CN111840562A

CN111840562A - Application of USP7 inhibitor in ABC-DLBCL treatment drug

Info

Publication number: CN111840562A
Application number: CN202010859627.8A
Authority: CN
Inventors: 吴圆圆; 范义辉
Original assignee: Nantong University
Current assignee: Nantong University
Priority date: 2020-08-24
Filing date: 2020-08-24
Publication date: 2020-10-30

Abstract

The invention discloses an application of a USP7 inhibitor in a medicine for treating ABC-DLBCL, which is used for researching the expression condition of USP7 in a DLBCL cell line, and then the database analysis shows that the USP7 small-molecule inhibitor is used for inhibiting the activity of USP7, the CCK-8 method is used for observing the influence of cell proliferation, PI staining and flow cytometry on ABC-type DLBCL cell apoptosis and the influence on the ABC-type DLBCL BCR signal path, USP7 is the most widely researched ubiquitin-specific protease at present, the research shows that USP7 participates in regulating the activity and the function of various protein substrates of cells, and USP7 abnormal expression or activity change has important effects on the occurrence and development of diseases and tumors, so that the USP7 inhibitor has good application prospect and market competitiveness, and has good promotion effect on the improvement of China's livelihood and society.

Description

Application of USP7 inhibitor in ABC-DLBCL treatment drug

Technical Field

The invention belongs to the field of medicines, and particularly relates to an application of a USP7 inhibitor in an ABC-DLBCL treatment medicine.

Background

Lymphomas are a group of monoclonal proliferative diseases originating from the lymphohematopoietic system, and are classified into Hodgkin's Lymphoma (HL) and non-Hodgkin's lymphoma (NHL), and about 8.4 million people are diagnosed as lymphomas each year in our country. non-Hodgkin's lymphoma (NHL) is a type of hematological malignancy with significant heterogeneity, divided into B-cell NHL and T/NK-cell NHL by histopathological type. Among the B-NHLs, the most common subtype is diffuse large B-cell lymphoma (DLBCL), with an incidence rate of 45.8% of NHLs. With the widespread use of the rituximab-based immunotherapy regimen (R-CHOP) for the treatment of DLBCL, more than half of patients are cured, but more than 30% of patients still face the risk of relapse, and patients who are not susceptible to R-CHOP chemotherapy will eventually die from DLBCL disease progression, with a 3-year survival rate of less than 60% for high-risk DLBCL cases, to be further improved.

Gene expression profiling and DNA sequencing have deepened our understanding of DLBCL biological properties in the years. DLBCL can be classified into two subtypes, namely, germinal center B-cell-like (GCB) DLBCL and activated B-cell-like (ABC) DLBCL, which have different cell signal activation transduction pathways and different prognosis after standard treatment. The GCB DLBCL is derived from a lymphoid follicle germinal center and expresses CD10, LMO2 and BCL 6. 30% -40% of GCB type DLBCL has t (14, 18), 30% has c-rel gene amplification, 20% can generate histone methyltransferase EZH2 gene mutation, and 10% PTEN gene deletion; EZH2 mediates the generation of GCB-type DLBCL in cooperation with BCL6 gene. The ABC type DLBCL is derived from the plasmablast stage of B cells, and is characterized by the gene pathology that the NF ⁃ kB pathway is continuously activated, so that the tumor cells are actively proliferated and are anti-apoptotic. Most ABC-type DLBCL rely on a chronically activated B-cell receptor (BCR) signaling pathway, which is mediated by B-cell receptors and downstream kinases. These kinases include SYK, PI3K, BTK, and PKC β. Mutations in CD79A/CD79B are present in about 20% of cases; 10% of ABC type DLBCL has CARD11 molecular mutation which can activate NF ⁃ kB pathway independently of upstream signals; deregulation of the negative regulator a20 encoding NF ⁃ kb can occur in 24% of ABC-type DLBCL. In addition, the most critical gene mutation, MYD88, in the TLRs signaling pathway can occur in 30% of ABC-type DLBCLs. However, the specific mechanism of induction of BCR signaling pathway activation remains to be elucidated. From the known data at present, the GCB type DLBCL has low malignancy degree and mainly depends on a PI3K/AKT/MTOR pathway, while the ABC type DLBCL has strong invasiveness and depends on a plurality of growth signal pathways, such as BCR/NF ⁃ kB, JAK-STAT signal pathways and the like. At present, most scholars consider that the prognosis of GCB type DLBCL is obviously better than that of ABC type GCB, and the GCB is an independent prognostic factor.

In recent years, targeted drugs directed to certain molecules in the signaling pathway have been developed in succession or have entered preliminary clinical trials, bringing new promise for the treatment of DLBCL. For example, the inhibitor Ibrutinib of BTK in BCR pathway obtains satisfactory curative effect in ABC type DLBCL which is difficult to cure, and phase I/II clinical research shows that the effective rate can reach 40%. However, because the BTK inhibitor only inhibits BTK molecules downstream of BCR signals, DLBCL especially uses ABC type DLBCL continuously activated by NF ⁃ kB to relate to the expression abnormality of a wide range of signal molecules, the BTK inhibitor only has curative effect on a small part of patients and is easy to generate drug resistance. Given the complexity of the molecular pathway network, a single targeted drug therapy may not be able to exert a long-term and comprehensive anti-tumor effect. Drugs that comprehensively inhibit activation of the BCR signaling pathway remain to be developed.

Ubiquitin-specific protease 7 (USP 7) is currently the most widely studied ubiquitin-specific protease, originally identified from the Herpes Simplex Virus (HSV) protein ICP0 interacting protein and hence also known as herpes virus-associated ubiquitin-specific protease (Hausp). USP7 can specifically hydrolyze ester bond, peptide bond or isopeptide bond formed between the C-terminal of ubiquitin and target protein, so that ubiquitin can be separated from the target protein, thereby affecting the stability, cell location and activity of the target protein. It has the effects of regulating cell cycle process, protein degradation, gene expression, DNA repair and apoptosis. The research finds that the USP7 is involved in the generation and progression of tumors through the interaction with oncoprotein and oncostatin. There are a number of reports in the literature of USP7 on tumorigenesis, which is manifested by its interaction with oncoproteins, deubiquitination to stabilize them, or by inhibition of oncogenic effects of oncoprotein function through interaction with oncoproteins, and a few reports in the literature that USP7 has tumor-inhibiting effects, mainly manifested by its interaction with oncoproteins to stabilize oncoproteins or USP7 to exert gene stability. USP7 is associated with a variety of tumors including lung, colon, breast, glioma, prostate cancer and has received much attention for its role in tumors. However, USP7 has a large difference in substrate and tumor-modulating pathways among different tumors.

Epigenetic regulation is a precise regulatory process involving DNA methylation and histone modification (methylation, acetylation, and phosphorylation). Studies have shown that USP7 can activate DNA methylation by modulating the activity of DNMT 1. USP7 promotes the development and progression of breast cancer by regulating the stabilization of histone demethylase PHF 8. These studies indicate the potential regulatory role of USP7 in histone modification, but the mechanism has not yet been elucidated. There are few reports of studies involving USP7 protein in lymphoma. The research results show that key proteins of USP7 and BCR signal pathway in ABC-DLBCL subtype are highly expressed. Inhibition of USP7 with a small molecule inhibitor of USP7 down-regulates the RNA levels of all key BCR signaling pathway molecules, thereby significantly inducing ABC-DLBCL cell death. The invention not only provides a new visual angle for understanding the pathogenesis of the ABC-DLBCL subtype, but also is expected to provide an effective strategy for the treatment of the ABC-DLBCL subtype.

Diffuse large B-cell lymphoma is the most common non-hodgkin lymphoma, with high incidence, more than 30% of patients are at risk of relapse, and patients insensitive to R-CHOP chemotherapy will eventually die of DLBCL disease progression, for high risk DLBCL cases, 3-year survival rates are still below 60%, to be further improved. Wherein the ABC type DLBCL is more invasive and malignant than other types. The continuous BCR signaling pathway chronic activation is the initiating factor of the ABC type diffuse large B cell lymphoma (ABC-DLBCL) and is also an important therapeutic target of the ABC-DLBCL. In recent years, targeted drugs directed to certain molecules in the signaling pathway have been developed in succession or have entered preliminary clinical trials, bringing new promise for the treatment of DLBCL. For example, the inhibitor Ibrutinib of BTK in BCR pathway obtains satisfactory curative effect in ABC type DLBCL which is difficult to cure, and phase I/II clinical research shows that the effective rate can reach 40%. However, because the BTK inhibitor only inhibits BTK molecules downstream of BCR signals, DLBCL especially uses ABC type DLBCL continuously activated by NF ⁃ kB to relate to the expression abnormality of a wide range of signal molecules, the BTK inhibitor only has curative effect on a small part of patients and is easy to generate drug resistance. Given the complexity of the molecular pathway network, a single targeted drug therapy may not be able to exert a long-term and comprehensive anti-tumor effect. Drugs that comprehensively inhibit activation of the BCR signaling pathway remain to be developed. USP7 is a ubiquitin-specific protease which is the most widely studied at present, and the research shows that USP7 is involved in regulating the activity and the function of various protein substrates of cells, and USP7 has an important role in the occurrence and the development of diseases and tumors due to abnormal expression or activity change. There are few reports of studies involving USP7 protein in lymphoma. Our earlier studies suggest that: compared with the GCB-DLBCL subtype, USP7 is highly expressed in ABC type DLBCL, and the expression difference indicates that the USP7 possibly participates in the generation of ABC-DLBCL. Our study uses USP7 as an entry point, uses USP7 small molecule inhibitors to inhibit USP7 activity, studies the lethality of the inhibitors on ABC type DLBCL cells, and defines the role of USP7 in ABC-DLBCL and determines whether the inhibitors can be used as targets for treating DLBCL.

Disclosure of Invention

The technical problem to be solved is as follows: in view of the above technical problems of risk of relapse and low survival rate, the present invention provides a USP7 inhibitor for use in the treatment of ABC-DLBCL drugs.

The technical scheme is as follows:

use of an inhibitor of USP7 in the treatment of ABC-DLBCL agents.

Further, the method comprises the following steps:

first step, USP7 expresses a relationship with ABC type DLBCL: determining whether USP7 is related to ABC type DLBCL, firstly researching the expression of USP7 in DLBCL cell line, and then analyzing the expression of USP7 in tumor tissue by a database;

secondly, whether USP7 participates in the occurrence and development of ABC type DLBCL and the specific role played by the same is determined: the method is characterized in that a USP7 small molecule inhibitor is used for inhibiting the activity of USP7, the effect of ABC DLBCL cell proliferation, PI staining and apoptosis detection by a CCK-8 method and the effect of the inhibitor on the ABC DLBCL BCR signal channel by Western blot and Real-time PCR detection are observed, and the specific implementation steps are as follows:

and (3) CCK-8 detection: taking cells in logarithmic growth phase, carrying out complete medium resuspension, and counting the cells; taking a sterile 96-well plate, and designing six P22077 concentration groups of 0uM, 0.5uM, 1uM, 5uM, 10uM and 20uM, wherein each concentration group comprises 4 auxiliary wells; inoculating 2 × 104cells to each well, adding complete culture medium containing the above-mentioned concentration of medicine to 200 μ l, continuously culturing for 48h at 37 deg.C under 5% CO2, adding 20 μ l CCK-8 reagent into each well, and incubating for 4 hr at 37 deg.C; taking out the 96-well plate, putting the 96-well plate into an enzyme-labeling instrument, setting the detection wavelength to be 450nm, and detecting and recording OD450 readings;

PI staining experiment: inoculating log-phase cells into a 96-well plate at 20000 cells/well, performing administration treatment, culturing for 24 or 48h, adding PI dye with final concentration of 50 ug/mL, dyeing in dark for 10min, and taking pictures by fluorescence microscope;

flow cytometry: inoculating each group of cells in logarithmic growth phase in a 6-well plate, and culturing for 24 hours; after resuspension, collecting cells in each hole in a single flow tube, centrifuging to remove supernatant, washing the cells for 2 times by PBS (phosphate buffer solution), slowly adding 1ml of 70% ethanol precooled at-20 ℃ into each tube, and standing overnight at 4 ℃; centrifuging to remove ethanol, washing cells with PBS, and centrifuging for 5 min; add 500. mu.l PI/RNase stabilizing Buffer to each tube to resuspend the cells, incubate at room temperature in the dark for 15min, and then check on the machine.

The influence of the inhibitor on the ABC type DLBCL BCR signal channel is detected by applying Real-time PCR and Western blot methods: treating ABC type DLBCL cells (HBL) with 10uM P22077, extracting mRNA and protein respectively, and detecting the mRNA level of target genes IL-6, MYC, BATF3 and IRF4 of BTK, PLCG2, PKC and CD79B and BCR-NFkB signal channels by a Real-time PCR method; the protein levels of BTK and PLCG2 were detected by Western blot method.

Further, the specific step of researching the expression condition of USP7 in the DLBCL cell line in the first step is to apply a Western blot method to detect the expression of USP7 protein in ABC type DLBCL and GCB type DLBCL cell lines, and the expression difference of USP7 in ABC type DLBCL compared with GCB-DLBCL subtype proves that USP7 participates in the generation of ABC-DLBCL.

Further, the database analysis in the first step showed high expression of USP7 in tumor tissues compared to paracarcinoma tissues.

Furthermore, the small molecule inhibitor P22077 of USP7 in the second step can obviously induce the apoptosis of DLBCL, particularly ABC-DLBCL (HBL-1, SU-DHL-2).

Has the advantages that:

1. abnormal expression or activity change of USP7 has important effect on the occurrence and development of diseases and tumors. Database analysis showed high expression of USP7 in tumor tissues compared to paracarcinoma tissues (see figure 1A, B). This difference in expression suggests that our USP7 may be involved in the development of tumors, including DLBCL.

2. Immunoblots showed that USP7 was highly expressed in type ABC DLBCL compared to the GCB-DLBCL subtype (see fig. 1C), suggesting that USP7 may be involved in the development of type ABC DLBCL.

3. Few reports are reported at present about the research of USP7 protein on lymphoma, and the development of corresponding targeted drugs aiming at ABC type DLBCL which is difficult to treat and easy to resist is imminent.

4. The small-molecule inhibitor P22077 USP7 can obviously induce DLBCL, particularly ABC-DLBCL (HBL-1, SU-DHL-2) to undergo apoptosis (see figure 2), and the discovery not only provides a new visual angle for understanding the pathogenesis of ABC-DLBCL subtype, but also is expected to provide an effective strategy for the development of ABC-DLBCL subtype therapeutic drugs.

5. USP7 small molecule inhibitor P22077 can significantly down-regulate ABC type DLBCL cell BCR signaling pathway key molecules (see fig. 3).

6. The method has good application prospect and market competitiveness, and has good promotion effect on improving the livelihood and society of China.

Drawings

FIG. 1 is a graph showing the expression level of USP7 in DLBCL in example 1 of the present application;

FIG. 2 is a graph of the activity of USP7 inhibiting induced apoptosis of DLBCL cells in example 1 of this application.

FIG. 3 is a graph showing that USP7 plays an important role in the up-regulation of key molecules of BCR signaling pathway of ABC-DLBCL cells in example 1 of the present application.

Detailed Description

The following examples illustrate specific steps of the present invention, but are not intended to limit the invention.

Terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified.

The invention is described in further detail below with reference to specific examples and with reference to data. It will be understood that these examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.

In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.

Example 1

The use of an inhibitor of USP7 in the treatment of ABC-DLBCL agents, comprising the steps of:

first step, USP7 expresses a relationship with ABC type DLBCL: determining whether USP7 is related to ABC type DLBCL, firstly researching the expression of USP7 in DLBCL cell lines, applying a Western blot method to detect the expression of USP7 protein in ABC type DLBCL and GCB type DLBCL cell lines, and comparing with the GCB-DLBCL subtype, the high expression of USP7 in ABC type DLBCL is higher, the expression difference proves that USP7 participates in the generation of ABC-DLBCL, and then the high expression of USP7 in tumor tissues is shown by database analysis compared with the paracancerous tissues.

As shown in fig. 1, the expression level of USP7 in DLBCL: a, picture A: TCGA database analysis showed that in 31 tumor tissues, USP7 expressed higher in cholangiocarcinoma, thymoma and DLBCL than normal tissues; and B, drawing: USP7 was highly expressed in DLBCL patients compared to the control group; and (C) diagram: compared with GCB-DLBCL subtype (BJAB), the ABC-DLBCL (HBL-1, SU-DHL-2, OCL-Ly 10) subtype has high USP7 expression.

Secondly, whether USP7 participates in the occurrence and development of ABC type DLBCL and the specific role played by the same is determined: USP7 small molecule inhibitor is used for inhibiting activity of USP7, the effect of ABC DLBCL cell proliferation, PI staining and apoptosis detection by a CCK-8 method and the effect of the inhibitor on the ABC DLBCLBCR signal pathway by Western blot and Real-time PCR detection are observed. The specific implementation steps are as follows:

As shown in figure 2, inhibition of USP7 activity induced apoptosis of DLBCL cells: CCK-8 (panel A), PI staining (panel B) and flow cytometry (panel C) results show that the USP7 small molecule inhibitor P22077 can obviously induce the apoptosis of DLBCL, especially ABC-DLBCL (HBL-1, SU-DHL-2).

As shown in FIG. 3, USP7 plays an important role in the upregulation of critical molecules of the BCR signaling pathway in ABC-DLBCL cells. Panel a, mRNA levels of BTK, PLCG2, PKC, and CD79B were significantly down-regulated after P22077 treatment. In panel B, protein levels of BTK and PLCG2 were also significantly down-regulated after P22077 treatment. C picture, after P22077 treatment, the BCR-NFkB signaling pathway target genes IL-6, MYC, BATF3 and IRF4 were significantly down-regulated.

Claims

1. Use of an inhibitor of USP7 in the treatment of ABC-DLBCL agents.

2. The use of an inhibitor of USP7 in the treatment of ABC-DLBCL according to claim 1, characterized by the steps of:

and (3) CCK-8 detection: taking cells in logarithmic growth phase, carrying out complete medium resuspension, and counting the cells; taking a sterile 96-well plate, and designing six P22077 concentration groups of 0uM, 0.5uM, 1uM, 5uM, 10uM and 20uM, wherein each concentration group comprises 4 auxiliary wells; inoculation of 2X 10 per well⁴cells, add complete medium containing the drug at the above concentrations to 200. mu.l, 37 ℃ with 5% CO₂Culturing for 48h under the condition, adding 20 μ l CCK-8 reagent into each well, and incubating for 4 hours at 37 ℃; taking out 96-well plate, placing into enzyme labeling instrument, setting detection wavelength at 450nm, detecting and recording OD450 readCounting;

flow cytometry: inoculating each group of cells in logarithmic growth phase in a 6-well plate, and culturing for 24 hours; after resuspension, collecting cells in each hole in a single flow tube, centrifuging to remove supernatant, washing the cells for 2 times by PBS (phosphate buffer solution), slowly adding 1ml of 70% ethanol precooled at-20 ℃ into each tube, and standing overnight at 4 ℃; centrifuging to remove ethanol, washing cells with PBS, and centrifuging for 5 min; adding 500 μ l PI/RNase stabilizing Buffer into each tube to resuspend cells, incubating at room temperature in dark place for 15min, and detecting on the machine;

3. The use of an inhibitor of USP7 in the treatment of ABC-DLBCL agents according to claim 2, wherein: the specific step of researching the expression condition of USP7 in the DLBCL cell line in the first step is to apply a Western blot method to detect the expression of USP7 protein in ABC type DLBCL and GCB type DLBCL cell lines, and the expression difference proves that USP7 participates in the generation of ABC-DLBCL compared with the expression difference of USP7 in ABC type DLBCL of a GCB-DLBCL subtype.

4. The use of an inhibitor of USP7 in the treatment of ABC-DLBCL agents according to claim 2, wherein: database analysis in the first step showed high expression of USP7 in tumor tissues compared to para-carcinoma tissues.

5. The use of an inhibitor of USP7 in the treatment of ABC-DLBCL agents according to claim 2, wherein: the USP7 small-molecule inhibitor P22077 can obviously induce DLBCL, particularly ABC-DLBCL (HBL-1, SU-DHL-2) to generate apoptosis.