GBM copper death marker ACP6 and application

Abstract

The invention discloses a GBM copper death marker ACP6 and application thereof, belongs to the technical field of biological medicines, aims to solve the problem of screening and identifying potential effective targets capable of being applied to copper death, takes a GBM cell line LN229 as a research object and CuET as a copper ion carrier, carries out CRISPR / dCas9 whole genome transcriptional activation library genetic screening, and carries out library screening, sequencing and RIGER analysis and verification, so as to achieve the purpose of screening and identifying the potential effective targets capable of being applied to copper death. A series of known and new candidate genes ACP6 for resisting CuET-induced GBM cell copper death are finally identified, the ACP6 is taken as a key candidate gene of copper ion carrier mediated GBM copper death, the ACP6 is further verified to be a key inhibition factor of copper ion carrier mediated GBM copper death, the copper death sensitivity of ES and CuET-induced GBM cells can be remarkably improved by knocking down / removing the ACP6, and the activity of the copper death is improved. And a new theoretical basis and an experimental basis are provided for clinically developing drug molecules for targeting tumor copper death.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the GBM copper death marker ACP6 and its applications. Background Technology

[0002] Gliomas are the most common primary malignant brain tumors, with glioblastoma (GBM) being the most aggressive and deadly, characterized by poor prognosis, high mortality, and high recurrence rates. For decades, GBM treatment has primarily relied on a combination of surgery and radiotherapy / chemotherapy, with a median survival of only 12 to 18 months, highlighting the urgent need for new treatment options. While the molecular basis of GBM has been extensively studied, targeted therapy remains ineffective in clinical practice, leading to a growing demand for effective anti-GBM drugs. Therefore, it is crucial to screen for new molecular biomarkers and drug targets for the diagnosis and treatment of GBM. Copper death, a novel cell death mechanism caused by copper overload, closely links copper-based metal ions to tumor metabolism, providing new insights and research strategies for anti-tumor drug therapy based on copper ion carriers. Therefore, further research is needed on how to apply targeting the copper death pathway as a novel anti-tumor research strategy to effective GBM treatment. However, due to the limited research on the mechanisms of copper death in the development of GBM, further identification of new key target molecules and elucidation of the molecular mechanisms by which target molecules mediate copper death in GBM are important research foundations for applying copper death-targeting strategies to GBM treatment. Summary of the Invention

[0003] To address the aforementioned problems, the purpose of this invention is to provide ACP6, a biomarker for copper death in GBM cells, and its applications. This invention uses the GBM cell line LN229 as the research object, and copper diethyldithiocarbamate-copper complex (CuET) and elesclomol (ES) as copper ion carriers. Genetic screening of CRISPR / dCas9 whole-genome transcriptional activation libraries was conducted. Through library screening, sequencing, and RIGER analysis and validation, a series of known and novel candidate genes resistant to CuET-induced copper death in GBM cells—ACP6—were ultimately identified. ACP6 was selected as a key candidate gene for copper ion carrier-mediated GBM copper death, and further validation confirmed that ACP6 is a key inhibitor of copper ion carrier-mediated GBM copper death, providing new theoretical and experimental basis for the clinical development of drugs targeting tumor copper death.

[0004] To achieve the above objectives, this application provides the following technical solution: The first aspect of the present invention provides the use of ACP6 as a biomarker in the preparation of reagents for assessing the sensitivity and / or prognosis of glioblastoma to immunotherapy.

[0005] In a preferred embodiment of this aspect, the immunotherapy is a copper ion carrier-induced copper death therapy for tumor cells, specifically, copper ion carriers CuET and ES induce copper death in GBM cells.

[0006] A second aspect of the invention provides the use of an ACP6 inhibitor in the preparation of a medicament for treating glioblastoma, wherein the ACP6 inhibitor can increase the sensitivity of glioblastoma to copper death synergistically regulated by the copper ion carriers CuET and ES.

[0007] In some preferred embodiments of this aspect, the ACP6 inhibitor is Plantamajoside and Isolugrandoside.

[0008] Compared with the prior art, the present invention has the following advantages: This invention is the first to discover that ACP6 is a key inhibitor of copper death in GBM cells mediated by copper ion carriers. By knocking down / removing ACP6, the sensitivity of GBM cells to copper death induced by ES and CuET can be significantly increased, thereby enhancing the efficacy of copper ion carriers in treating tumors. Attached Figure Description

[0009] Figure 1 Analysis of genetic screening results for the CRISPR / dCas9 whole-genome transcription activation library; where A is a schematic diagram of genetic screening for the CRISPR / dCas9 whole-genome transcription activation library; BC are the sgRNA sequencing quality control results; D is the TOP4 sgRNAs in the forward screening analysis after CuET treatment; E is the MAGeCK algorithm analysis verifying that the counts of the three sgRNAs of ACP6 were significantly increased after CuET treatment compared with the control group.

[0010] Figure 2This study analyzed the results indicating that ACP6 is a key factor mediating the copper death sensitivity of GBM cells. Specifically, A represents the viability of ACP6-overexpressing LN229 cells treated with different concentrations of ES using CCK-8 assays; B represents the viability of ACP6-overexpressing LN229 cells treated with different concentrations of CuET using CCK-8 assays; C represents the viability of ACP6-overexpressing T98G cells treated with different concentrations of ES using CCK-8 assays; D represents the viability of ACP6-overexpressing T98G cells treated with different concentrations of CuET using CCK-8 assays; E represents the viability of ACP6-knockout LN229 cells treated with different concentrations of ES using CCK-8 assays; F represents the viability of ACP6-knockout LN229 cells treated with different concentrations of CuET using CCK-8 assays; G represents the viability of ACP6-knockout LN229 cells treated with copper ion chelator TTM combined with different concentrations of ES using CCK-8 assays; and H represents the viability of ACP6-knockout LN229 cells treated with copper ion chelator TTM combined with different concentrations of CuET using CCK-8 assays.

[0011] Figure 3 The ACP6 inhibitors, Plantamajoside and Isolugrandoside, synergistically regulate GBM cell death using copper ion carriers. AB shows the surface and 2D diagrams of the binding pattern between Plantamajoside and Human ACP6 protein. C shows the inhibition of ACP6 protein expression in LN229 cells by different concentrations of Plantamajoside using Western blot. D shows the interaction between Human ACP6 protein and Plantamajoside using SPR. E shows the effect of Plantamajoside on LN229 cell viability using CCK8 assay. FG shows the synergistic effect of Plantamajoside, Isolugrandoside, and copper ion carrier ES on LN229 cell viability. Detailed Implementation

[0012] Cells and reagents Human glioblastoma cells (LN229) were purchased from Wuhan Pronosei Biotechnology Co., Ltd., and cultured in DMEM medium with 10% fetal bovine serum and 1% penicillin-streptomycin. Anti-ACP6, Anti-GAPDH, and HRP-conjugated Goat Anti-Rabbit IgG (H+L) were purchased from Wuhan Sanying Biotechnology Co., Ltd. The Easy PAGE color rapid gel preparation kit was purchased from Seven Innovations (Beijing) Biotechnology Co., Ltd. SDS-PAGE electrophoresis buffer, transfer buffer, and TBS powder were from Wuhan Saiweier Biotechnology Co., Ltd. Primary antibody dilution buffer was purchased from Shanghai Beyotime Biotechnology Co., Ltd. SuperSignal™ West Pico PLUS chemiluminescent substrate was purchased from Thermo Fisher Scientific (China) Co., Ltd. Enhanced CCK-8 kit was from Shandong Cisco Biotechnology Co., Ltd.

[0013] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0014] The first aspect of this invention provides the application of ACP6 as a biomarker in the preparation of reagents for assessing the sensitivity and / or prognosis of glioblastoma to immunotherapy, wherein the immunotherapy is a copper ion carrier-induced copper death therapy for tumor cells, specifically, copper ion carriers CuET and ES inducing copper death in GBM cells. The second aspect of this invention provides the application of ACP6 inhibitors in the preparation of drugs for treating glioblastoma, wherein the ACP6 inhibitors can increase the sensitivity of glioblastoma cells to copper death synergistically regulated by copper ion carriers CuET and ES. The ACP6 inhibitors are Plantamajoside and Isolugrandoside.

[0015] Example 1: Screening key candidate inhibitors of copper ion carrier-mediated copper death in GBM To identify novel copper death suppressor genes, this project used the GBM cell line LN229 as the research subject, CuET as the copper ion carrier, and conducted genetic screening of CRISPR / dCas9 whole-genome transcriptional activation libraries. The specific screening strategy is as follows: Figure 1As shown in Figure A. This study constructed a genome-level SAM sgRNA lentiviral library containing 56,762 sgRNAs. Through library screening, sequencing, and RIGER analysis and validation, a series of known and novel candidate genes resisting CuET-induced copper death in GBM cells were finally identified. sgRNA sequencing results showed that the plasmid and genome library construction were well performed, and the sequenced sequences accounted for 99.664% of the entire genome (…). Figure 1 (BC). After CuET treatment, positive selection analysis showed that the TOP4 sgRNA contained ATP7A, MT4, MT1X, and ACP6 ( Figure 1 (D). Literature review revealed that ATP7A, MT4, and MT1X are all involved in copper metabolism and copper death, and are important molecules regulating copper death. To explore new target molecules for copper death, and given that ACP6 is located in mitochondria, its clinicopathological significance and functional role in human cancer remain unclear, this study focused on ACP6. Further MAGeCK algorithm analysis confirmed that the counts of the three sgRNAs of ACP6 were significantly increased after CuET treatment compared to the control group (D). Figure 1 (E).

[0016] Based on a series of literature reviews and experimental analyses, this project proposes to use ACP6 as a key candidate gene for copper ion carrier-mediated copper death in GBM.

[0017] Example 2: Verification that ACP6 is a key inhibitory factor mediating copper ionophore-induced GBM cell death. To further verify whether ACP6 is a key inhibitor of copper ion transporter-mediated copper death in GBM cells, GBM cells were used as the research object, and ES and CuET were used as copper ion transporters. The effect of ACP6 expression on copper ion transporter-induced GBM cell death was detected by CCK8 assay. The results showed that, compared with the control group, overexpression of ACP6 significantly resisted ES and CuET-induced copper death in LN229 and T98G cells. Figure 2 In the context of AD), knocking down or eliminating ACP6 significantly increased the sensitivity of GBM cells to copper death induced by ES and CuET, and the copper ion chelator TTM could alleviate the sensitivity of GBM cells to copper death. Figure 2 (EH). Therefore, ACP6 is a key inhibitor of copper death in GBM ionophores mediated by copper ionophores.

[0018] The specific process of the CCK8 experiment described above is as follows: Cells with good growth and a growth density of 80% were digested with trypsin to obtain a cell pellet. The cells were then resuspended in 1 mL of complete culture medium to form a cell suspension. The cell counting chamber was removed, and the cells were gently mixed. 10 μL of the cell suspension was then used for cell counting, with approximately 8000 cells per well seeded into a 96-well plate at a seeding volume of 100 μL. The cells were cultured overnight. After cell attachment, the cells were treated with the appropriate drugs according to experimental requirements. After treatment, the old culture medium was aspirated from the cells, and the cells were washed twice with PBS to prevent residual old culture medium from reacting with the subsequently added CCK8 solution and affecting the experimental results. Pure culture medium and CCK8 solution were added to each well at a ratio of 10:1. The 96-well plate was incubated for 1–4 h, and the color change of the solution was observed every 30 minutes. When a significant change in color intensity was observed between the untreated group (blank group) and the cell-treated group (where normal cell lines served as the control group, and ACP6-overexpressing, knocked-down, or knocked-out cell lines served as the experimental group), the absorbance at 450 nm was measured using a microplate reader. Cell viability calculation method: (Experimental group absorbance - Blank group absorbance) / (Control group absorbance - Blank group absorbance) × 100%.

[0019] Example 3: Screening and validation of ACP6 inhibitors. Plantamajoside is a potential drug candidate molecule for enhancing GBM copper death sensitivity. To enhance the efficacy of copper ion carriers in treating tumors, drugs were screened targeting ACP6 to increase the synergistic effect of copper ion carriers in treating GBM. The 3D structures of 69.1K compounds were compared with the 3D structure of Human ACP6 using a high-throughput screening (HTVS) mode in the Glide module. The results showed that the hydroxyl group of Plantamajoside can form five hydrogen bonds with PHE355 / PRO203 / GLN206 / TYR106 / ASP254 of the Human ACP6 protein, and the benzene ring of Plantamajoside can form one π-π interaction with PHE355 of Human ACP6. Figure 3 (A, B). Western blot analysis revealed that plantamajoside reduced ACP6 expression in a concentration-dose dependent manner. Figure 3 (C). The affinity signal value (KD) between the compound and the ACP6 protein was determined by screening and analysis using multi-concentration SPR technology; KD=5.15E-06. Figure 3 (D), therefore, Forsythoside 1 can bind to the protein ACP6. Further studies found that Plantamajoside kills LN229 cells in a dose-dependent manner, with an IC50 of 248.18 μM (D). Figure 3Further research revealed that plantamajoside and isolugrandoside synergistically regulate the sensitivity of GBM copper death with the copper ion carrier ES. Figure 3 (FG).

[0020] This study provides new research directions and ideas for revealing the mechanism by which ACP6 resists copper death, and further provides new theoretical basis and experimental foundation for the development of drug molecules that target tumor copper death in clinical practice.

[0021] It is understood that the above specific description of the present invention is only for illustrating the present invention and is not limited to the technical solutions described in the embodiments of the present invention. Those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention to achieve the same technical effect; as long as the use needs are met, they are all within the protection scope of the present invention.

Claims

1. Application of ACP6 as a biomarker in the preparation of reagents for evaluating the sensitivity and / or prognosis of glioblastoma to immunotherapy.

2. The application according to claim 1, characterized in that, The immunotherapy described is a copper ion carrier-induced copper death therapy for tumor cells.

3. The application of ACP6 inhibitors in the preparation of drugs for treating glioblastoma, characterized in that, ACP6 inhibitors can increase the sensitivity of glioblastoma cells to synergistic regulation of copper death by the copper ion carriers CuET and ES.

4. The application according to claim 3, characterized in that, The ACP6 inhibitors are Plantamajoside and Isolugrandoside.

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