Use of usp4 inhibitors in the manufacture of a product for the treatment of leukemia

Abstract

The application discloses application of a USP4 inhibitor in preparation of a leukemia treatment product, and belongs to the technical field of biological medicines. The USP4 inhibitor can inhibit the activity of USP4, weaken the protein synthesis of leukemia cells, inhibit the growth of leukemia cells, and further prevent the pathogenic process of leukemia, so that the treatment effect of leukemia is achieved. The application of the USP4 inhibitor in the preparation of the leukemia treatment product is disclosed for the first time.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of USP4 inhibitors in the preparation of products for treating leukemia. Background Technology

[0002] Leukemia is a common hematologic malignancy, listed as one of the top ten most common cancers in my country. Acute leukemia is significantly more prevalent than chronic leukemia (approximately 5.5:1), with acute myeloid leukemia (AML) being the most common type. Finding effective treatment strategies for AML is crucial for alleviating the pressure on my country's healthcare system. AML is challenging to treat and has a high relapse rate. Protein synthesis plays a vital role in leukemia cell growth. USP4 protein, a deubiquitinating enzyme, plays a significant role in the development of various tumors, such as breast cancer, colon cancer, and lung cancer. Therefore, research on inhibiting USP4 activity to reduce protein synthesis in leukemia cells, thereby inhibiting cell growth and halting the progression of leukemia, has significant clinical application value. Summary of the Invention

[0003] The purpose of this invention is to provide a method for inhibiting leukemia cell protein synthesis and weakening leukemia cell growth by inhibiting USP4 protein activity, thereby enabling the application of USP4 inhibitors in the preparation of products for treating leukemia.

[0004] The USP4 inhibitor of this invention includes Vialinin A, the structure of which is shown in formula (I): Formula (I).

[0005] The inventors of this application discovered that USP4 protein levels are elevated in leukemia patients. By knocking down USP4 gene expression and reducing USP4 activity in leukemia cells, the growth of leukemia cells can be inhibited. Furthermore, the USP4 protein activity inhibitor Vialinin A exhibits concentration-dependent inhibition of leukemia cell growth. Inhibition of USP4 activity significantly reduces protein synthesis in leukemia cells and prolongs the survival time of leukemia model mice, indicating that USP4 inhibitors can be used for clinical prevention and treatment of leukemia.

[0006] The USP4 inhibitors involved in this invention include Vialinin A (but are not limited to this one inhibitor), and their use in the preparation of products for treating leukemia is disclosed for the first time. Moreover, they can significantly reduce protein synthesis in human chronic myeloid leukemia cells and acute monocytic leukemia cells. There is no possibility that other compounds could provide any inspiration for this, and they have outstanding substantive characteristics. It is obviously a significant advancement for the prevention and treatment of leukemia. Attached Figure Description

[0007] Figure 1 To analyze the expression level of USP4 protein in human leukemia samples (LAML) using data from public databases (GEPIA databases); Figure 2 Figure showing the cell growth results after knocking down the USP4 gene in the human chronic myeloid leukemia cell line K562. Figure 3 Figure showing the cell growth results after knocking down the USP4 gene in the human acute monocytic leukemia cell line MV4-11. Figure 4 The image shows the growth of human chronic myeloid leukemia cell line K562 cells after 72 hours of treatment with Vialinin A. Figure 5 The image shows the cell growth of human acute monocytic leukemia cell line MV4-11 after 72 hours of treatment with Vialinin A. Figure 6 The results of analyzing protein synthesis in K562 cells, a chronic myeloid leukemia cell line with knocked-down USP4 gene, after incorporation with OP-Puro staining. Figure 7 The results of protein synthesis analysis in MV4-11 cells, a human acute monocytic leukemia cell line with knocked-down USP4 gene, after incorporation with OP-Puro staining. Figure 8 The image shows the results of analyzing protein synthesis in human chronic myeloid leukemia cell line K562 after OP-Puro staining following treatment with Vialinin A. Figure 9 The image shows the results of analyzing protein synthesis in MV4-11 cells after incorporating Vialinin A into the human acute monocytic leukemia cell line MV4-11 and staining with OP-Puro. Figure 10 The survival curve of mice with MLL-AF9 fusion gene-induced leukemia induced by Vialinin A treatment is shown in the figure. Figure 11 Survival curve results of a mouse model of leukemia induced by the MLL-AF9 fusion gene with USP4 gene knockout. Detailed Implementation

[0008] The present invention will be further described below with reference to specific embodiments and accompanying drawings, but the embodiments do not constitute a limitation of the present invention. No limitation in any form is permitted. Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field.

[0009] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0010] Example 1: Analysis of USP4 protein expression levels in leukemia patients using public database data.

[0011] Using data from public databases (GEPIA databases), the inventors of this application compared and analyzed the expression levels of USP4 protein in human leukemia sample data by searching the databases for expression levels in normal and leukemia patient samples. The results are as follows: Figure 1 As shown, the expression level of USP4 protein was significantly increased in human leukemia samples, suggesting that elevated USP4 protein levels may play an important role in the development and progression of human leukemia, and that inhibiting USP4 activity could be used for the treatment of human leukemia.

[0012] Example 2: Knockdown of the USP4 gene in human chronic myeloid leukemia cell line K562 and human acute monocytic leukemia cell line MV4-11.

[0013] Human chronic myeloid leukemia (CML) cell line K562 and human acute monocytic leukemia (AML) cell line MV4-11 were grown in 1640 medium containing 10% FBS. The expression level of USP4 gene in the cells was knocked down using USP4-targeting ShRNA, thus reducing USP4 activity. KML-knocked CML cells and AML cells were grown in 1640 medium containing 10% FBS for 0, 24, 48, and 72 hours. The number of viable cells in both CML cells was counted under a microscope using placental blue staining and a cell counting chamber to analyze cell growth. Results are as follows: Figure 2 and 3 The results showed that knocking down USP4 gene expression and reducing USP4 activity in cells could inhibit the growth of human chronic myeloid leukemia cell line K562 and human acute monocytic leukemia cell line MV4-11. This demonstrates that inhibiting USP4 activity can suppress the growth of leukemia cells.

[0014] Example 3: Human chronic myeloid leukemia cell line K562 and human acute monocytic leukemia cell line MV4-11 were treated with Vialinin A.

[0015] Human chronic myeloid leukemia (CML) cell line K562 and human acute monocytic leukemia (AML) cell line MV4-11 were grown in 1640 medium containing 10% FBS and treated with 0, 2, 5, and 10 μM Viialin A for 0, 24, 48, and 72 hours. The number of viable cells in both CML and AML cells was counted under a microscope using a cell counting chamber after placental blue staining. The growth of CML and AML cells treated with various concentrations of Viialin A was analyzed. Results are as follows: Figure 4 and 5 The results showed that Vialinin A exhibited concentration-dependent inhibition of the growth of human chronic myeloid leukemia cell line K562 and human acute monocytic leukemia cell line MV4-11. This demonstrates that Vialinin A inhibits USP4 activity and can suppress the growth of leukemia cells.

[0016] Example 4: OP-Puro was incorporated into K562 cells (a chronic myeloid leukemia cell line with knocked-down USP4 gene) and MV4-11 cells (a human acute monocytic leukemia cell line).

[0017] Protein synthesis is crucial for the growth and proliferation of leukemia cells and plays a vital role in leukemia treatment. To further verify whether inhibiting USP4 activity can suppress leukemia cell growth by inhibiting protein synthesis, O-Propargyl-Puromycin (OP-Puro), a puromycin analog that can be incorporated into newly synthesized proteins, was used to label newly synthesized proteins in cells. The level of OP-Puro incorporation into cells was analyzed by fluorescence colorimetry, and its fluorescence intensity was detected by flow cytometry, enabling the detection of cellular protein synthesis levels.

[0018] OP-Puro was incorporated into K562 cells (a chronic myeloid leukemia cell line with knocked-down USP4 gene) and MV4-11 cells (a human acute monocytic leukemia cell line). The OP-Puro was then fluoresced using an OP-Puro chromogenic kit. Flow cytometry was used to detect the fluorescence intensity of OP-Puro in both K562 and MV4-11 cells with knocked-down USP4 gene, thus analyzing protein synthesis in these cells. Results are as follows: Figure 6 and 7The results showed that knocking down the USP4 gene to reduce USP4 protein levels in cells significantly reduced the fluorescence intensity of OP-Puro incorporated into human chronic myeloid leukemia cell line K562 and human acute monocytic leukemia cell line MV4-11. This demonstrates that knocking down the USP4 gene to reduce USP4 levels in cells can inhibit protein synthesis in leukemia cells.

[0019] Example 5: Incorporation of OP-Puro into human chronic myeloid leukemia cell line K562 / human acute monocytic leukemia cell line MV4-11 cells when treating with Vialinin A.

[0020] USP4 activity was inhibited using Viialin A (10 μM), and then incorporated into cells via OP-Puro. The OP-Puro-incorporated cells were fluoresced using an OP-Puro chromogenic kit. Flow cytometry was used to detect the fluorescence intensity of OP-Puro-incorporated cells in Viialin A-treated human chronic myeloid leukemia cell line K562 and human acute monocytic leukemia cell line MV4-11, analyzing the protein synthesis status of these cells. Results are as follows: Figure 8 and 9 The results showed that treatment with Vialinin A significantly reduced the fluorescence intensity of OP-Puro incorporated into human chronic myeloid leukemia cell line K562 and human acute monocytic leukemia cell line MV4-11. This demonstrates that inhibiting USP4 activity using Vialinin A can suppress protein synthesis in leukemia cells.

[0021] Example 6: MLL-AF9 fusion gene-induced leukemia model mice were treated with Vialinin A.

[0022] Mice with leukemia induced by the MLL-AF9 fusion gene were administered Vialinin A via intraperitoneal injection at a dose of 5 mg / kg / 2 days, while the control group received an equal volume of phosphate buffer containing the drug solvent. The survival time of the leukemia model mice was statistically analyzed. Results are as follows: Figure 10 The results showed that treatment with Vialinin A to inhibit USP4 activity significantly prolonged the survival time of mice with leukemia.

[0023] Example 7: USP4 gene knockout in a mouse model of leukemia induced by the MLL-AF9 fusion gene.

[0024] Wild-type mouse leukemia cells expressing the MLL-AF9 fusion gene or USP4 gene knockout mouse leukemia cells expressing the MLL-AF9 fusion gene were transplanted into CD45.1 mice to construct MLL-AF9 fusion gene-induced wild-type leukemia mouse models and MLL-AF9 fusion gene-induced USP4 gene knockout leukemia mouse models, respectively. Survival time of the mice was statistically analyzed after transplantation to investigate the effect of USP4 gene knockout on reducing USP4 activity in cells on the survival time of leukemia model mice. Results are as follows: Figure 11 The results showed that USP4 gene knockout, which reduces USP4 activity in cells, significantly prolongs the survival time of leukemia model mice.

[0025] The above results confirm that inhibiting USP4 activity can suppress the growth of leukemia cells and delay the progression of leukemia in mice. Reducing USP4 activity using USP4 inhibitors can be used for the prevention and treatment of leukemia in clinical practice.

[0026] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The use of USP4 inhibitors in the preparation of drugs for treating human chronic myeloid leukemia and / or human acute monocytic leukemia, characterized in that, The USP4 inhibitor is Vialinin A, and the structure of Vialinin A is shown in formula (I): Formula (I) states that the inhibitor inhibits the activity of the USP4 protein, thereby inhibiting the synthesis of leukemia cell proteins and the growth of leukemia cells, ultimately enabling its application in leukemia treatment drugs.

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