Application of KDM5B inhibitor in preparation of medicine for treating EBV-related epithelial tumor

Abstract

The invention provides application of a KDM5B inhibitor in preparation of a medicine for treating EBV (Epstein-Barr Virus) related epithelial tumors. The invention can be used for prognosis monitoring and precise treatment of EBV-related epithelial tumor patients clinically.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the use of a KDM5B inhibitor in the preparation of a drug for treating EBV-related epithelial tumors. Technical Background

[0002] Epstein-Barr virus (EBV) is a ubiquitous herpes virus that plays a key role in the progression of a variety of human tumors, especially EBV-related epithelial tumors, which account for about 80% of all EBV-related malignant tumors. EBV-related epithelial tumors mainly include nasopharyngeal carcinoma and EBV-positive gastric cancer. The incidence of nasopharyngeal carcinoma has significant regional specificity, mainly occurring in southern China and Southeast Asia. Studies have shown that about 98% of nasopharyngeal carcinomas are related to EBV infection, and EBV infection is an early event in the development of nasopharyngeal carcinoma. The incidence of EBV-positive gastric cancer accounts for about 10% of all gastric cancers. Compared with EBV-negative gastric cancer, EBV-positive gastric cancer has unique cancer-promoting molecular characteristics, including high-frequency mutations in PIK3CA and high genomic methylation. Moreover, EBV-positive gastric cancer is poorly differentiated histologically compared with EBV-negative gastric cancer, indicating that EBV plays an important role in the malignant progression of EBV-positive gastric cancer.

[0003] Although the relationship between EBV and these tumors has been confirmed, especially by detecting EBV molecular expression in tumors and increased plasma EBV DNA levels in patients during disease progression; however, the specific molecular mechanism by which EBV drives the development of these tumors and its prospects for clinical translational application remain to be explored.

[0004] Currently, chemoradiotherapy combined with surgery is still the first-line treatment strategy for patients with EBV-related epithelial tumors. With the advancement of diagnostic and treatment technologies such as magnetic resonance imaging (MRI), intensity-modulated radiation therapy (IMRT), synchronous chemoradiotherapy, and immunotherapy, the overall prognosis of EBV-related epithelial tumors has been significantly improved. However, problems such as disease recurrence, metastasis, and treatment resistance are still key factors limiting the improvement of prognosis, especially for advanced patients, whose survival rate and quality of life are still low. Therefore, it is urgent to discover new EBV-related epithelial tumor markers to achieve more accurate prognostic assessment and use them as potential therapeutic targets for patients with EBV-related epithelial tumors. The key role of EBV in the malignant progression of epithelial tumors makes it an ideal therapeutic target. Blocking the molecular mechanism by which EBV promotes the progression of epithelial tumors is expected to provide a new direction for the treatment of EBV-related epithelial tumors, thereby further improving the prognosis of patients. Summary of the invention

[0005] In view of the defects of the prior art, the object of the present invention is to provide a new tumor marker for EBV-related epithelial tumors and its application, so as to solve the problems raised in the above background technology.

[0006] To achieve the above technical objectives, the present invention provides the use of a KDM5B inhibitor in the preparation of a drug for treating EBV-related epithelial tumors.

[0007] In a preferred embodiment, the EBV-related epithelial tumor is nasopharyngeal carcinoma or EBV-positive gastric cancer.

[0008] In a preferred embodiment, the KDM5B inhibitor specifically targets KDM5B and inhibits KDM5B by chelating iron.

[0009] In a preferred embodiment, the KDM5B inhibitor is selected from any one or more of AS-8351 (CAS No.: 796-42-9), KDM5B-IN-4 (CAS No.: HY-149091), GSK467 (CAS No.: 1628332-52-4), PBIT (CAS No.: 2514-30-9), CPI-455 (CAS No.: 1628208-23-0), but is not limited thereto. Any reagent that can specifically inhibit KDM5B, especially a small molecule compound, belongs to the "KDM5B inhibitor" referred to herein.

[0010] The expression level of KDM5B in EBV-related epithelial tumor tissues and cells was detected using single-cell transcriptome sequencing and whole transcriptome sequencing methods, and it was found that KDM5B was significantly highly expressed in EBV-positive tumor tissues and cells.

[0011] The expression of KDM5B in EBV-related epithelial tumors can be detected by immunohistochemical staining of paraffin-embedded wax block sections of tumor tissue. It was found that KDM5B was widely and abundantly expressed in tumor cells, and the copy number of EBV DNA in patient plasma was significantly correlated with the high expression of KDM5B. The high expression of KDM5B was significantly correlated with the poor prognosis of patients.

[0012] KDM5B can promote the proliferation of tumor cells in EBV-positive tumor cells. This pro-proliferation effect can be inhibited by KDM5B inhibitors. Therefore, KDM5B can be used as an important target for the treatment of EBV-related epithelial tumors, and KDM5B inhibitors can be used to treat EBV-related epithelial tumors.

[0013] It can be seen that the expression level of KDM5B is related to the tumor proliferation and formation of EBV-related epithelial tumors, and is significantly correlated with the clinical prognosis of patients with EBV-related epithelial tumors. It can be used for prognosis monitoring and precise treatment of patients with EBV-related epithelial tumors in clinical practice, and has high clinical translational value. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Shown are: (A) the expression level of KDM5B in EBV-high and EBV-low tumor cell populations in single-cell transcriptome sequencing data of nasopharyngeal carcinoma; (B) the expression level of KDM5B in non-infected (NC) and EBV-infected (EBV-P) cells in whole transcriptome sequencing data of nasopharyngeal carcinoma and gastric cancer cells.

[0015] Figure 2 The following are shown: (A) Immunohistochemical staining experiments detected the expression of KDM5B in paraffin-embedded wax block sections of NPC tumor tissue; (B) The results of KDM5B immunohistochemical staining and the EBV copy number in the patient's plasma of 120 NPC patients were analyzed, and it was found that the number of patients with high KDM5B expression in the EBV DNA copy number-positive patient group was higher; (C) Survival analysis of KDM5B immunohistochemical staining results and patient prognostic information of 120 NPC patients showed that the higher the KDM5B expression level in the patient's tumor tissue, the worse the patient's prognosis.

[0016] Figure 3 It is shown that: (A) KDM5B was stably knocked down in EBV-positive epithelial tumor cells using a shRNA lentiviral packaging system targeting KDM5B; (B) The effect of KDM5B on the proliferation ability of tumor cells was detected by a plate cloning experiment, and knocking down KDM5B significantly inhibited the proliferation of tumor cells.

[0017] Figure 4 The figure shows the in vivo tumorigenesis experiment after KDM5B was stably knocked down in EBV-positive epithelial tumor cells using the shRNA lentiviral packaging system targeting KDM5B to detect the effect of KDM5B on the tumorigenesis ability of tumor cells. (A) The tumor volumes of the control group and the KDM5B knockdown group in the mouse experiment were measured respectively; (B) The transplanted tumors obtained by sampling the control group and the KDM5B knockdown group in the mouse experiment and the final statistical tumor weight.

[0018] Figure 5The following are shown: (A) The proliferation ability of EBV-positive epithelial tumor cells treated with control solvent and KDM5B inhibitor was detected by plate cloning experiment. The clone formation ability of cells in the KDM5B inhibitor-treated group was significantly lower than that in the control group; (B) The tumor volumes of EBV-positive nasopharyngeal carcinoma PDX mice (NPC PDX#1, NPC PDX#2) treated with control solvent and KDM5B inhibitor by intraperitoneal injection, and the control group and KDM5B inhibitor group in the AGS-EBV cell subcutaneous tumor formation experiment were measured respectively; (C) The transplanted tumors collected from the control group and KDM5B inhibitor group in the mouse experiment and the final statistically obtained tumor weights.

[0019] Figure 6 Immunohistochemical staining experiments show Figure 5 The expression of proliferation marker Ki-67 in paraffin-embedded wax block sections of mouse tumor tissue obtained in the figure. The left figure is the staining result, and the right figure is the statistical bar graph, indicating that the Ki-67 positivity rate in tumor tissue sections of the KDM5B inhibitor-treated group was significantly lower than that of the control group. DETAILED DESCRIPTION

[0020] In order to more clearly understand the above-mentioned objects, features and advantages of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific examples. It should be understood by those skilled in the art that the examples are only to help understand the present invention and should not be regarded as specific limitations of the present invention; unless otherwise specified, the sequencing and analysis methods used in the following examples are conventional methods.

[0021] definition

[0022] As used herein, "EBV-associated epithelial tumors" refers to nasopharyngeal carcinoma and EBV-positive gastric cancer.

[0023] As used herein, "EBV-positive gastric cancer" refers to gastric cancer in which the presence of EBV molecules in tumor tissues was confirmed by EBER in situ hybridization.

[0024] As used herein, "KDM5B inhibitor" refers to a small molecule compound that specifically targets KDM5B and inhibits KDM5B by chelating iron. "KDM5B inhibitor" can be selected from any one or more of AS-8351 (CAS No.: 796-42-9), KDM5B-IN-4 (CAS No.: HY-149091), GSK467 (CAS No.: 1628332-52-4), PBIT (CAS No.: 2514-30-9), CPI-455 (CAS No.: 1628208-23-0), but is not limited thereto.

[0025] Example 1: KDM5B is highly expressed in EBV-positive tumor tissues and cells and its high expression is associated with poor prognosis of patients

[0026] A total of 10 nasopharyngeal carcinoma tumor tissue samples from the Sun Yat-sen University Cancer Center were collected for single-cell transcriptome sequencing. In short, the nasopharyngeal carcinoma tumor tissue samples were first enzymatically digested into single-cell suspensions, and then the commercial single-cell transcriptome library kit (10x Genomics) was used to load the living single cells and construct libraries. Finally, the obtained cDNA library was subjected to high-throughput sequencing to obtain sequence files.

[0027] The results are as follows Figure 1 As shown. The obtained single-cell transcriptome sequencing data were analyzed to obtain tumor cell subpopulations with high and low EBV molecule expression. Among them, KDM5B was significantly overexpressed in the nasopharyngeal carcinoma tumor cell subpopulation with high EBV expression ( Figure 1 , A).

[0028] On the other hand, an in vitro EBV infection model was used, that is, the EBV-transformed lymphocyte line AKATA-EBV-GFP that integrated the EBV genome was expanded and cultured to induce intracellular viral lysis and release to prepare EBV suspension, and then the EBV suspension was used to contact the cells for infection; the nasopharyngeal carcinoma cells (S26, HK-1, provided by the Cancer Center of Sun Yat-sen University) and gastric cancer cells (AGS, ATCC NO.CRL-1739) before and after EBV infection were subjected to whole transcriptome sequencing, that is, the total cell RNA was extracted and purified using the Ribo-Zero Magnetic Kit (Illumina) to remove ribosomal RNA, and then the commercial whole transcriptome kit TruSeq RNA Library Prep Kit (Illumina) was used for library construction, and finally the obtained library was subjected to high-throughput sequencing to obtain the sequence file.

[0029] The obtained whole transcriptome sequencing data showed that KDM5B expression was significantly increased in EBV-infected group (EBV-P) cells ( Figure 1 , B).

[0030] We further collected 120 NPC tumor tissue wax block sections from the Sun Yat-sen University Cancer Center for immunohistochemical staining. In brief, the tumor tissue sections were dewaxed, hydrated, antigen repaired, oxidase removed, and blocked. After overnight incubation with KDM5B antibody (Sigma, HPA027179), the sections were stained and sealed. Pathologists were invited to read and score the sections to evaluate the expression of KDM5B in the tissues ( Figure 2 , A), combined with the patient's plasma EBV DNA copy number level and patient prognosis information, it was found that patients with positive plasma EBV DNA copy number had higher KDM5B expression levels ( Figure 2, B), while patients with high KDM5B expression had a worse survival prognosis ( Figure 2 , C), which suggests that KDM5B expression is associated with the malignant progression of EBV-related epithelial tumors and affects the survival of patients.

[0031] Example 2: Expression of KDM5B in EBV-related epithelial tumors is beneficial for tumor cell proliferation in vitro and tumorigenesis in vivo

[0032] The shRNA lentiviral packaging system targeting KDM5B was used to construct EBV-positive nasopharyngeal carcinoma cell lines (HONE1-EBV, CNE2-EBV) and gastric cancer (AGS-EBV) cell lines with stable knockdown of KDM5B. The corresponding shRNA plasmids (control plasmid, KDM5B knockdown plasmid 1, KDM5B knockdown plasmid 2) and lentiviral packaging plasmids were transfected into 293T cells in a ratio (corresponding shRNA plasmid: psPAX2: pMD2.G = 1:0.75:0.3), and then the culture supernatant of 293T cells cultured for 48 hours was collected to infect the target cells (HONE1-EBV, CNE2-EBV, AGS-EBV cells) to obtain stable cell lines.

[0033] In brief, the corresponding shRNA plasmids mentioned above were constructed as follows:

[0034] The control sequence (5'-AATTCAAAAATTCCTGGAACAATTGCTTTTACTCGAGTAAAAGCAATTGTTCCAGGAA-3') and the shRNA sequence targeting KDM5B (5'-CCGGAAGCGTATCCGTTTGGA ACAACTCGAGTTGTTCCAAACGGATACGCTTTTTTTG-3'; 5'-AATTCAAAAAAAGCGTAT CCGTTTGGAACAACTCGAGTTGTTCCAAACGGATACGCTT-3') were ligated into the enzyme-digested PLKO.1 plasmid through the restriction sites (Age1, EcoR1) to construct the control plasmid, KDM5B knockdown plasmid 1 and KDM5B knockdown plasmid 2.

[0035] WB experimental results showed that the shRNA lentiviral packaging system targeting KDM5B stably knocked down KDM5B in EBV-positive epithelial tumor cells ( Figure 3 , A). Using EBV-positive nasopharyngeal carcinoma (HONE1-EBV, CNE2-EBV) and gastric cancer (AGS-EBV) cell lines with stable knockdown of KDM5B, in vitro plate clone formation experiments were performed to detect the effect of KDM5B on the proliferation ability of tumor cells. The results showed that the cloning ability of tumor cells with knockdown of KDM5B was significantly reduced ( Figure 3, B), indicating that KDM5B knockdown can effectively attenuate the in vitro proliferation ability of EBV-positive epithelial tumor cells.

[0036] Four-week-old male BALB / c nude mice were subcutaneously injected (100ul / injection point) with a mixture of matrix gel (100ul PBS containing 5x 10 6 The target cells, the cell suspension and the volume ratio of matrix gel were 4:1) CNE2-EBV and AGS-EBV cells with shRNA knockdown of KDM5B or control group. The tumor size of the mice was measured with a caliper every three days, and the formula V = 0.52*L*W was used. 2 The tumor volume was calculated and the mouse tumor volume growth curve was drawn, where L was the longest diameter of the tumor and W was the shortest diameter.

[0037] The results of in vivo tumorigenesis experiments showed that the tumor volumes formed by CNE2-EBV and AGS-EBV cells in the KDM5B knockdown group were significantly smaller than those in the control group ( Figure 4 , A), The statistical results of transplanted tumors and tumor weights obtained from the control group and KDM5B knockdown group in the mouse experiment showed that the growth rate of mouse tumors formed by KDM5B knockdown tumor cells was significantly reduced ( Figure 4 , B). This indicates that KDM5B knockdown can effectively inhibit the tumorigenicity of EBV-positive epithelial tumor cells in vivo.

[0038] Example 3: Therapeutic regimen targeting KDM5B inhibits tumor progression of EBV-positive epithelial tumors in mice

[0039] HONE1-EBV, CNE2-EBV, and AGS-EBV cells (from the Cancer Center of Sun Yat-sen University) were treated with the KDM5B inhibitor AS-8351 (CAS No. 796-42-9) at a working concentration (10 μM) for 24 hours, and then a cell growth counting experiment was performed. Cells treated with blank solvent (DMSO) were used as controls for plate clone formation experiments. The experimental results showed that the clone formation ability of cells in the KDM5B inhibitor treatment group was significantly lower than that of the control group, and targeted inhibition of KDM5B significantly inhibited the in vitro proliferation ability of EBV-related epithelial tumor cells ( Figure 5 , A).

[0040] About 4-5 weeks old NOD / Scid mice were used for EBV-positive nasopharyngeal carcinoma PDX (human tumor xenografts, NPCPDX#1, NPC PDX#2) inoculation experiments. EBV-positive nasopharyngeal carcinoma tissue masses were divided into blocks of about 10 mm using a tissue separation knife. 3 / piece, and then implanted into the abdomen of mice according to the experimental plan. 3 The mice were randomly divided into experimental group and control group.

[0041] Male BALB / c nude mice aged about 4-5 weeks were used for the subcutaneous tumor formation experiment of EBV-positive gastric cancer cells AGS-EBV. AGS-EBV cells were mixed with Matrigel (100ul PBS containing 5x 10 6 The target cells were inoculated by subcutaneous injection (100ul / injection point) with a cell suspension and matrix gel volume ratio of 4:1. 3 The mice were randomly divided into experimental and control groups.

[0042] Then, the control group mice were intraperitoneally injected with the control solvent (DMSO), and the experimental group mice were intraperitoneally injected with the KDM5B inhibitor (AS-8351), with an injection dose of 25 mg / kg, injected every other day; the tumor changes and the growth status of the mice were continuously observed and recorded. The tumor size of the mice was measured with a vernier caliper every three days, and the formula V = 0.52*L*W was used. 2 Calculate the tumor volume and draw the mouse tumor volume growth curve (where L is the longest diameter of the tumor and W is the shortest diameter). The mouse tumor volume measurement results are as follows Figure 5 (B) As shown. Before the tumor volume of mice reached the ethical limit, the mice used in the experiment were euthanized. The tumors and corresponding tumor weights of the treated mice are shown in Figure 5 As shown in (C), the tumor volume of mice treated with KDM5B inhibitor was significantly reduced.

[0043] Paraffin sections made from tumor tissue were taken for immunohistochemical staining to detect the proliferation marker Ki-67 and count the positive rate of Ki-67 in each group. The experimental results showed that the Ki-67 positive rate of tumors in mice treated with KDM5B inhibitors was significantly reduced ( Figure 6 ), further confirmed that targeting KDM5B significantly inhibited tumor growth, and KDM5B is expected to become a new epigenetic therapeutic target in EBV-related epithelial tumors in the clinic in the future.

Claims

1. Application of KDM5B inhibitors in the preparation of drugs for treating EBV-related epithelial tumors.

2. The use according to claim 1, characterized in that: The EBV-related epithelial tumor is nasopharyngeal carcinoma or EBV-positive gastric cancer.

3. The use according to claim 1, characterized in that: The KDM5B inhibitor specifically targets KDM5B and inhibits KDM5B by chelating iron.

4. The use according to claim 1, characterized in that: The KDM5B inhibitor is selected from any one or more of AS-8351, KDM5B-IN-4, GSK467, ​​PBIT, and CPI-455.

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