Application of PWWP2B as a target in the preparation of drugs for the treatment of leukemia

Abstract

This invention relates to the application of PWWP2B as a target in the preparation of drugs for the treatment of leukemia, belonging to the field of biomedical technology. To address the problem that traditional AML treatment targets lack tumor specificity and fail to meet clinical needs, this invention provides the application of PWWP2B as a target in the preparation of drugs for the treatment of leukemia. This invention demonstrates that PWWP2B is highly expressed in acute myeloid leukemia (AML) cells, and as a molecular marker, it can accurately distinguish AML from healthy individuals or non-leukemia patients. In vivo and in vitro experiments demonstrate that knocking out PWWP2B can significantly inhibit the proliferation and colony formation of AML cells, promote their differentiation and apoptosis, and inhibit the activity of leukemia cells in patients. This invention also reveals for the first time a novel use of the small molecule compound EZM0414 in the treatment of AML and its synergistic effect when used in combination with methyltransferase inhibitors.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of PWWP2B as a target in the preparation of drugs for the treatment of leukemia. Background Technology

[0002] Acute myeloid leukemia (AML) is a malignant hematologic malignancy affecting the bone marrow, with a five-year survival rate still below 50%. Commonly used chemotherapy drugs for AML include anthracyclines, cytarabine, and targeted therapies. However, high rates of chemotherapy resistance and relapse in AML lead to numerous treatment failures. Drug resistance often results from drug-induced gene mutations, epigenetic alterations, or microenvironmental protection. Furthermore, traditional diagnostic and therapeutic targets lack tumor specificity and are prone to damaging normal hematopoietic cells, failing to meet clinical needs. Therefore, developing novel tumor-specific targets is a key direction for improving AML treatment.

[0003] The PWWP2B (PWWP Domain Containing 2B) gene is a protein-coding gene. Previous studies have reported that PWWP2B can form stable NuRD complexes with deacetylase subunits such as MTA1 / 2 / 3, HDAC1 / 2, and RBBP4 / 7, affecting chromatin remodeling and downstream gene transcription, and playing an important role in biological functions such as DNA repair. However, the functional role of PWWP2B in the formation and development of other cancers, including leukemia, remains unclear. Summary of the Invention

[0004] To address the problem that traditional AML diagnostic and therapeutic targets lack tumor specificity and fail to meet clinical needs, this invention provides the application of PWWP2B as a target in the preparation of leukemia therapeutic drugs.

[0005] The technical solution of this invention:

[0006] Application of PWWP2B gene expression inhibitors in the preparation of drugs for the treatment of leukemia.

[0007] Furthermore, the PWWP2B gene expression inhibitor is an artificially designed small nucleic acid gene silencing agent.

[0008] Furthermore, the artificially designed small nucleic acid gene silencing agent is sgRNA, shRNA, or siRNA, wherein the sequence of sgRNA is shown in SEQ ID No:3, SEQ ID No:4, or SEQ ID No:5, the sequence of shRNA is shown in SEQ ID No:6, SEQ ID No:7, or SEQ ID No:8, and the sequence of siRNA is shown in SEQ ID No:11 or SEQ ID No:12.

[0009] Furthermore, the PWWP2B gene expression inhibitor is a small molecule compound EZM0414, with the chemical formula C. 22 H 29 FN4O2, structural formula is

[0010] .

[0011] Furthermore, the leukemia treatment drug is a combination of the small molecule compound EZM0414 and decitabine.

[0012] Furthermore, the leukemia treatment drug is a combination of the small molecule compound EZM0414 and azacitidine.

[0013] Furthermore, the leukemia treatment drug is a combination of a man-designed small nucleic acid gene silencing agent and the small molecule compound EZM0414. The man-designed small nucleic acid gene silencing agent is a targeted therapy sensitizer of the small molecule compound EZM0414. The man-designed small nucleic acid gene silencing agent is sgRNA, shRNA, or siRNA. The sequence of the sgRNA is shown in SEQ ID No:3, SEQ ID No:4, or SEQ ID No:5. The sequence of the shRNA is shown in SEQ ID No:6, SEQ ID No:7, or SEQ ID No:8. The sequence of the siRNA is shown in SEQ ID No:11 or SEQ ID No:12.

[0014] Furthermore, the leukemia mentioned is acute myeloid leukemia.

[0015] The beneficial effects of this invention are:

[0016] This invention confirms that PWWP2B is specifically highly expressed in acute myeloid leukemia (AML) cells. Therefore, it can be used as a molecular marker to accurately distinguish AML from healthy individuals or non-leukemia patients by detecting the mRNA expression level of the PWWP2B gene. Furthermore, high expression of PWWP2B is closely related to disease progression and prognosis, and can serve as an important indicator for monitoring treatment efficacy and predicting relapse, providing a scientific basis for the development of personalized treatment plans. The development of diagnostic agents, kits, or detection methods based on PWWP2B will greatly improve the diagnostic efficiency and accuracy of AML.

[0017] This invention experimentally demonstrates that PWWP2B knockout significantly inhibits the proliferation and colony formation of acute myeloid leukemia cells, while promoting their differentiation and apoptosis. In a mouse model of leukemia, PWWP2B knockout effectively inhibited the growth and spread of leukemia cells and prolonged the survival of mice.

[0018] This invention reveals a novel application of the small molecule compound EZM0414 in the treatment of acute myeloid leukemia (AML). In vitro and in vivo experiments demonstrated that EZM0414 effectively inhibits the proliferation and colony formation of AML cells, promotes cell differentiation and apoptosis, and also inhibits the activity of bone marrow cells in patients. Furthermore, knockout of PWWP2B significantly enhances the sensitivity of AML cells to EZM0414. This invention also confirms that the combination of EZM0414 with commonly used clinical demethylase inhibitors (decitabin / azacitidine) can enhance patients' sensitivity to decitabine, providing a new treatment option for AML. Attached Figure Description

[0019] Figure 1 The image shows a comparison of PWWP2B expression levels in healthy donors and AML patients from different databases in Example 1. A: TCGA_AML, B: GSE33223, C: GSE15061, D: BeatAML.

[0020] Figure 2This is a comparison of PWWP2B expression levels in healthy donors and AML patients with different gene fusion types from the BeatAML database in Example 1. A: Healthy donors (35 cases), B: Acute myeloid leukemia with RUNX1-RUNX1T1 (15 cases), C: Acute myeloid leukemia with CBFB-MYH11 (40 cases), D: Acute myeloid leukemia with myelodysplastic syndromes (143 cases), E: Acute myeloid leukemia with DEK-NUP214 (3 cases), F: Unspecified acute myeloid leukemia (66 cases), G: Acute myeloid leukemia with RPN1-EV11 (12 cases), H: Acute myeloid leukemia with... Acute myeloid leukemia of MLLT3-MLL (11 cases), I: Acute monocytic leukemia (23 cases), J: Acute myeloid leukemia with NPM1 mutation (166 cases), K: Acute mixed phenotype leukemia (T cell / myeloid, non-specific type) (5 cases), L: Acute myelomonocytic leukemia (20 cases), M: Acute myeloid leukemia with mature form (12 cases), N: Acute myeloid leukemia without mature form (17 cases), O: Very poorly differentiated acute myeloid leukemia (13 cases), P: Acute myeloid leukemia with CEBPA mutation (20 cases), Q: Treatment-related acute myeloid leukemia (t-AML) (53 cases), R: Other (11 cases);

[0021] Figure 3 This is a comparison of PWWP2B expression levels in healthy donors and AML patients with different malignant grades and prognoses from different databases in Example 1. A: TCGA_AML, B: GSE6891, C: BeatAML;

[0022] Figure 4 This is a comparison chart of PWWP2B expression levels in patients at different stages of AML development from different databases in Example 1. A: BeatAML, B: TCGA_AML;

[0023] Figure 5 This is a comparison of survival curves for AML patients in different databases with high and low PWWP2B expression in Example 1. A: TCGA_AML, B: TCGA_AML, C: GSE6891 227999_at, D: GSE6891 238051_x_at;

[0024] Figure 6 The image shows the WB diagram of PWWP2B protein expression in each group of AML leukemia cell lines in Example 2. A: Kasumi-1, B: SKNO1.

[0025] Figure 7The image shows a comparison of cell counts in AML cells with PWWP2B knocked out by sgRNA in each group in Example 2. A: Kasumi-1, B: SKNO1, C: MOLM13, D: THP1.

[0026] Figure 8 The image shows a comparison of cell differentiation in AML cells with PWWP2B knocked out by sgRNA in each group in Example 2. A: Giemsa staining images of Kasumi-1 and SKNO1, B: CD11b percentage of Kasumi-1 and SKNO1, C: Giemsa staining images of MOLM13 and THP1, and D: CD11b percentage of MOLM13 and THP1.

[0027] Figure 9 The images show a comparison of clonal formation in AML cells with PWWP2B knockout of sgRNA in each group in Example 2. A: Kasumi-1 and SKNO1 clones, B: Kasumi-1 and SKNO1 colony counts, C: MOLM13 and THP1 clones, D: MOLM13 and THP1 colony counts.

[0028] Figure 10 The following are comparative graphs of apoptosis in AML cells with PWWP2B knockout of sgRNA in each group in Example 2: A: Annexin V / PI scatter plot, B: Kasumi-1 apoptotic cell percentage, C: SKNO1 apoptotic cell percentage.

[0029] Figure 11 This is a comparison of mRNA and protein expression levels in AML cells with shRNA knockdown of PWWP2B and those with PWWP2B overexpression in Example 2. A: mRNA expression level, B: PWWP2B protein expression.

[0030] Figure 12 This is a comparison of cell counts in AML cells with shRNA knockdown of PWWP2B and those with overexpression of PWWP2B in Example 2. A: shPW_1 group, B: shPW_2 group;

[0031] Figure 13 The image shows a comparison of AML cells with shRNA knockdown of PWWP2B and those with overexpression of PWWP2B in Example 2. A: Photograph of colony formation; B: Number of cell colonies.

[0032] Figure 14 The images show a comparison of in vivo fluorescence and total luminescence intensity on the ventral and dorsal sides of the control and interference mouse models in Example 3. A: Ventral fluorescence image, B: Dorsal fluorescence image, C: Ventral total luminescence intensity, D: Dorsal total luminescence intensity.

[0033] Figure 15 This is a comparison of the survival rate, spleen, and abdominal mass of the control and interference mouse models in Example 3. A: Survival rate; B: Spleen and abdominal mass of the mice.

[0034] Figure 16 This is a comparison of the mRNA expression level and cell count of pwwp2b mice in the control group and interference group in Example 3. A: mRNA expression level, B: cell count.

[0035] Figure 17 This is a comparison image of clone formation in the control group and interference group mice in Example 3. A: Photograph of clone formation, B: Number of clones formed;

[0036] Figure 18 This is a comparison of mRNA expression levels, protein expression, and growth rate of bone marrow cells from leukemia patients in the control and interference groups in Example 3. A: mRNA expression level, B: protein expression, C: growth rate.

[0037] Figure 19 The figures are: A: Comparison of DNA methylation levels between patients with high and low PWWP2B expression in the TCGA_AML database in Example 4, and the results of the correlation analysis between PWWP2B expression and patient DNA methylation levels.

[0038] Figure 20 The results of Dot blot analysis at 5mC in AML patients and Kasumi-1 cells after silencing PWWP2B in Example 4 are shown. A: Bone marrow cells from AML patients, B: Kasumi-1 cells.

[0039] Figure 21 The image shows the whole-genome methylation sequencing (WGBS) results after PWWP2B knockout in Kasumi-1 cells in Example 4. A: 5mC trend chart, B: 5mC level distribution chart.

[0040] Figure 22 The image shows the mass spectrometry analysis results of PWWP2B based on immunoprecipitation (IP) and proximity labeling (BioID) technology in Example 4.

[0041] Figure 23 The image shows the results of immunoprecipitation of PWWP2B and UHRF1 in Kasumi-1 and SKNO1 cells in Example 4. A: Kasumi-1-PWWP2B, B: Kasumi-1-UHRF1, C: SKNO1-PWWP2B, D: SKNO1-UHRF1.

[0042] Figure 24The images show the immunofluorescence results of PWWP2B and UHRF1, DNMT1, and H3K36me3 in Example 4, respectively. A: UHRF1, B: DNMT1, C: H3K36me3;

[0043] Figure 25 The results of cut & run of PWWP2B and cut & tag of UHRF1 in Kasumi-1 in Example 4 are: A: heatmap of binding site distribution, B: pie chart of binding site distribution characteristics.

[0044] Figure 26 The image shows a comparison of H3K36me3 protein levels in AML cells treated with different concentrations of EZM0414 in Example 5. A: Kasumi-1, B: SKNO1, C: MOLM13, D: THP1.

[0045] Figure 27 The image shows a comparison of cell counts after treating AML cells with different concentrations of EZM0414 in Example 5. A: Kasumi-1, B: SKNO1, C: MOLM13, D: THP1.

[0046] Figure 28 Comparison of colony formation results of AML cells treated with different concentrations of EZM0414 in Example 5: A: Kasumi-1 colony formation and number; B: SKNO1 colony formation and number.

[0047] Figure 29 Comparison of Giemsa staining and flow cytometry detection of CD11b in AML cells treated with different concentrations of EZM0414 in Example 5; A: Kasumi-1, B: SKNO1, C: MOLM13, D: THP1;

[0048] Figure 30 This is a comparison of apoptosis levels in AML cells treated with different concentrations of EZM0414 in Example 5. A: Kasumi-1, B: SKNO1, C: MOLM13, D: THP1.

[0049] Figure 31 Comparative images of clonogenic formation in bone marrow cells of AE9a leukemia mice treated with different concentrations of EZM0414 in Example 5; A: Clone formation photograph, B: Clone number comparison, C: Giemsa staining photograph;

[0050] Figure 32 Comparison of cell viability of bone marrow cells from AML patients treated with different concentrations of EZM0414 in Example 5; A: Comparison of cell viability detected by CTG; B: Giemsa staining image;

[0051] Figure 33The following are cell count results of shEV and shPWWP2B AML cells treated with EZM0414 in Example 6: A: Cell count results of EZM0414 treatment at different times; B: Cell count results of EZM0414 treatment at different concentrations.

[0052] Figure 34 Comparative images of colony formation in AML cells treated alone and in combination with shPWWP2B and EZM0414 in Example 6; A: Photograph of colony formation; B: Comparison of the number of colonies formed;

[0053] Figure 35 This is a comparison of apoptosis levels in AML cells treated alone and in combination with shPWWP2B and EZM0414 in Example 6. A: Annexin V / PI scatter plot, B: Comparison of apoptotic cell percentage.

[0054] Figure 36 The diagram shows the synergistic tumor suppression analysis of AML cells treated with the combined demethylation inhibitor (DAC / AZA) and EZM0414 in Example 7. A: DAC+EZM0414 in Kasumi-1, B: DAC+EZM0414 in SKNO1, C: DAC+EZM0414 in MOLM13, D: DAC+EZM0414 in THP1, E: AZA+EZM0414 in Kasumi-1, F: AZA+EZM0414 in SKNO1.

[0055] Figure 37 This is a comparison of the synergistic tumor suppression test results of bone marrow cells from AML patients treated with decitabine and EZM0414 in Example 7. A: AML patient 1, B: AML patient 2, C: AML patient 3, D: AML patient 4. Detailed Implementation

[0056] The technical solution of the present invention will be further described below with reference to embodiments, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention. In the following embodiments, the process equipment or apparatus not specifically specified are all conventional equipment or apparatus in the art. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commercially available; unless otherwise specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0057] In all embodiments of this invention, bone marrow cell samples from AML patients were collected by the Department of Hematology, First Affiliated Hospital of Harbin Medical University. Written informed consent was obtained from all subjects, and the research protocol was approved by the Ethics Committee for Scientific and Clinical Research of the First Affiliated Hospital of Harbin Medical University. All animal studies were conducted in accordance with the guidelines approved by the Animal Experiment Ethics Committee of the First Affiliated Hospital of Harbin Medical University.

[0058] Statistical analysis methods for each embodiment of the present invention:

[0059] Graphpad Prism 8.0 was used to plot the experimental data. Quantitative data are expressed as mean ± standard deviation. Unpaired t-tests were used between two groups, one-way ANOVA was used between multiple groups, and repeated measures ANOVA was used between different groups at the same time point. P < 0.05 was considered statistically significant (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

[0060] Example 1

[0061] This embodiment verifies that the expression of the PWWP2B gene is significantly increased in acute myeloid leukemia, and also verifies the relationship between PWWP2B expression and the prognosis of leukemia patients; the higher the PWWP2B expression, the worse the prognosis of the patient.

[0062] First, this embodiment analyzed the expression of the PWWP2B gene in healthy individuals and AML patients using four leukemia databases: TCGA_AML, GSE33223, GSE15061, and BeatAML. The results are as follows: Figure 1 As shown, in four different public databases, PWWP2B expression was significantly elevated in AML patients compared to healthy individuals. This embodiment also analyzed PWWP2B expression in healthy donors and patients with different types of AML, with results as follows: Figure 2 As shown, PWWP2B expression was significantly elevated in patients with various types of AML compared to healthy individuals. These results suggest that PWWP2B may serve as a potential indicator for the diagnosis of AML.

[0063] This embodiment further analyzed the correlation between PWWP2B expression and the malignancy of AML, and analyzed its expression in patients with different malignancy levels and different stages of development. Figure 3 As shown, the expression of PWWP2B mRNA in AML patients with different genetic risks from three databases was analyzed. The results showed that the expression of PWWP2B in patients with intermediate and high genetic risk was higher than that in low-risk AML patients. Figure 4 The expression levels of PWWP2B at different stages of AML development, and the effects of PWWP2B on recovery after treatment, such as... Figure 4 The relief group of A, Figure 4 After treatment with B, the expression level of PWWP2B in the acute myeloid leukemia group decreased significantly.

[0064] This embodiment further analyzed the survival curves of AML patients in the high and low expression groups of PWWP2B in the TCGA_AML and GSE6891 databases. The results are as follows: Figure 5 As shown. Figure 5 A represents disease-free survival in the TCGA_AML database, with a hazard ratio (HR) of 1.564 (1.008–2.427) and a p-value of 0.0404. Figure 5 B represents the total lifetime of the TCGA_AML database, with a hazard ratio (HR) of 1.472 (1.017 - 2.131) and a p-value of 0.0375. Figure 5 C represents the event-free lifetime of the GSE6891 227999_at database, with a hazard ratio (HR) of 1.38 (1.04-1.83) and a p-value of 0.0026. Figure 5 D represents the event-free lifetime of the GSE6891 238051_x_at database, with a hazard ratio (HR) of 1.51 (1.13-2.03) and a p-value of 0.0005. Figure 5 C and Figure 5 The number of at-risk individuals in the low-expression and high-expression groups of D is shown in Table 1.

[0065] Table 1

[0066]

[0067] like Figure 5 As shown in Table 1, AML patients with high expression of PWWP2B have significantly shorter survival.

[0068] The prognostic results of the PWWP2B gene in multiple independent clinical cohorts (including databases such as GEO, TCGA, and TARGET), the hazard ratios and their 95% confidence intervals calculated by the multivariate Cox proportional hazards model, are shown in Table 2.

[0069] Table 2

[0070]

[0071] Table 2 data shows that prognostic analyses of multiple independent clinical cohorts demonstrate the risk of PWWP2B high expression in AML patients. Combined with... Figure 4 , Figure 5 As shown in Table 2, high PWWP2B expression levels are closely related to reduced survival rates in leukemia patients.

[0072] The data comparison above shows that PWWP2B is specifically highly expressed in AML cells, confirming that PWWP2B can serve as a potential indicator for the clinical diagnosis of acute myeloid leukemia (AML), assessment of malignancy, and clinical prognosis. Using it as a molecular marker, the mRNA expression level of the PWWP2B gene can be detected to accurately diagnose AML. Furthermore, high expression of PWWP2B is closely related to disease progression and prognosis, and can serve as an important indicator for monitoring treatment efficacy and predicting relapse, providing a scientific basis for the development of personalized treatment plans. The development of diagnostic agents, kits, or detection methods based on PWWP2B will greatly improve the diagnostic efficiency and accuracy of AML.

[0073] Furthermore, this embodiment provides a leukemia diagnostic preparation containing reagents for detecting the mRNA expression level of the PWWP2B gene. The reagents for detecting the mRNA expression level of the PWWP2B gene include primers for detecting the PWWP2B gene:

[0074] The sequence of the upstream primer is shown in SEQ ID No:1: 5'-GGGACATCGTCTGGGGTAAG-3';

[0075] The sequence of the downstream primer is shown in SEQ ID No:2: 5'-TCGGAGAACCAAACCACGAG-3';

[0076] Example 2

[0077] This embodiment verifies that PWWP2B gene knockout inhibits the occurrence and development of acute myeloid leukemia.

[0078] To investigate the function of the PWWP2B gene, this embodiment knocked out PWWP2B in four leukemia cell lines, including leukemia cells with AML1-ETO fusion type: Kasumi-1 and SKNO1; and leukemia cells with MLL-AF9 fusion type: MOLM13 and THP1.

[0079] The method for constructing a stable PWWP2B gene knockout cell line is as follows:

[0080] (1) Cell culture: HEK293T cells were cultured in DMEM cell culture medium containing 10% FBS, and leukemia cell lines were cultured in RPMI 1640 cell culture medium containing 10% FBS. Both culture media were supplemented with 1% penicillin and streptomycin. After the cells and culture medium were thoroughly mixed, they were placed in cell culture dishes. Finally, the culture dishes were placed in a cell culture incubator at a constant temperature of 37°C and containing 5% carbon dioxide for culture.

[0081] (2) Vector, virus packaging and establishment of stable cell lines

[0082] The sgRNA sequences targeting different regions of the PWWP2B gene and the control LacZ sequence are as follows:

[0083] sgPW_1 shown in SEQ ID No:3: 5'-TGACAGGGGACTCATCGACC-3';

[0084] SEQ ID No:4 shows sgPW_2: 5'-GGACCATCAAGCGCACGCGG-3';

[0085] SEQ ID No:5 shows sgPW_3: 5'-ACGACCTGCTCCACCCGCAC-3';

[0086] LacZ shown in SEQ ID No:13: 5'-TGCGAATACGCCCACGCGAT-3'.

[0087] The base sequences shown above were chemically synthesized by Anhui General Biotechnology Co., Ltd., with restriction enzyme sites introduced at both ends. sgPW_1, sgPW_2, sgPW_3, and LacZ were cloned into the lentiviral vector lentiCRISPRv2 (Addgene) using BsmBI enzyme. In the establishment of stable cell lines, the lentiviral expression vectors, packaging plasmids pMD2.G and psPAX2 were simultaneously transfected into HEK293T cells using PEI transfection reagent. The supernatant viral solution was collected at 48h and 72h after transfection, filtered, and 8 μg / mL polybrene was added to infect the target AML cells. Positive cells were selected with the corresponding puromycin 48h after infection.

[0088] (3) Western spectroscopy was used to detect protein expression levels in stable cell lines.

[0089] Collect the precipitate from each group of stable transfected cells, wash once with PBS, and prepare cell lysis buffer using RIPA and protease inhibitors to ensure complete cell lysis. Determine the total protein concentration according to the BCA kit instructions. Add the same mass of protein to 5X SDS loading buffer based on the calculated protein concentration and boil at 95°C for 5 minutes. Select a suitable gel (7.5%, 10%, or 12.5%) and calculate the required protein volume for 20-30 μg of protein based on the calculated protein concentration. Load the sample. After loading, perform electrophoresis at a constant voltage of 80 V. Once the marker bands are clearly separated, perform electrophoresis at a constant voltage of 120 V. Remove the glass plate to expose the gel, cut the PVDF membrane, and activate it by soaking it in methanol. First, place a layer of filter paper, then place the gel in the middle of the filter paper, cover with the activated PVDF membrane, and finally cover with another layer of filter paper. Place the gel in the electroporation tank. Fill with pre-cooled electroporation buffer, maintain a constant current of 300 mA, and electroporate for 2 hours. After electroporation, the membrane was incubated in 5% skim milk at room temperature for 1 hour. After washing the membrane with TBST for 30 minutes, it was incubated overnight with primary antibody at 4°C in a shaker. The PVDF membrane was then removed, washed with TBST for 30 minutes, and incubated with secondary antibody at room temperature for 1 hour. After washing the membrane again with TBST for 30 minutes, it was placed on an exposure unit, ECL developer was added, the exposure time and sensitivity were set, the membrane was exposed, and the image was saved.

[0090] The results are as follows Figure 6 As shown, compared with the control group LacZ, the expression of PWWP2B protein in the PWWP2B knockout AML leukemia cell lines in each group almost disappeared.

[0091] This embodiment further conducts functional experiments on AML cells after PWWP2B knockout:

[0092] I. Cell Counting Experiment

[0093] Collect stable cells from each group, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend in 1 ml of complete culture medium, and aspirate 30 μl for cell counting. Add an equal volume of 0.4% trypan blue staining solution to the 30 μl cell suspension and vortex thoroughly. Use a 10 μl pipette to transfer the mixture into a cell counting chamber and count under low magnification. The results are shown in the figure. Figure 7 .

[0094] II. Giemsa staining and flow cytometry for cell differentiation detection

[0095] Collect stable cells from each group, smear the suspension onto a glass slide, fix with anhydrous methanol and dry, add working solution for staining, gently rinse with pH 7.0 buffer, and then air dry the slides for microscopic observation.

[0096] Collect stable cells from each group, wash the cells twice with pre-chilled PBS, and centrifuge at 500g, 4℃ for 5 minutes. Resuspend the cells in 100μl PBS, add 5μl CD11b antibody, mix well, and incubate at room temperature for 15 minutes. After incubation, centrifuge to remove the supernatant, resuspend the cell pellet in PBS, and analyze as soon as possible. Results are shown below. Figure 8 .

[0097] III. Soft agar colony formation experiment

[0098] Prepare 3% agarose, autoclave, and place in a 42℃ water bath. Preheat the complete culture medium in a 37℃ water bath. Mix 3% agarose and preheated complete culture medium at a 1:3 ratio to achieve an agarose concentration of 0.75%. After thorough mixing, add 1.5 ml of the mixture to each well of a six-well plate to prepare the lower gel. Allow to solidify at room temperature. Next, mix 3% agarose and preheated complete culture medium at a 1:7 ratio to achieve an agarose concentration of 0.36%. After thorough mixing, prepare the upper gel. Add 5000 cells per well according to the stable cell concentration obtained from cell counting for each group. Mix thoroughly and spread on the lower agar, adding 2 ml per well. Gently place the six-well plate in a cell culture incubator. Add culture medium every 3 days, 200-300 μl each time. After 10-14 days, add 300 μl of dye to each well, incubate overnight at 37℃, count the number of colonies formed, and photograph the results. See [see attached image]. Figure 9 .

[0099] IV. Flow Cytometry Detection of Apoptosis

[0100] Observe cell state under a microscope before collecting cells. Collect stable cells from each group and centrifuge at 500g, 4℃ for 5 minutes. Discard the supernatant, add pre-chilled PBS to wash cells, centrifuge at 500g, 4℃ for 5 minutes, and wash 2-3 times. According to the apoptosis detection instructions, discard the supernatant and resuspend cells in 100μl of pre-chilled Annexin V binding buffer. Add 5μl Annexin V-FITC and 5μl PI respectively, and mix gently. Incubate at room temperature in the dark for 15 minutes. After incubation, add 400μl of pre-chilled Annexin V binding buffer to the mixture. Place the samples on ice in the dark and detect apoptosis within 1 hour using flow cytometry. Results are shown in the figure. Figure 10 .

[0101] from Figures 7-10 As can be seen, PWWP2B knockout inhibited cell proliferation and colony formation, and promoted cell differentiation and apoptosis. Therefore, this embodiment confirms the anti-cancer function of PWWP2B knockout through the above cell experiments.

[0102] To further evaluate the cancer-promoting function of PWWP2B, this embodiment first constructed a stable cell line with PWWP2B knockdown using an shRNA plasmid targeting the PWWP2B UTR region. Then, by transfecting the PWWP2B knockdown cell line with a plasmid overexpressing the PWWP2B CDS region, changes in cell function were observed.

[0103] In this embodiment, the shRNA targeting the UTR region of PWWP2B mRNA was synthesized by Anhui General Biotechnology Co., Ltd., and the specific sequence is as follows:

[0104] shPW_1 shown in SEQ ID No:6: 5'-GGAAACCGGGCTTCAACAGTA-3';

[0105] SEQ ID No:7 shows shPW_2: 5'-ACCGGGCTTCAACAGTACAAG-3';

[0106] The human PWWP2B CDS fragment was synthesized by Anhui General Biotechnology Co., Ltd., and its sequence is shown in SEQ ID No:14. shPW_1 and shPW_2 were cloned into the lentiviral vector pLKO.1 (Addgene) using EcoRI and Age I, respectively. The lentiviral expression vector, packaging plasmid pMD2.G, and psPAX2 were simultaneously transfected into HEK293T cells using PEI transfection reagent. Supernatant viral fluid was collected at 48 h and 72 h post-transfection, filtered, and then infected with 8 μg / mL polybrene to infect Kasumi-1 cells. 48 h after infection, stable PWWP2B knockdown cells were selected using the appropriate antibiotic (G418).

[0107] The PWWP2B CDS fragment encoding the human gene was cloned into the pLVX_puro lentiviral vector using Xho I and EcoRI to construct an overexpression plasmid for the PWWP2B CDS region. The lentiviral expression vector, packaging plasmid pMD2.G, and psPAX2 were simultaneously transfected into HEK293T cells using PEI transfection reagent. Supernatant viral fluid was collected at 48 h and 72 h post-transfection, filtered, and then infected with 8 μg / mL polybrene to achieve stable PWWP2B knockdown. Stable transfected cells were selected using puromycin 48 h after infection.

[0108] The results are as follows Figure 11 As shown, PCR and Western blotting verified the restoration of PWWP2B expression. Figure 12 , Figure 13The results showed that the restoration of PWWP2B expression led to the recovery of cell proliferation and colony formation capabilities. These results further confirm that PWWP2B plays a pro-cancer role in leukemia and can serve as a potential target for leukemia treatment.

[0109] Example 3

[0110] This embodiment verifies that PWWP2B gene interference inhibits the occurrence and development of leukemia in vivo.

[0111] To investigate the effect of interfering with PWWP2B expression on the growth and spread of leukemia cells in vivo, this embodiment constructs a leukemia cell-derived xenograft mouse model (CDX) by injecting luciferase-tagged leukemia cells into the tail vein of immunodeficient mice. Finally, the growth and spread of leukemia cells in mice are tracked using a small animal imaging system.

[0112] The method for constructing a leukemia cell-derived xenograft mouse model (CDX) is as follows:

[0113] Following the method described in Example 2, viruses packaged with pLKO plasmids containing the shPW_1 sequence shown in SEQ ID No:6 and the shPW_2 sequence shown in SEQ ID No:7 were used to infect Kasumi-1 cells carrying luciferase. After screening with antibiotics (G418), the luminescence intensity was verified by in vitro bioluminescence imaging (BLI). Subsequently, 1×10 6 ~3×10 6 Luciferase-positive cells in the logarithmic growth phase were injected via tail vein into 6-8 week old immunodeficient mice (NCG). The EV control group consisted of mice that received intravenous injection of PWWP2B cells without Kasumi-1 knockout.

[0114] These cells were allowed to grow in mice, and leukemia cells were observed to spread and proliferate within the mice after 1-3 weeks. The cells typically expand in the bloodstream or specific sites such as the spleen and bone marrow, thus mimicking human leukemia. Tumor growth was monitored weekly using a small animal in vivo imaging system (IVIS), 10 minutes after intraperitoneal injection of 150 mg / kg D-fluorescein (dissolved in PBS). Results are shown below. Figure 14 Compared with the control group, the growth and spread rate of PWWP2B-interfered cells in mice was significantly reduced.

[0115] This embodiment statistically analyzed the survival time of three groups of mice, such as... Figure 15 As shown in Figure A, the results indicate that the survival time of mice affected by PWWP2B interference was significantly prolonged. Figure 15 B shows that after dissection, the spleen volume of mice in the PWWP2B interference group was significantly smaller. (Summary) Figure 14 , Figure 15 The results showed that PWWP2B interference significantly affected the growth and spread of leukemia cells in vivo and the survival of mice. This further confirms that targeting PWWP2B is a feasible strategy for treating leukemia.

[0116] Based on this, this embodiment also establishes an ex vivo operated xenograft mouse model of leukemia:

[0117] Bone marrow cells were harvested from adult wild-type (WT)C57 mice. Mouse-derived pwwp2b was knocked down using the shpw_1 sequence (SEQ ID No:9) and shpw_2 sequence (SEQ ID No:10), and AE9a was infected with lentivirus. Successfully infected mouse bone marrow cells were screened and transplanted into immunodeficient mice. The growth and spread of the treated cells in the mice were observed.

[0118] SEQ ID No:9 shows shpw_1:5'-AGAAGACAGCGCTGTCATAG-3';

[0119] SEQ ID No:10 shows shpw_2:5'-GCAAACAGAAACAGGGCTTTC-3';

[0120] like Figure 16 As shown in Figure A, the designed targeted mouse pwwp2b successfully interfered with its expression in cells. Figure 16 B Figure 17 The figures show cell counts and colony formation results after pwwp2b interference in mice. In summary, these results indicate that interfering with pwwp2b expression in leukemia mice can inhibit cell proliferation and colony formation. These results confirm the feasibility of targeting pwwp2b for the treatment of mouse leukemia.

[0121] To further evaluate the effect of targeting PWWP2B on leukemia patients, this embodiment used siRNA to interfere with PWWP2B expression in bone marrow cells of leukemia patients. The specific sequence of the siRNA is as follows:

[0122] SEQ ID No:11 shows siPW_1: 5'-CGGUCAUCAAGAUCUCCUAtt-3';

[0123] SEQ ID No:12 shows siPW_2: 5'-CCUGCUGGACUGCACGAAAtt -3';

[0124] To comply with WIPO ST.26 standards, uracil in RNA is represented by T replacing U at positions 4, 7, 13, 15, and 18 at the 5' end, as shown in SEQ ID NO:11, and uracil in RNA is represented by T replacing U at positions 3, 6, and 11 at the 5' end, as shown in SEQ ID NO:12.

[0125] like Figure 18 As shown in Figures AB, PCR and Western blotting verified that the designed siPWWP2B significantly interfered with the expression of PWWP2B. Figure 18 The results showed that bone marrow cell proliferation was slowed in leukemia patients after PWWP2B expression interference. These results further confirm the credibility of targeted PWWP2B therapy for leukemia in clinical practice.

[0126] Based on the above in vivo and in vitro experimental results, PWWP2B is confirmed as a potential target for the treatment of leukemia.

[0127] Example 4

[0128] This embodiment investigates the mechanism by which PWWP2B gene knockout inhibits the occurrence and development of leukemia.

[0129] To investigate the mechanism by which PWWP2B knockout inhibits the development and progression of leukemia cells, this embodiment analyzes DNA methylation data from TCGA_AML, such as... Figure 19 As shown in Figure A, AML patients were divided into two groups based on PWWP2B expression. Patients with high PWWP2B expression had significantly higher DNA methylation levels than those with low PWWP2B expression. Figure 19 B showed that PWWP2B expression was positively correlated with the patient's DNA methylation level in AML patients.

[0130] PWWP2B in AML patient bone marrow cells and Kasumi-1 cells were silenced using siPW_1 and siPW_2, respectively. Dot blot analysis was performed on the silenced cells, and the results are as follows: Figure 20 As shown, Dot blot confirmed that DNA methylation levels decreased after PWWP2B knockout.

[0131] Whole-genome methylation sequencing of Kasumi-1 cells after PWWP2B knockout yielded the following results: Figure 21 As shown, whole-genome methylation sequencing results reveal the specific levels of methylation decrease.

[0132] To further investigate the reasons for the changes in DNA methylation levels caused by PWWP2B knockout, this embodiment employed immunoprecipitation-mass spectrometry and biotin-nearest neighbor labeling-mass spectrometry techniques, such as... Figure 22As shown in Table 3, mass spectrometry detected the interaction between PWWP2B and DNA methylation-related proteins UHRF1 and DNMT1.

[0133] Table 3

[0134]

[0135] In this example, PWWP2B and UHRF1 were co-precipitated in Kasumi-1 and SKNO1 cells, and the results are as follows: Figure 23 As shown, the results of the immunoprecipitation further confirmed the protein interaction between PWWP2B and UHRF1.

[0136] Immunofluorescence of PWWP2B with UHRF1, DNMT1, and H3K36me3 yielded the following results: Figure 24 As shown, PWWP2B is colocalized in the nucleus with UHRF1, DNMT1 and H3K36me3, respectively. Figure 25 The cut & run results of PWWP2B and the cut & tag results of UHRF1 show that PWWP2B and UHRF1 can bind to gene promoters.

[0137] Based on the above results, this embodiment confirms that PWWP2B can influence gene transcription levels by recruiting UHRF1 and DNMT1 to gene promoters.

[0138] Example 5

[0139] This example examines the efficacy of EZM0414 in treating acute leukemia.

[0140] PWWP2B is a specific recognizer of H3K36me3. The small molecule compound EZM0414 can specifically inhibit H3K36me3 levels. In this example, Kasumi-1 and SKNO1 cells were treated with EZM0414 at concentrations of 0, 200, and 500 nM, while MOLM13 and THP1 cells were treated with EZM0414 at concentrations of 0, 500, 1000, and 2000 nM. The inhibitory effect of different concentrations of EZM0414 on H3K36me3 levels in AML cells was investigated, and the results are as follows: Figure 26 As shown, EZM0414 can inhibit H3K36me3 expression in a concentration-dependent manner.

[0141] This embodiment further investigated the effects of EZM0414 on AML cells in cell proliferation, colony formation, cell differentiation, and apoptosis. The results are as follows: Figures 27-30 As shown, EZM0414 can inhibit AML cell proliferation and colony formation, and promote cell differentiation and apoptosis in a concentration-dependent manner.

[0142] This example investigated the anticancer effect of EZM0414 on mouse leukemia cells. AE9a leukemia mouse bone marrow cells were treated with EZM0414 at concentrations of 0, 1000, and 2000 nM for 7 days (cell concentration 100,000 / mL). The results are as follows: Figure 31 As shown, EZM0414 can inhibit the clonogenic ability of mouse leukemia cells and promote their differentiation. These results indicate that EZM0414 has an anti-cancer effect on mouse leukemia cells.

[0143] Next, this example examines the inhibitory effect of EZM0414 on bone marrow cells of AML patients.

[0144] AML patient bone marrow cells were treated with EZM0414 at concentrations of 0, 1000, 2000, 4000, and 8000 nM for 7 days at a cell concentration of 10,000 / 100 μL. Cell viability was detected using CellTiter-Glo (CTG) reagent after 7 days, and the data were obtained and analyzed using an ELISA reader.

[0145] The specific method for the chemiluminescence cell viability assay (CTG) is as follows:

[0146] Prepare the CTG solution in advance. Use a pipette to thoroughly mix the treated cells in a 96-well plate. Add 50 μl of the mixture to a light-protected 96-well plate. Prepare a mixture of 20 μl CTG solution / well and 30 μl PBS solution / well, mix thoroughly, and add 50 μl of the CTG mixture to each well. Incubate on a shaker at room temperature for 5 minutes, then blow off any bubbles and let stand for 5 minutes. Perform analysis.

[0147] like Figure 32 As shown in Figure A, EZM0414 significantly inhibited the activity of bone marrow cells in AML patients, and the inhibitory effect gradually increased with increasing EZM0414 concentration. Figure 32 Results B indicate that EZM0414 can promote the differentiation of bone marrow cells in AML patients. Figure 32 The results indicate that EZM0414 has potential therapeutic effects on AML patients.

[0148] In summary Figures 27-32 The results showed that EZM0414 has a significant anti-cancer effect on AML, and this effect is concentration-dependent. EZM0414 may provide a new treatment option for AML.

[0149] Example 6

[0150] This embodiment examines the effect of PWWP2B interference on the sensitivity of EZM0414.

[0151] To investigate the combined effects of PWWP2B interference and EZM0414, this embodiment constructed PWWP2B interference group cells using shPW_1 (SEQ ID No: 6), shPW_2 (SEQ ID No: 7), and shPW_3 (SEQ ID No: 8), respectively. The control group and PWWP2B interference group cells were treated with concentrations of 0, 40, 100, and 200 nM for 9 days, and with 200 nM EZM0414 for 0, 3, 6, 9, and 12 days. The concentration of the PWWP2B interference group cells was 10,000 / mL.

[0152] shPW_3 shown in SEQ ID No:8: 5'-ACTGAGATTTAATCGTAAGAA-3';

[0153] The results are as follows Figure 33 As shown, compared with the control group cells, the PWWP2B interference group cells showed significantly enhanced sensitivity to EZM0414. Figure 34 , Figure 35 The results of clonogenic assays and apoptosis detection showed that the combined effect of PWWP2B interference and EZM0414 on inhibiting AML cells was stronger than that of single-factor treatment. These results confirm that PWWP2B interference can enhance the sensitivity of AML cells to EZM0414.

[0154] Example 7

[0155] This example investigated the synergistic effect of EZM0414 and commonly used clinical demethylase inhibitors (decitabine / azacitidine).

[0156] To further optimize the clinical treatment efficacy of AML and enhance the anti-cancer effect of EZM0414, this embodiment used EZM0414 at concentrations of 0, 250, 500, 1000, 2000, 4000, and 8000 nM, and decitabine (DAC) at concentrations of 0, 31.25, 62.5, 125, 250, 500, and 1000 nM, or azacitidine (AZA) at concentrations of 0, 250, 500, 1000, 2000, and 4000 nM, to treat AML cells at a cell concentration of 1000 / mL. Cell viability was detected by CTG.

[0157] Figure 36 The ZIP synergy scores were 18.51, 11.19, 19.32, 10.78, 9.93 and 20.52, respectively, indicating that EZM0414 and DAC / AZA can synergistically suppress tumors in different AML cells (ZIP Synergy Score>10).

[0158] Next, in this embodiment, DAC was used in combination with DMSO and 4 μM EZM0414 at concentrations of 0, 31.25, 62.5, 125, 250, 500, 1000, and 2000 nM in bone marrow cells from four AML patients. The results are as follows... Figure 37 As shown in Table 4, CTG results indicate that EZM0414 treatment enhances the sensitivity of AML patients to decitabine.

[0159] Table 4

[0160]

[0161] In conclusion, the combination of EZM0414 and commonly used clinical demethylase inhibitors (decitabine / azacitidine) can provide a new option for the clinical treatment of AML.

Claims

1. Use of a PWWP2B gene expression inhibitor in the preparation of a drug for the treatment of leukemia, characterized in that, The leukemia is acute myeloid leukemia, and the PWWP2B gene expression inhibitor is an artificially designed small nucleic acid gene silencing agent or a small molecule compound EZM0414; the artificially designed small nucleic acid gene silencing agent is sgRNA, shRNA or siRNA, the sequence of the sgRNA is shown in SEQ ID No: 3, SEQ ID No: 4 or SEQ ID No: 5, the sequence of the shRNA is shown in SEQ ID No: 6, SEQ ID No: 7 or SEQ ID No: 8, and the sequence of the siRNA is shown in SEQ ID No: 11 or SEQ ID No: 12; the chemical formula of the small molecule compound EZM0414 is C 22 H 29 FN4O2, and the structural formula is 。 2. The use of the PWWP2B gene expression inhibitor according to claim 1 in the preparation of a medicament for the treatment of leukemia, characterized in that, The leukemia treatment drug is a combination of a small molecule compound EZM0414 and decitabine.

3. The use of the PWWP2B gene expression inhibitor according to claim 1 in the preparation of a medicament for the treatment of leukemia, wherein the PWWP2B gene expression inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof. The leukemia treatment drug is a combination of a small molecule compound EZM0414 and azacitidine.

4. The use of the PWWP2B gene expression inhibitor according to claim 1 in the preparation of a medicament for the treatment of leukemia, characterized in that, The leukemia treatment drug is a combination of an artificially designed small nucleic acid gene silencing preparation and a small molecule compound EZM0414, wherein the artificially designed small nucleic acid gene silencing preparation is a targeted treatment sensitizer for the small molecule compound EZM0414, and the artificially designed small nucleic acid gene silencing preparation is an shRNA, and the sequence of the shRNA is shown in SEQ ID No: 6, SEQ ID No: 7 or SEQ ID No: 8.

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