosis, treatment effect evaluation or prognosis evaluation of acute T lymphocytic leukemia.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The present invention has discovered for the first time through experiments that the expression of USP20 in T-ALL is significantly increased, and is negatively correlated with the survival of T-ALL patients, and reveals that USP20 can be used as a new T-ALL regulatory factor, which not only provides a new theoretical basis for the pathogenesis of T-ALL, but more importantly, provides a new approach for the development of T-ALL drugs. Therefore, the deubiquitinase USP20 can be used as a marker molecule for T-ALL diagnosis, and USP20 can be used as a target molecule to screen new T-ALL therapeutic drugs. At the same time, USP20 can also be used as a detection indicator to evaluate T-ALL therapeutic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1To identify the SE-regulated gene USP20 in T-ALL, which is highly expressed in T-ALL and associated with poor prognosis. A shows the H3K27ac Chip-seq analysis of 6 T-ALL cell lines and 7 T-ALL patients, as well as the data of 6 T-ALL patients in the GEO dataset, to screen potential super enhancer (SE)-driven genes in the USP family. B shows that an enriched region was identified near USP20, indicating that it is highly likely to be regulated by super enhancers. C shows that the mRNA expression of USP20 in T-ALL patients is significantly higher than that in bone marrow samples of healthy donors. D shows that the data of the GEO dataset GSE13159 also show that the mRNA expression of USP20 in T-ALL is significantly higher than that in non-leukemic individuals and healthy bone marrow samples. E shows that knocking down BRD4 in T-ALL cells leads to a decrease in the expression level of USP20 mRNA. F shows that the clinical data of our center show that T-ALL patients with higher expression levels of USP20 have a lower survival rate.

[0016] Figure 2 The hockey stick graph shows super-enhancer-associated genes (including USP20) in six T-ALL cell lines.

[0017] Figure 3 Hockey stick graphs show super-enhancer-associated genes (including USP20) in 6 T-ALL patients.

[0018] Figure 4 CCLE database and Blood Spot analysis showed that USP20 was overexpressed in T-ALL cell lines compared with other cancer cells.

[0019] Figure 5 , A shows that knocking down BRD4 in T-ALL cells leads to decreased expression of USP20 protein level. B shows that the application of BRD4 inhibitor in T-ALL cells leads to decreased expression of USP20 protein level. C shows that Western blot technology is used to detect the expression of USP20 in T-ALL cells, AML cells and peripheral blood T cells of children with T-ALL.

[0020] Figure 6Figure 2 is the regulation of USP20 by related super enhancers. A is the ATAC and H3K27ac signals of the USP20 gene location reflected by ChIP-seq gene mapping. The figure shows Hi-C interactions in T-ALL and marks the three candidate enhancer regions of USP20 (E1, E2, and E3). B is the evaluation of the efficiency of Jurkat and J.gamma1 cells transfected with dual luciferase reporter vectors containing NC or USP20 enhancer components. C is a schematic diagram showing the use of dCas9 fused to a transcriptional repressor (KRAB) to inhibit the constitutive enhancer of USP20. D is the evaluation of the survival rate of Jurkat and J.gamma1 cells after transfection with non-targeting negative control and E3-targeting sgRNAs by CCK8 detection to evaluate cell viability. E shows the Western blot results of Jurkat and J.gamma1 cells after transfection with sgRNA-dCas9 vectors. F shows the USP20 mRNA expression levels in Jurkat and J.gamma1 cells after transfection with sgRNA-dCas9 vectors.

[0021] Figure 7 White light films and fluorescent images were taken to assess the survival of Jurkat and J.gamma1 cells after transfection with non-targeting negative control and E3-targeting sg RNA.

[0022] Figure 8 Figure 1 shows the analysis of the regulation of USP20 expression by key transcriptional regulatory factors. A is an IGV graph of CUT&Tag showing the presence of a high peak H3K27ac modification at the USP20 gene locus in the T-ALL cell line (J.gamma1). B is the detection of the activity of the E3 luciferase reporter gene after knocking down RUNX1 and ETS1 in T-ALL cell lines (Jurkat and J.gamma1). C is a qRT-PCR analysis showing the changes in the mRNA level of USP20 after knocking down a single key transcription factor (RUNX1 and ETS1) in T-ALL cell lines (Jurkat and J.gamma1). E is a scatter plot showing that the transcription levels of USP20, RUNX1, and ETS1 are positively correlated in T-ALL cases (GSE13159 n=165, GSE26713 n=117).

[0023] Fig. 9 The pie chart shows the distribution of DNA binding sites of different antibodies in J.gamma1 cell line found by CUT&Tag experiment. A is RUNX1 antibody / J.gamma1 cell line. B is ETS1 Antibody / J.gamma1 cell line. C is ERG antibody / J.gamma1 cell line. D is ETV6 antibody / J.gamma1 cell line.

[0024] Fig.10 The pie chart shows the distribution of DNA binding sites of different antibodies in J.gamma1 and 6T-CEM cell lines found in the CUT&Tag experiment. A is ELF1 Antibody / J.Gamma1 cell line. B is ERG Antibody / 6T-CEM cell line. C is ETV6 Antibody / 6T-CEM cell line.

[0025] Fig.11 The changes in USP20 mRNA expression levels after knocking down key transcriptional regulatory factors (ELF1, ERG, ETV6) in T-ALL cells (Jurkat and J.gamma1).

[0026] Fig.12 Knockdown of USP20 can inhibit the proliferation and development of T-ALL both in vitro and in vivo. A is the knockdown level of USP20 in cells verified by Western blotting. B is the qRT-PCR detection of USP20 mRNA levels in Jurkat, Molt4, and J.gamma1 cells infected with sh-NC, sh-USP20#2, and sh-USP20#3. C is the CCK-8 method to detect the proliferation rate of Jurkat, Molt4, and J.gamma1 cells after knockdown of USP20. D is the flow cytometry detection of the apoptosis level of Jurkat, Molt4, and J.gamma1 cells infected with sh-NC, sh-USP20#2, and sh-USP20#3. E is the Western blot detection that after knockdown of USP20, the cleavage of PARP and cleaved caspase-3 in T-ALL cells increased. F is a schematic diagram of the experimental design. G is the fluorescence imaging of the USP20 knockdown group and the control group obtained on days 19, 29, 36, and 43. H is a histogram showing the bioluminescent signal values ​​of the two groups of mice at different time points. I is compared with the control group, the sh-USP20 treatment group showed a longer survival period.

[0027] Fig.13 To knock down USP20 and inhibit the proliferation and development of T-ALL in vitro. A is a clone formation experiment of Jurkat, Molt4 and J.gamma1 cells transfected with sh-USP20#2, sh-USP20#3 and sh-NC. B is the change of apoptosis level of Jurkat, Molt4 and J.gamma1 cells after infection detected by flow cytometry.

[0028] Fig.14Knockdown of USP20 inhibits the proliferation and development of T-ALL in vitro. A shows the microscopic changes of Jurkat, Molt4 and J.gamma1 cells after knockdown of USP20. B shows the cleavage of cleaved caspase-8 in T-ALL cells after knockdown of USP20 detected by Western blotting. C shows the changes in the cell cycle of Jurkat, Molt4 and J.gamma1 cells after knockdown of USP20 detected by flow cytometry after PI staining.

[0029] Fig.15 To knock down USP20 and inhibit the proliferation and development of T-ALL in vivo. A is the biological images of liver, spleen and bone tissues in two groups. The bar graph shows the luminescent signal values ​​of the liver, spleen and bones. B is that the number of human CD45-positive cells in the liver, spleen and bone marrow with USP20 knockdown was significantly lower than that in the control group. C is the different sizes of liver and spleen in sh-NC and sh-USP20 mice. D is that there was no significant change in the weight of the two groups of mice. E is the H&E staining analysis of the mouse liver, spleen and bone marrow, showing that the tumor cells in the USP20 knockdown group were significantly reduced compared with the control group. F is the Ki67 staining of the mouse liver, spleen and bone marrow.

[0030] Fig.16 The USP20 inhibitor GSK2643943A can inhibit the proliferation and progression of T-ALL both in vitro and in vivo. A is the effect of GSK2643943A in T-ALL cells and T cells detected by CCK-8 assay, and it was found that GSK2643943A had a strong inhibitory effect on the growth of tumor cells in three T-ALL cell lines. B is a Western blot assay showing that PARP, cleaved caspase-3 and C-MYC proteins in Jurkat, 6T-CEM and J.gamma1 cells were stressed after GSK2643943A treatment, while the USP20 protein level remained unchanged. C is a flow cytometry assay showing that GSK2643943A treatment of Jurkat and J.gamma1 cells can induce more cell apoptosis in a dose-dependent manner. D is the application of GSK2643943A in Jurkat and J.gamma1 cells, and PI staining shows changes in the cell cycle. E is a schematic diagram of the experimental design. F shows that there are differences between the GSK2643943A group and the vehicle group in bioluminescence imaging on days 15, 22, 29, and 36. G shows that the bioluminescence signal values ​​of the two groups of mice at different time points. H shows that the GSK2643943A group showed a longer survival period compared with the untreated group. I shows that the number of human CD45-positive cells in the liver and spleen was significantly reduced after GSK2643943A treatment compared with the untreated sample.

[0031] Fig.17 The USP20 inhibitor GSK2643943A inhibits the proliferation and development of T-ALL in vitro. A is the flow cytometry detection, which found that GSK2643943A induced more apoptotic cells in Jurkat, Molt4 and J.gamma1 after treatment with Jurkat, Molt4 and J.gamma1, and it was dose-dependent. B is the PI staining detection after GSK2643943A treatment, which found that the cell cycle of Jurkat, Molt4 and J.gamma1 changed.

[0032] Fig.18 The USP20 inhibitor GSK2643943A inhibits the proliferation and development of T-ALL in vivo. A and B are biological images of liver, spleen and bone tissues of two groups of mice. The bar graph shows the bioluminescent signal values ​​of the liver, spleen and bones. C shows that compared with the control group, the human CD45-positive cells in the bone marrow of the GSK2643943A-treated group were significantly reduced. D shows the liver and spleen of the GSK2643943A group and the control group. E shows that there was no significant change in the body weight of the two groups of mice. F is the H&E staining analysis of the liver, spleen and bone marrow of the GSK2643943A group and the control group. G is a representative IHC Ki67 staining of the liver, spleen and bone marrow of mice.

[0033] Fig.19 H&E staining analysis of the kidneys and intestines of mice in the GSK2643943A group and the control group.

[0034] Fig. 20 To analyze the molecular mechanism by which USP20 inhibits T-ALL cell proliferation and progression. A is the differentially expressed genes between the USP20 knockdown and untreated groups found from RNA-seq data through volcano plot analysis; up-regulated and down-regulated genes are represented in red and blue, respectively. B is a KEGG pathway enrichment study. C is a GSEA diagram showing that T-ALL cells treated with USP20 knockdown have gene enrichment in the MYC, hypoxia, and glycolysis signaling pathways. D is a Heatmap showing the expression levels of MYC, hypoxia, and glycolysis-related genes in J.gamma1 cells treated with USP20 knockdown. E is a q PCR test that found that the expression levels of genes ODC1, LDHA, and VEGFA were reduced. F is the downregulation of C-MYC in WB analysis after knocking down USP20 in J.gamma1 cells.

[0035] Fig.21 Western blotting analysis showed that C-MYC expression was downregulated after knockdown of USP20 in Jurkat and Molt4 cells.

[0036] Fig. 22USP20 promotes the progression of T-ALL by deubiquitinating HIF1A. A shows the identification of 16 potential target proteins of USP20 using mass spectrometry and software analysis. The top 10 genes were ranked according to unique peptides, coverage (%) and intensity, with HIF1A ranking first. B shows the expression of USP20, Flag and HIF1A proteins in Jurkat and J.gamma1 cells overexpressing USP20 after immunoprecipitation with Flag antibody, and WB detection. C shows the amount of USP20, HIF1A and β-actin proteins in Jurkat and J.gamma1 cells stably expressing sh-NC, sh-USP20#2 and sh-USP20#3 by WB detection. D shows the mRNA levels of USP20 and HIF1A in Jurkat and J.gamma1 cells stably expressing sh-NC, sh-USP20#2 and sh-USP20#3, detected by qRT-PCR. E: Protein levels of USP20 and HIF1A were detected by Western Blot in Jurkat and J.gamma1 cells stably expressing sh-NC, sh-USP20#2, and sh-USP20#3. F: Jurkat and J.gamma1 cells overexpressing USP20 (flag-USP20) or stably expressing an empty vector (flag) were treated with 100 μg / ml CHX for the indicated time periods. Then, the protein levels of USP20, HIF1A, and β-actin were evaluated by Western blotting. G: HEK-293FT cells transfected with Flag-USP20, GFP-HIF1A, Ub (wild type), Ub-K48 (only lysine 48 on the Ub molecule), and Ub-K63 (only lysine 63 on the Ub molecule) were exposed to MG132 (20 μM) 6 h before harvesting. The ubiquitination level of HIF1A was then detected.

[0037] Fig.23 Candidate substrates of USP20 were analyzed using the software (Ubibroser).

[0038] Fig.24USP20 promotes the progression of T-ALL by deubiquitinating HIF1A. A shows 16 potential target proteins of USP20 obtained by mass spectrometry and software analysis. The last 6 genes are sorted by specific peptide chain, coverage [%] and intensity. B shows a two-dimensional peak diagram describing a peptide sequence of HIF1A protein. C shows immunoprecipitation using Flag antibody in Jurkat and J.gamma1 cells expressing USP20, and WB identifies CTNNB1 protein. D shows Western blotting detection of USP20, CTNNB1 and β-actin expression in Jurkat and J.gamma1 cells stably expressing sh-NC, sh-USP20#2 and sh-USP20#3. E shows qRT-PCR detection of USP20 and CTNNB1 mRNA expression in Jurkat and J.gamma1 cells. F shows the effect of GSK2643943A on the expression of USP20, HIF1A and β-actin protein levels in Jurkat and J.gamma1 cells detected by Western blotting. G shows Jurkat and J.gamma1 cells stably expressing sh-NC, sh-USP20#2 and sh-USP20#3 were treated with 100 μg / ml CHX at the indicated time points. Changes in the levels of USP20, HIF1A and β-actin proteins were detected by Western blotting.

[0039] Fig.25 The pie chart shows the distribution of DNA binding sites of different antibodies in J.gamma1 cells found in the CUT&Tag experiment. A is USP20 Antibody / J.Gamma1 cells. B is HIF1A antibody / J.gamma1 cells. C is the IGV map of CUT&Tag showing the H3K27Ac signal, ATAC signal, and USP20 and HIF1A gene site signals of the T-ALL cell line (J.gamma1), showing potential target genes of USP20 and HIF1A, such as PKM, CASP9, and DLG4.

[0040] Fig.26USP20 regulates gene transcription through HIF1A. A is a heat map generated by CUT&Tag study showing that USP20 and HIF1A are contained in the J.gamma1 cell line. B is the distribution of HIF1A and USP20 peaks within the ±1kb window of the USP20 binding site. C is a CUT&Tag IGV map showing the H3K27ac signal, ATAC signal and gene locus signal of USP20 and HIF1A in the T-ALL cell line (J.gamma1), highlighting potential target genes such as SLC2A1, PER1, CIC, MAZ, PKM, CASP9 and DLG4. DETAILED DESCRIPTION

[0041] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0042] The invention discloses the regulatory mechanism of super enhancer-regulated deubiquitinase USP20 in T-ALL and its application as a drug target.

[0043] The experimental means and experimental operations required for the invention are given below.

[0044] 1. Cell Culture

[0045] Human leukemia cell lines J.gamma1, 6T-CEM, MOLT-4, and Jurkat were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd., and HUT78 and CCRF-CEM were from the Chinese Academy of Sciences Cell Bank. Cells were cultured in RPMI1640 medium containing 10% FBS in a humidified environment, 5% CO2, and 37°C cell culture incubator. In addition, HEK293FT cells were derived from the Chinese Academy of Sciences Cell Bank and cultured in DMEM medium containing 10% FBS. All cell lines were authenticated by short tandem repeat (STR) analysis and were regularly tested using the MycoAlert kit to ensure that there was no mycoplasma contamination.

[0046] 2. Lentivirus Packaging and Infection

[0047] The total mass of the target plasmid and the packaging plasmid (psPAX2; pMD2.G) was 15 μg, and they were co-transfected into 293FT cells at a ratio of 4:3:1 using a polyethyleneimine (PEI) reagent (49553-93-7, Sigma-Aldrich, USA). After 6 hours of transfection, fresh complete DMEM medium was replaced, and the viral supernatant was collected after 48 hours of culture and filtered through a syringe filter (0.45 μm). T-ALL cells were then co-cultured with lentivirus containing 1 μg / ml Polybrene (Sigma-Aldrich) for 24 hours. Stable T-ALL cell lines were selected by 1 μg / ml puromycin.

[0048] To achieve stable overexpression of USP20, the USP20 CDS sequence with a Flag tag was cloned into the pLVX-EF1α-IRES-puro lentiviral vector (General Biosystems (Anhui) Co., Ltd., China). T-ALL cells were then infected with the lentivirus and selected by puromycin to obtain a T-ALL cell line overexpressing USP20.

[0049] 3. CRISPR-mediated USP20 silencing

[0050] To generate stable knockout cell lines using the Cas9 / CRISPR system, Jurkat and J.gamma1 cells were first transfected with lentivirus carrying the Cas9 gene, followed by selection with puromycin. sgRNA targeting USP20 was cloned into a lenti-crispr plasmid synthesized by IGE Biotechnology Co., Ltd. (Guangzhou, China), and Cas9-expressing cells were transfected with lentiviral particles containing sgRNA-USP20 or non-targeting control sgRNA (sgNC).

[0051] 4. RNA Purification, cDNA Generation, and qRT-PCR

[0052] Total RNA was extracted from cell samples using FastPure Cell / Tissue Total RNA Isolation Kit V2 (RC112-01, Vazyme, China) according to the instructions. Then, 2 μg of total RNA was reverse transcribed to generate cDNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, California, USA). Quantitative real-time PCR was performed using 480 Green I Master mix (Cat. No. 04887352001; Roche, Germany) was performed on a LightCycler 480 Real-Time System (Roche). The cycle threshold (Ct) values ​​of samples and controls were normalized to GAPDH as an internal reference, and then the enrichment fold of relative gene expression was determined using the 2–ΔΔCT method. The relative expression of each gene was based on the average of three replicates.

[0053] 5. Western blotting analysis

[0054] The collected cells were washed with PBS and then lysed using RIPA lysis buffer (Beyotime, China) containing protease and phosphatase inhibitors. After sonication, the quantitatively matched protein samples were separated by SDS-PAGE gel and transferred to a PVDF membrane (CST, USA). The membrane was blocked with TBST solution (1×TBS, 0.1% Tween20) containing 5% skim milk powder for 1.5 hours at room temperature. Then, the membrane was incubated with the primary antibody overnight at 4°C. The next day, the PVDF membrane was incubated with the secondary antibody at room temperature, followed by treatment with ECL luminescent solution (Yeasen, China) and visualized by AI600 Image Gel Imaging Analyzer (GE, USA).

[0055] 6. Cell Proliferation Assay

[0056] After the puromycin resistance screening, the cells of the experimental and control groups were plated at 3×10 3 Cells were seeded in 96-well plates at a density of 10 cells / mL. CCK-8 reagent (APExBIO, K1018-1, USA) was added to each well on days 1, 3, and 5 to assess cell proliferation. The absorbance was measured at a wavelength of 450 nm using a spectrophotometer to quantify the cell activity of the two groups at designated time intervals. To determine the IC50 (half-maximal inhibitory concentration) of GSK2643943A, cells (1×10 4 Cells / well) were treated with different concentrations of GSK2643943A (MCE, hay-111458) ranging from 30 nM to 4 μM for 24 h, and each concentration was repeated at least three times for each cell line.

[0057] 7. Soft agar colony formation assay

[0058] The bottom agar gel was prepared with 1.2% agarose gel and 2×RPMI 1640 medium (YuChun, YC-1010) and 20% FBS. After the gel solidified at room temperature, 100 μl of cell suspension of sh-NC group and sh-USP20 group was added at 5×10 3 / ml concentration was inoculated into the soft agar medium of a 6-well plate. The upper agar gel was prepared with 0.7% agarose gel and 2×RPMI 1640 medium, and 20% fetal bovine serum was added. The 6-well soft agar plate was placed in a 37°C, 5% CO2 incubator for culture. After about 2 weeks, it was fixed with 4% paraformaldehyde (Beyotime, P0099) for 24 hours, then stained with Giemsa (Beyotime, C0131), and the number of clones was counted.

[0059] 8. Cell Apoptosis Detection

[0060] After puromycin selection, cells were initially washed with cold 1× PBS buffer. Then, samples were suspended in 1× Annexin V binding buffer and labeled with FITC-Annexin V antibody and PI solution according to the instructions in the FITC-Annexin V Apoptosis Kit Manual (Cat. No. 556547, BD Biosciences, USA). Beckman Gallios TM Cell apoptosis was assessed by flow cytometry (Beckman, Krefeld, Germany).

[0061] 9. Dual luciferase reporter gene assay

[0062] The USP20 super enhancer (USP20-SE) was divided into three different parts to construct three luciferase expression vectors, labeled E1-E3, and each vector (0.5 μg) was transfected into Jurkat cells together with 0.05 μg of Renilla luciferase (used as a normalization control) using Lipofectamine LTX reagent (Thermo Fisher, USA). 48 hours after transfection, luciferase reporter gene activity was assessed using the Dual Luciferase Reporter Gene Assay System (E1910, Promega, USA), which simultaneously measures the activities of firefly and Renilla luciferase. To adjust for the variation of enhancer fragments in transfected cells, the calculated ratio of firefly to Renilla luciferase activity was normalized.

[0063] 10. Cell Cycle Analysis

[0064] After the cells were collected, they were rinsed with cold PBS and then fixed in 75% ethanol overnight. Subsequently, cell cycle analysis was performed using a cell cycle and apoptosis analysis kit (C1052, Bio-Time, China). Cell flow cytometry (Beckman Gallios TM , Beckman) to analyze the cell cycle distribution.

[0065] 11. RNA Sequencing and Data Processing

[0066] RNA-seq analysis followed the protocol provided by BGI Technology Service Co., Ltd. (Wuhan, China). First, total RNA was reverse transcribed to generate complementary DNA (cDNA) for the construction of sequencing libraries. The libraries were sequenced on an appropriate platform, and the resulting raw reads were filtered to remove low-quality reads, adapters, and other artifacts to obtain clean reads. These clean reads were then aligned to the reference genome using the HISAT framework to determine their mapping positions. Gene expression levels were quantified using the per kilobase per million reads exon model (FPKM). Differentially expressed genes (DEGs) were identified by DESeq2 analysis, with significance thresholds of P < 0.05 and |log2 Fold change|> 0.5. Gene set enrichment analysis (GSEA) was performed using the R / Bioconductor clusterProfiler package, and the wikipathways collection (version 2023.1) from the Molecular Signatures Database (MSigDB) was used.

[0067] 12. In vivo experiments

[0068] All animal experiments were approved and permitted by the Animal Care and Use Committee of the Children's Hospital of Soochow University (CAM161 SU-AP#: JP-2018-1). Female NSG mice aged 4–6 weeks were purchased from Shanghai South Model Biotechnology Co., Ltd.

[0069] To investigate the function of USP20 in vivo, NSG mice were randomly divided into two groups. J-gamma1 cell suspensions expressing sh-NC or sh-USP20 (100 μl PBS per mouse, containing 2 × 10 6 cells) were injected into the tail vein. D-luciferin sodium salt (115144-35-9, GOLDBIO, USA) was injected intraperitoneally on days 19, 29, 36, and 43 after tail vein injection, and then small animal in vivo imaging scanning was performed using the Berthold imaging system (Germany) to measure the maximum standard mean radiometric value of mouse tumors. The liver, spleen, and bone tissues of the sh-NC group and sh-USP20 group were collected by CO2 inhalation method for euthanasia mice for subsequent experimental procedures, including flow cytometry analysis (using CD45 detection), paraffin embedding, hematoxylin-eosin (H&E) staining, and immunohistochemistry.

[0070] In order to study the function of GSK2643943A in vivo, a batch of 4-6 week-old female NSG mice were purchased and randomly divided into two groups. Each mouse was injected with 2×10 6J-gamma1 cell suspension of 100 μl PBS. 10 days after tail vein injection, D-luciferin sodium salt was injected intraperitoneally, and the fluorescence signal was displayed by in vivo imaging scanning. 2 days later, the experimental group was intraperitoneally injected with 15 mg / kg of GSK2643943A, and the control group was intraperitoneally injected with 15 mg / kg of HS15 (solvent of GSK2643943A) was administered once daily for 10 times (2 days apart). Imaging was then continued using the NightOWL in vivo imaging system on days 15, 22, 29, and 36. Mice were euthanasia by CO2 inhalation, and liver, spleen, kidney, and intestinal tissues were collected from the experimental and control groups for subsequent experiments.

[0071] 13.CUT&Tag experiment

[0072] CUT&Tag experiments were performed on J.gamma1 cells using the CUT&Tag assay kit (TD903-01, Vazyme) according to the instructions. 6 Cells were treated with nuclear extraction buffer for 10 minutes. After centrifugation, the cell pellet was resuspended in 500 μl of 0.1% formaldehyde solution and incubated at room temperature for 2 minutes to initiate the cross-linking reaction, which was then terminated by adding 2.5 M glycine. The resulting cell pellet was collected after centrifugation and resuspended in 100 μl of wash buffer. Activated ConA beads were then added for further incubation, followed by the addition of primary antibodies (including USP20, HIF1A, and IgG as a control) and incubated overnight at 4°C. After overnight incubation, 50 μl of secondary antibody diluted to 1:100 was added to each sample and then incubated for 1 hour at room temperature with rotation. The cell-bead complex was then gently washed three times with 200 μl Dig-wash buffer and then incubated with pA / G-Tnp. The samples were then fragmented and DNA extracted. PCR products were purified by adding 2× volume of VAHTS DNA cleaning beads (Vazyme Biotech, #N411) and incubated at room temperature for 5 minutes. The beads were washed twice with 200 μl of fresh 80% ethanol and eluted in 22 μl ddH2O. All CUT&Tag libraries were sequenced by Shanghai Bohe Biotechnology Co., Ltd.

[0073] 14. Co-immunoprecipitation

[0074] Jurkat and J.gamma1 cells transfected with USP20-Flag overexpression vector were lysed using lysis buffer containing NP-40 (P0013F, Betech, China) and protease inhibitors. After incubation at 4°C for 30 min, the lysate was centrifuged in a refrigerated centrifuge to collect the supernatant. 50 μl of the lysate was mixed with 5× loading buffer and boiled at 100°C for 10 min to denature the protein as the input group. The remaining supernatant was divided into two parts, protein A / G beads (B23202, Bimake, China) were added, and then Flag antibody (F1804, Sigma, USA) or IgG antibody (ab172730, abcam, USA) was added, respectively, and rotated overnight at 4°C. The next day, the samples were centrifuged at 8200g to remove the supernatant. The precipitate was washed five times with pre-cooled TBST. The pellet was then sent for mass spectrometry analysis (PTM BIO, Hangzhou, China) or resuspended in 2× loading buffer in preparation for subsequent WB detection.

[0075] The relevant results of the present invention are given below

[0076] 1. Identification of super enhancer (SE) regulatory gene USP20 in T-ALL using data analysis methods, and the correlation between high expression of USP20 in T-ALL and poor prognosis

[0077] To identify genes associated with SEs in T-ALL, we reanalyzed H3K27ac ChIP-seq data from six T-ALL cell lines (Jurkat, J.gamma1, CCRF, MOLT3, MOLT4, PF382) and seven T-ALL patients. Using ROSE software, we mapped the SE distribution of these 13 samples. Among the USP family genes, we found that USP4, USP5, USP7, USP20, USP36, USP44, and USP49 overlapped with or were surrounded by SEs in multiple samples ( Figure 1 Middle A, Figure 2 and Figure 3 ). Further analysis of H3K27ac ChIP-seq samples from six T-ALL patients in the GEO dataset GSE155339 revealed that USP20 is highly likely to be a SE-driven gene ( Figure 1 A). Notably, USP20 was identified as a possible SE driver gene in more than 90% of the samples. A consistent pattern of USP20 loci was observed in six T-ALL cell lines, seven T-ALL patients, and three T cell samples (GSE18927), but not in CD34+HSCs (GSE231486). Figure 1Middle B). Data from the GEO dataset GSE26713 showed that USP20 mRNA expression in T-ALL patients was significantly higher than that in normal bone marrow (NBM) ( Figure 1 In addition, data from GSE13159 showed that USP20 mRNA expression in T-ALL patients was significantly higher than that in bone marrow samples from non-leukemic and healthy individuals ( Figure 1 Analysis of the Cancer Cell Line Encyclopedia (CCLE) database showed that the expression level of USP20 mRNA in T-ALL cell lines was higher than that in other tumor cell lines ( Figure 4 BloodSpot data also confirmed the high expression of USP20 in T-ALL ( Figure 4 B), indicating that the high specific expression of USP20 in T-ALL may play an important role. After knocking down the super enhancer regulatory gene BRD4 in T-ALL cells, it was found that USP20 was reduced at both protein and mRNA levels ( Figure 1 Middle E, Figure 5 A), similar results were obtained using a BRD4 inhibitor ( Figure 5 Western blot analysis showed that USP20 levels were high in peripheral blood T lymphocytes of children with T-ALL and in T-ALL and AML cell lines ( Figure 5 C). Based on the clinical data of our center, we analyzed the survival rates of 24 children with low USP20 expression and 54 children with high USP20 expression in T-ALL. The results showed that the survival rate of the high expression group was significantly lower than that of the low expression group, and the difference was statistically significant (P<0.05) ( Figure 1 In conclusion, our study identified for the first time that USP20 is a SE-regulated gene that is specifically overexpressed in T-ALL. Furthermore, elevated USP20 expression is associated with poor prognosis in children with T-ALL, highlighting its important pathogenic role.

[0078] 2. USP20 expression is regulated by super enhancers

[0079] During tumorigenesis and progression, super-enhancers change in composition and function and may become potential therapeutic targets. Acetylation of histone H3 lysine 27 (H3k27ac) is one of the commonly used indicators for super-enhancer identification. We reanalyzed the publicly available T-ALL Hi-C data to confirm the association between the USP20 enhancer and the promoter. These connections were confirmed by Figure 2 The connecting lines above the ChIP-seq profile in A are shown. Within the USP20-SE region, we identified three candidate enhancers (E1-E3) and a negative control region ( Figure 6These candidate enhancers were cloned into the pGL3-promoter luciferase reporter vector and transfected into Jurkat and J.gamma1 cell lines. Luciferase reporter assays showed that E3 exhibited the strongest luciferase activity, consistent with its high H3K27ac signal ( Figure 6 Based on these findings, we concluded that the E3 enhancer is the major active unit of USP20 SE. To further validate the transcriptional function of this enhancer component, we used CRISPR interference technology, in which sgRNAs targeted the dCas9 / KRAB complex to specifically inhibit the USP20 E3 cis-regulatory element ( Figure 6 In Jurkat and J.gamma1 cells transfected with sgRNA-dCas9 / KRAB, inhibition of USP20 SE activity led to a significant decrease in cell growth, whereas Jurkat and J.gamma1 cells transfected with the sgRNA control plasmid had no significant effect on cell proliferation ( Figure 6 Middle D, Figure 7 In addition, sgRNA targeting E3 significantly reduced the expression of USP20 at both protein and mRNA levels ( Figure 6 These results indicate that the expression of USP20 in T-ALL is regulated by super enhancers.

[0080] 3. Analysis of the regulation of USP20 expression by key transcription factors

[0081] We observed that ATAC-seq and H3K27ac signals were highly consistent, indicating that transcriptional activity is highly correlated with chromatin plasticity ( Figure 8 In addition, we further investigated whether this positive correlation was related to the binding of T-ALL key transcription factors (including RUNX1, ETS1, ETV6, ELF1 and ERG, etc.) to the USP20-SE region. Using CUT&Tag technology, we used antibodies to specifically bind to target proteins to enrich DNA fragments that interact with them, and constructed high-resolution DNA sequencing libraries to investigate the binding of these key transcription factors to DNA in J.gamma1 cells. The results showed that there were multiple enrichment sites of key transcription factors in the USP20-SE region, among which the enhancer E3 region showed the most significant enrichment ( Figure 8 Middle A, Fig. 9 and 10). To confirm whether T-ALL CRC TFs directly promote USP20 expression through SE, we knocked down each key transcription factor and then transfected the pGL3-promoter luciferase reporter vector containing the USP20-E3 enhancer element into Jurkat and J.gamma1 cells. Consistent with our hypothesis, knockdown of TFs significantly reduced USP20-E3 luciferase activity ( Figure 8 In addition, at the mRNA level, the loss of any one of the transcription factors consistently and significantly downregulated the expression of USP20 ( Figure 8 Middle C, Fig.11 ). Analysis of expression data from T-ALL patient samples in the GEO database (GSE13159, GSE26713) showed that there was a significant positive correlation between the expression level of USP20 and the expression levels of key transcription factors RUNX1 and ETS1 ( Figure 8 These results suggest that RUNX1 and ETS1 cooperatively regulate USP20 transcription in T-ALL by directly binding to its SE region and enhancing its plasticity.

[0082] 4. Knockdown of USP20 inhibits the proliferation and development of T-ALL in vitro and in vivo

[0083] To investigate the function of USP20 in T-ALL, we transfected USP20 shRNA (sh-USP20#1, sh-USP20#1, and sh-USP20#3) and control sh-NC in three T-ALL cell lines (Jurkat, J.gamma1, and Molt4). Compared with the control group, cells transfected with USP20 shRNA (#2 and #3) showed a significant decrease in USP20 expression at both the protein and RNA levels, as determined by Western blot and qRT-PCR analysis ( Fig.12 In the soft agar assay, knockdown of USP20 significantly reduced the colony-forming ability of T-ALL cells ( Fig.13 In addition, in the cell proliferation experiment detected by CCK8, the USP20 knockdown group significantly inhibited cell proliferation compared with the control group (P<0.001) ( Fig.12 Middle C).

[0084] Apoptosis was assessed by flow cytometry, showing that USP20 deficiency induced apoptosis in Jurkat, Molt4, and J.gamma1 cell lines ( Fig.12 Middle D, Fig.13 In addition, compared with the sh-NC group, the USP20 knockdown group showed significant growth inhibition, leading to cell death ( Fig.14Consistent with these findings, Western blot analysis showed downregulation of USP20 expression, along with apoptotic proteins PARP and Caspase-3 ( Fig.12 Middle E), Caspase-8 ( Fig.14 Cell cycle detection showed that USP20 knockdown led to cell cycle arrest, with a significant increase in G2 / M phase cells and a decrease in G1 / S phase cells, thereby inhibiting cell proliferation ( Fig.14 Middle C).

[0085] In vivo, J.gamma1 cells transfected with sh-USP20 or sh-NC were injected into NSG mice via tail vein. J.gamma1 cells were labeled with luciferase ( Fig.12 Middle F). The fluorescence signal level of the sh-USP20 group was significantly lower than that of the sh-NC group ( Fig.12 Middle G). The histogram of tumor light flux showed that the sh-USP20 group had a significant decrease compared with the sh-NC group ( Fig.12 Middle H, Fig.15 Fluorescence imaging of mouse liver, spleen, and bone samples showed that the tumor burden was significantly reduced in the USP20 knockdown group ( Fig.15 Survival analysis showed that sh-USP20 mice had a significant median survival advantage ( Fig.12 The proportion of CD45+ cells in the bone marrow and liver / spleen of the sh-USP20 group was significantly reduced ( Fig.15 B), and two sets of white light images of the liver and spleen were obtained at the same time ( Fig.15 No significant difference was observed in the body weight of the two groups of mice ( Fig.15 In addition, H&E staining showed that the number of tumor cells in the USP20 knockdown group was significantly reduced ( Fig.15 These findings were further supported by immunohistochemical staining for Ki67, showing that USP20 knockdown had an inhibitory effect on tumor development in liver, spleen, and bone marrow tissues ( Fig.15 In summary, the consistent results from in vitro and in vivo studies indicate that USP20 knockdown has an antitumor effect on T-ALL.

[0086] 3.5 USP20 inhibitor GSK2643943A inhibited the proliferation and development of T-ALL cells in vitro and in vivo

[0087] The small molecule inhibitor GSK2643943A can inhibit the deubiquitination activity of USP20 and affect cell proliferation and apoptosis. Our results showed that different concentration gradients of GSK2643943A acted on two CD8+T cell samples and T-ALL cell lines, among which four T-ALL cells were strongly inhibited, and their cell viability curves and IC50 were as follows: Fig.16 As shown in A. In Jurkat, 6T-CEM and J.gamma1 cell lines, the expression of C-MYC and the cleavage of apoptotic proteins were dose-dependent, the cleavage of PARP and caspase-3 gradually increased in a dose-dependent manner, but the expression level of USP20 protein remained unchanged ( Fig.16 In addition, flow cytometry analysis showed that the proportion of apoptotic cells increased in a dose-dependent manner after GSK2643943A treatment ( Fig.16 Middle C, Fig.17 GSK2643943A also caused cell cycle arrest in T-ALL cell lines by increasing the proportion of cells in the G2 phase and decreasing the proportion of cells in the G1 phase ( Fig.16 Middle D, Fig.17 (B).

[0088] To investigate the anti-tumor function of the USP20 inhibitor GSK2643943A in mice, a T-ALL mouse model was established using J.gamma1 cells stably expressing luciferase ( Fig.16 When GSK2643943A was administered to mice, the fluorescence signal intensity was significantly lower than that of the control group ( Fig.16 In addition, GSK2643943A-treated mice showed significantly reduced infiltration in the liver, spleen, and bone marrow compared with the control group ( Fig.18 A and B). Comparison of the survival time between the two groups showed that the life span of the GSK2643943A-treated group was significantly prolonged ( Fig.16 In addition, the proportion of CD45+ cells in the bone marrow, liver, and spleen of the GSK2643943A-treated group was significantly reduced compared with the control group ( Fig.16 Middle I, Fig.18 White light photos of the liver and spleen of the two groups of mice showed that there was no statistically significant difference in body weight between the two groups ( Fig.18 Middle D, Fig.18 Middle E). HE staining of liver, spleen, bone marrow, intestine and kidney samples of mice showed a significant reduction in tumor cells in the GSK2643943A-treated group. It is worth noting that no pathological changes were observed in the kidneys after treatment, and intestinal damage also appeared to be mild ( Fig.18 Middle F, Fig.19 In addition, the Ki67-positive cells in the liver, spleen, and bone marrow of the GSK2643943A-treated group were significantly reduced ( Fig.18Middle G). Together with the fact that there was no significant change in the weight of the mice, this suggests that GSK2643943A causes less damage to the organs. In summary, this suggests that GSK2643943A has an inhibitory effect on the tumorigenesis of T-ALL cells in mice, and that GSK2643943A inhibits the proliferation and development of T-ALL in vitro and in vivo, similar to the effect of USP20 knockdown.

[0089] 6. Analysis of the molecular mechanism of USP20 inhibiting T-ALL cell proliferation and development

[0090] To investigate the potential downstream genes of USP20 in T-ALL, we performed RNA-seq analysis using J.gamma1 cells from the control and USP20 knockdown groups to identify downstream genes that may be regulated by USP20 in T-ALL. The results showed that in the USP20 knockdown group, a total of 588 genes were downregulated and 502 genes were upregulated (log2FoldChange≥0.5, FDR<0.05) ( Fig. 20 Hallmark gene set enrichment analysis (GSEA) revealed that hypoxia, glycolysis, and MYC target genes were significantly enriched in the USP20 knockdown group and the control group ( Fig. 20 Heatmaps showing changes in gene expression related to MYC, hypoxia, and glycolysis pathways after USP20 knockdown ( Fig. 20 D). MYC target genes are essential for the survival of T-ALL. There is a close relationship between hypoxia and the glycolytic pathway. Under hypoxic conditions, cells regulate gene expression by activating hypoxia-inducible factors (HIFs), which promote the expression of genes involved in the glycolytic pathway. This pathway produces ATP and metabolites to maintain cell survival and function under hypoxic conditions. T-ALL cells have a high proliferation capacity and an increased demand for energy and metabolites, so hypoxia and the glycolytic pathway play an important role in maintaining tumor cell survival and proliferation. qRT-PCR results showed that after USP20 knockdown, the expression levels of genes related to MYC, hypoxia and glycolytic pathways, such as ODC1, LDHA and PGK1, were significantly reduced ( Fig. 20 In addition, Western blot analysis showed that the expression level of C-MYC was also reduced after USP20 knockdown ( Fig. 20 Middle F, Fig.21 ).

[0091] 3.7 USP20 promotes the development of T-ALL by modifying HIF1A through deubiquitination and regulating gene transcription through HIF1A

[0092] Through mass spectrometry and software analysis, we hypothesized 16 potential target proteins of USP20, including HIF1A, PLK1, and CTNNB1. Among them, HIF1A and CTNNB1 are more likely to be USP20 targets based on unique peptides, coverage, and intensity ( Fig. 22 Middle A, Fig.23 , Fig.24 A and B). Combining RNA-seq data analysis and literature review, we focused on the relationship between USP20 and HIF1A. First, USP20 was overexpressed in Jurkat and J.gamma1 cells, and Co-IP experiments revealed that there was an interaction between USP20 and HIF1A or CTNNB1 ( Fig. 22 Middle B, Fig.24 In addition, knockdown of USP20 in Jurkat, J.gamma1, and 6T-CEM cells resulted in a significant decrease in HIF1A or CTNNB1 protein levels, while mRNA levels were not affected ( Fig. 22 C and D, Fig.24 In addition, treatment with the USP20 inhibitor GSK2643943A resulted in a decrease in HIF1A protein levels with increasing concentrations ( Fig.24 We hypothesized that USP20 could stabilize HIF1A by regulating its ubiquitination, and that the proteasome inhibitor MG-132 could reverse the downregulation of HIF1A protein levels caused by USP20 knockdown ( Fig. 22 In addition, our results also showed that USP20 knockdown accelerated the degradation of HIF1A after cycloheximide (CHX) treatment in Jurkat and J.gamma1 cells, while USP20 overexpression prolonged its half-life ( Fig. 22 Middle F, Fig.24 Middle G). These results indicate that USP20 stabilizes HIF1A by inhibiting its proteasomal degradation. As a deubiquitinase, USP20 may maintain the stability of HIF1A by inhibiting K48-linked polyubiquitination. To test this hypothesis, we co-expressed Flag-USP20, GFP-HIF1A, and Ub (wild type), Ub-K48 (containing only lysine 48 on the ubiquitin molecule), and Ub-K63 (containing only lysine 63 on the ubiquitin molecule) in 293FT cells. Western blot results showed that the ubiquitination level of HIF1A protein in cells was high ( Fig. 22 Middle G, lane 1), while USP20 overexpression significantly reduced the ubiquitination of HIF1A ( Fig. 22In addition, compared with K63-linked polyubiquitination, USP20 overexpression significantly reduced K48-linked polyubiquitination on HIF1A ( Fig. 22 G, lanes 3 and 4). These results suggest that USP20 stabilizes HIF1A by inhibiting K48-linked polyubiquitination. In addition, we performed Cut&Tag experiments to analyze the regulatory roles of USP20 and HIF1A in T-ALL ( Fig.25 The results showed that the DNA binding site of USP20 in J.gamma1 cells significantly overlapped with the HIF1A binding site ( Fig.26 Middle A, Fig.26 Middle B), further verified that USP20 can regulate gene transcription through HIF1A and regulate the expression of genes such as SLC2A1, PER1, CIC, MAZ, PKM, CASP9 and DLG4 ( Fig.26 Middle C, Fig.25 Middle C).

Claims

1. Use of an inhibitor of the deubiquitinase USP20 protein or its encoding gene in the preparation of a drug for treating acute T-lymphocytic leukemia.

2. The use according to claim 1, characterized in that The inhibitor is selected from the following substances: a. a substance that inhibits the enzymatic activity of the deubiquitinating enzyme USP20; or / and b. A substance that reduces or blocks the expression of a gene encoding the deubiquitinating enzyme USP20.

3. The use according to claim 2, characterized in that: The substance that inhibits the enzymatic activity of the deubiquitinating enzyme USP20 is an antibody or inhibitor GSK2643943A of the deubiquitinating enzyme USP20 protein.

4. The use according to claim 2, characterized in that: The substance that reduces or blocks the expression of the gene encoding the deubiquitinating enzyme USP20 is siRNA, antisense nucleic acid or CRISPR / Cas gene editing system for knocking out the gene encoding the deubiquitinating enzyme USP20.

5. Use of a substance for detecting the expression amount of deubiquitinating enzyme USP20 in the preparation of a kit for diagnosis, treatment effect evaluation or prognosis evaluation of acute T lymphocytic leukemia.

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