The Role of Targeting CYP3A5 in Glioma Treatment

Abstract

The present invention discloses the role of targeting CYP3A5 in the treatment of glioma. The present invention provides a biomarker CYP3A5 related to glioma, which can be used for the diagnosis, prognosis judgment, prevention and / or treatment of glioblastoma, and can help clinicians effectively treat glioma patients, having important clinical application value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to the role of targeting CYP3A5 in the treatment of glioma. Background Art

[0002] Glioblastoma (GBM) is the most common and lethal malignant brain tumor. Even with the implementation of standard treatments including surgery, radiotherapy, and chemotherapy, the median overall survival of newly diagnosed GBM is still short at present, being 15 - 17 months. Glioblastoma stem cells (GSCs) promote tumor phenotypic plasticity and heterogeneity and are the cause of tumorigenesis, development, and treatment resistance. GSCs maintain self-renewal through molecular signaling pathways such as Notch and receptor tyrosine kinases, the latter being growth factor receptors that activate the PI3K-AKT or STAT signaling cascades. Temozolomide (TMZ) treatment generates evolutionary selection pressure, leading to the proliferation of drug-resistant GSCs, which are characterized by enhanced DNA repair ability. However, the mechanisms by which GSCs maintain self-renewal and promote chemoresistance have not been fully elucidated.

[0003] CYP3A5 is a member of the cytochrome P450 (CYP) enzyme superfamily and is involved in drug metabolism and lipid synthesis. As a monooxygenase, P450 uses reduced NAD (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) as an electron donor (NAD(P)H + O2 + R → NAD(P)+ + RO + H2O). The role of CYP3A5 in tumors has a background. It has been reported that CYP3A5 plays a tumor suppressor role in hepatocellular carcinoma through the mTORC2 / Akt signaling pathway. On the other hand, CYP3A5 is considered to mediate treatment resistance in pancreatic ductal adenocarcinoma. Nevertheless, the role of CYP3A5 in GBM progression is unclear. Summary of the Invention

[0004] To make up for the deficiencies of the prior art, the purpose of the present invention is to provide a biomarker CYP3A5 related to glioblastoma, which can be used for the diagnosis, prognosis judgment, prevention, and / or treatment of glioblastoma.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides a biomarker related to glioblastoma.

[0007] Further, the biomarker is CYP3A5.

[0008] Further, the biomarker can be used for the diagnosis, prognosis judgment, prevention, and / or treatment of glioblastoma.

[0009] Furthermore, the expression level of the biomarker CYP3A5 is significantly increased in glioblastoma.

[0010] In the present invention, the term "biomarker" refers to a biomolecule that exists in an individual at different concentrations and can be used to predict the cancer status of the individual. Biomarkers can include, but are not limited to, nucleic acids, proteins, and their variants and fragments. A biomarker can be DNA that contains all or part of the nucleic acid sequence encoding the biomarker or the complement of such a sequence. Biomarker nucleic acids useful in the present invention are considered to include DNA and RNA that contain all or part of the sequence of any nucleic acid sequence of interest.

[0011] In a specific embodiment of the present invention, the biomarker includes a gene, the protein encoded thereby, and its homologs, mutations, and isoforms. The term encompasses full-length, unprocessed biomarkers, as well as any form of biomarker derived from processing in a cell. The term encompasses naturally occurring variants of the biomarker (e.g., splice variants or allelic variants).

[0012] A second aspect of the present invention provides a product for early diagnosis or predicting the prognosis of glioblastoma.

[0013] Furthermore, the product includes a reagent for detecting the expression level of the biomarker CYP3A5 described in the first aspect of the present invention.

[0014] Furthermore, the product includes a chip, a kit, a test strip, and a high-throughput sequencing platform.

[0015] Furthermore, the reagent for detecting the expression level of the biomarker CYP3A5 in the test sample includes a reagent for detecting the expression level of the biomarker CYP3A5 mRNA in the test sample and / or a reagent for detecting the expression level of the protein and / or polypeptide encoded by the biomarker CYP3A5 in the test sample.

[0016] Furthermore, the reagent for detecting the expression level of the biomarker CYP3A5 mRNA in the test sample includes a probe that specifically recognizes the biomarker CYP3A5 and / or a primer that specifically amplifies the biomarker CYP3A5.

[0017] Furthermore, the reagent for detecting the expression level of the protein and / or polypeptide encoded by the biomarker CYP3A5 in the test sample includes an antibody that specifically binds to the biomarker CYP3A5 and / or an antibody fragment and / or an affinity protein.

[0018] In the present invention, the chip includes a gene chip and / or a protein chip. The gene chip includes: a solid-phase carrier; and oligonucleotide probes orderly immobilized on the solid-phase carrier, and the oligonucleotide probes specifically correspond to a part or all of the sequences shown by CYP3A5. The protein chip includes: a solid-phase carrier; and antibodies or ligands orderly immobilized on the solid-phase carrier, and the antibodies or ligands can specifically bind to the CYP3A5 protein.

[0019] Specifically, suitable probes can be designed according to the gene of the present invention, immobilized on a solid-phase carrier to form an "oligonucleotide array". The "oligonucleotide array" refers to an array having addressable positions (i.e., positions characterized by distinguishable, accessible addresses), and each addressable position contains a characteristic oligonucleotide connected thereto. According to needs, the oligonucleotide array can be divided into multiple sub-arrays.

[0020] "Probe" is intended to include a nucleic acid oligomer or aptamer that specifically hybridizes with a target sequence in a nucleic acid or its complement under conditions that promote hybridization, thereby allowing detection of the target sequence or its amplified nucleic acid. The detection can be direct (i.e., produced by a probe directly hybridizing with the target or amplified sequence) or indirect (i.e., produced by a probe hybridizing with an intermediate molecular structure connecting the probe and the target or amplified sequence). The "target" of a probe generally refers to a sequence in the amplified nucleic acid sequence that specifically hybridizes with at least part of the probe sequence through standard hydrogen bonds or "base pairing". The probe can be labeled or unlabeled. The probe can be produced by molecular cloning of a specific DNA sequence or can be synthesized. Those skilled in the art of the present invention can easily determine various primers and probes that can be designed and used in the context of the present invention.

[0021] In the present invention, the kit can be used to detect the expression of the CYP3A5 gene or protein. Preferably, the preparation or kit further contains a marker for labeling the RNA sample and a substrate corresponding to the marker. In addition, the kit can further include various reagents required for RNA extraction, PCR, hybridization, color development, etc., including but not limited to: extraction solution, amplification solution, hybridization solution, enzyme, control solution, color development solution, washing solution, etc. In addition, the kit also includes an instruction manual and / or chip image analysis software.

[0022] The components of the kit can be packaged in the form of an aqueous medium or in a lyophilized form. Suitable containers in the kit generally include at least one vial, test tube, flask, bottle, syringe or other container, in which one component can be placed and preferably can be appropriately aliquoted. When there is more than one component in the kit, the kit will generally also include a second, third or other additional container in which the additional components are placed separately. However, different combinations of components can be included in one vial. The kit of the present invention will generally also include a container for containing the reactants, sealed for commercial sale. Such a container can include a molded or blow-molded plastic container in which the required vials can be retained.

[0023] In the present invention, the term "sample" is used in its broadest sense. It is intended to include any tissue or material derived from a living or dead human being, which may include the biomarker of the present invention. In a specific embodiment of the present invention, the sample can be tumor or glioma tissue.

[0024] The third aspect of the present invention provides a pharmaceutical composition for treating glioblastoma.

[0025] Furthermore, the pharmaceutical composition includes an inhibitor targeting the biomarker CYP3A5 described in the first aspect of the present invention.

[0026] Furthermore, the inhibitor refers to any substance that can reduce the expression of the nucleic acid encoding CYP3A5, reduce the level of CYP3A5 protein, or inhibit the activity of CYP3A5, preferably a substance that reduces the activity of CYP3A5 protein, reduces the stability of CYP3A5 gene or protein, down-regulates the expression of CYP3A5, reduces the effective action time of CYP3A5 protein, or inhibits the transcription and translation of CYP3A5.

[0027] Preferably, the inhibitor includes Cobicistat.

[0028] Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipient.

[0029] Preferably, the pharmaceutical composition is temozolomide (TMZ) and Cobicistat.

[0030] More preferably, the ratio of Cobicistat to TMZ is 2:1.

[0031] In the present invention, the inhibitor includes, but is not limited to: nucleic acid inhibitors, protein inhibitors, proteolytic enzymes, protein-binding molecules; and combinations thereof. Among them, the nucleic acid inhibitor is selected from: an interfering molecule that targets CYP3A5 or its transcript and can inhibit the expression or gene transcription of the CYP3A5 gene, including: shRNA (small hairpin RNA), small interfering RNA (siRNA), dsRNA, microRNA, or a construct capable of expressing or forming the shRNA, small interfering RNA, dsRNA, microRNA. The protein-binding molecule is selected from: a substance that specifically binds to the CYP3A5 protein, such as an antibody or ligand capable of inhibiting the activity of the CYP3A5 protein; a competitor for the ligand-binding site of CYP3A5, including a CYP3A5 receptor and its ligand-binding fragment, a soluble truncated CYP3A5 receptor, a soluble CYP3A5 receptor fusion protein, such as a CYP3A5 fusion protein containing the Fc portion of an IgG immunoglobulin, a ligand fusion protein; peptidomimetic; peptide inhibitor; small molecule compound; and combinations thereof.

[0032] In the present invention, the "pharmaceutically acceptable carrier" refers to a carrier for administering a therapeutic agent, including various excipients and diluents. This term refers to such pharmaceutical carriers: they are not necessarily the active ingredient itself and have no excessive toxicity after administration. Suitable carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable carriers in the composition may contain liquids, such as water, saline, buffer solutions. Additionally, auxiliary substances may also be present in these carriers, such as fillers, lubricants, glidants, wetting agents or emulsifiers, pH buffering substances, etc. The carrier may also contain a cell (host cell) transfection reagent.

[0033] The present invention can use a variety of methods well known in the art to administer the inhibitor or its coding gene, or its pharmaceutical composition to a mammal. Including but not limited to: oral administration, subcutaneous injection, intramuscular injection, transdermal administration, topical administration, implantation, sustained-release administration, etc.; preferably, the administration method is oral administration.

[0034] Preferably, gene therapy means can be adopted. For example, the inhibitor of CYP3A5 can be directly administered to the subject by methods such as injection; or, the expression unit carrying the inhibitor of CYP3A5 (such as an expression vector or a virus, etc., or siRNA or shRNA) can be delivered to the target site through a certain route and made to express the active CYP3A5 inhibitor, and the specific situation depends on the type of the inhibitor, and these are all well known to those of ordinary skill in the art.

[0035] The pharmaceutical composition of the present invention can also be used in combination with other drugs for treating glioblastoma, and other therapeutic compounds can be administered simultaneously with the main active ingredient, or even in the same composition simultaneously.

[0036] The pharmaceutical composition of the present invention can also be administered to other therapeutic compounds in a separate composition or in a dosage form different from the main active ingredient. A partial dose of the main ingredient can be administered simultaneously with other therapeutic compounds, while other doses can be administered separately. During the treatment process, the dosage of the pharmaceutical composition of the present invention can be adjusted according to the severity of the symptoms, the frequency of recurrence, and the physiological response to the treatment regimen.

[0037] The fourth aspect of the present invention provides an application.

[0038] Furthermore, the application includes any one of the following:

[0039] 1) The application of a reagent for detecting the expression level of the biomarker CYP3A5 described in the first aspect of the present invention in a sample in the preparation of a product for early diagnosis or prognosis judgment of glioblastoma;

[0040] 2) The application of the pharmaceutical composition described in the third aspect of the present invention in the preparation of a product for preventing and / or treating glioblastoma;

[0041] 3) The application of a reagent for detecting the expression level of the biomarker CYP3A5 described in the first aspect of the present invention in a sample in the preparation of a product for evaluating and / or predicting the sensitivity of glioblastoma patients to TMZ. Preferably, in glioblastoma patients who are non-responsive to TMZ treatment, the expression level of CYP3A5 is significantly higher than that of patients who are responsive to TMZ treatment;

[0042] 4) The application of an inhibitor targeting the biomarker CYP3A5 described in the first aspect of the present invention in the preparation of a drug for sensitizing glioblastoma patients to TMZ.

[0043] Furthermore, the reagent for detecting the expression level of the biomarker CYP3A5 in the sample includes a reagent for detecting the expression level of the biomarker CYP3A5 mRNA in the sample, and / or a reagent for detecting the expression level of the protein and / or polypeptide encoded by the biomarker CYP3A5 in the sample.

[0044] Furthermore, the reagent for detecting the expression level of the biomarker CYP3A5 mRNA in the sample includes a probe that specifically recognizes the biomarker CYP3A5, and / or a primer that specifically amplifies the biomarker CYP3A5.

[0045] Furthermore, the reagent for detecting the expression level of the protein and / or polypeptide encoded by the biomarker CYP3A5 in the sample includes an antibody that specifically binds to the biomarker CYP3A5, and / or an antibody fragment, and / or an affinity protein.

[0046] Furthermore, the sample includes tissue and body fluid.

[0047] Further, the product includes a chip, a kit, a test strip, and a microarray.

[0048] In certain embodiments, as used herein, the determination of "protein expression level", "gene expression", or "gene expression level" includes, but is not limited to, the determination of the corresponding RNA, protein, or peptide level (or a combination thereof). The present invention is not limited to the specific methods and reagents for determining the protein, peptide, or RNA level, and all such methods and reagents are well known in the art.

[0049] In the present invention, the reagents for detecting the CYP3A5 expression level are well known in the art. Such reagents suitable for the present invention are commercially available or can be conventionally prepared by methods well known to those skilled in the art.

[0050] In the present invention, a microarray refers to a solid-phase support having a flat surface, which has a nucleic acid array. Each member in the array contains the same copy of an oligonucleotide or polynucleotide immobilized on a spatially defined region or site, and the regions or sites do not overlap with the regions or sites of other members in the array; that is, the regions or sites are discrete in space. In addition, the spatially defined hybridization sites can be "addressable" because their positions and the identities of the immobilized oligonucleotides are known or predetermined (e.g., known or predetermined before their use). Usually, the oligonucleotide or polynucleotide is single-stranded and is usually covalently linked to the solid-phase support at the 5'-end or 3'-end. The density of the nucleic acids containing non-overlapping regions in the microarray is usually greater than 100 / cm 2 , more preferably greater than 1000 / cm 2 .

[0051] In the present invention, the terms "subject", "patient", and "individual" are used interchangeably herein and refer to warm-blooded animals, such as mammals. The term includes, but is not limited to, domestic animals, rodents (e.g., rats and mice), primates, and humans. Preferably, the term refers to humans.

[0052] The fifth aspect of the present invention provides a method for regulating the CYP3A5 expression level in glioblastoma cells.

[0053] Further, the method includes the following steps: introducing a substance that inhibits the expression or activity of STAT3, or promotes the phosphorylation of STAT3, into glioblastoma.

[0054] Further, when a substance that inhibits the expression or activity of STAT3 is introduced into glioblastoma, the CYP3A5 expression level decreases; when a substance that promotes the phosphorylation of STAT3 is introduced into glioblastoma, the CYP3A5 expression level increases.

[0055] In the present invention, the term "STAT3" refers to a gene located on chromosome 17, and the encoded protein is a member of the STAT protein family, which plays an important role in various biological processes such as cell proliferation, survival, differentiation, angiogenesis, and immune regulation. The STAT3 protein consists of approximately 770 amino acids and has six functionally conserved domains, namely the amino-terminal domain, coiled-coil domain, DNA-binding domain, linker domain, SRC homology 2 domain, and carboxyl-terminal transactivation domain.

[0056] In a specific embodiment of the present invention, we found that in glioblastoma, STAT3 can regulate the expression of CYP3A5. Specifically, after knocking out STAT3, the expression level of CYP3A5 decreases, while after activating STAT3 or promoting STAT3 phosphorylation, the expression level of CYP3A5 increases.

[0057] The sixth aspect of the present invention provides a method for inhibiting the proliferation of glioblastoma cells.

[0058] Furthermore, the method includes the following steps: introducing an inhibitor of the biomarker CYP3A5 described in the first aspect of the present invention into glioblastoma cells. Optionally, the inhibitor includes a substance that inhibits the expression or activity of STAT3.

[0059] In the context of the present invention, the terms "comprising", "including", "having", "containing", or "involving" are inclusive or open-ended and do not exclude other unlisted elements or method steps. The term "consisting of" is considered a preferred embodiment of the term "comprising". If a group is defined in the text as including at least a certain number of embodiments, this should also be understood as disclosing a group preferably consisting only of these embodiments.

[0060] Advantages and beneficial effects of the present invention:

[0061] The biomarker CYP3A5 provided by the present invention is a potential therapeutic target for glioblastoma, which plays an important role in the diagnosis, prognosis, treatment, and chemotherapy resistance of glioblastoma, and can help clinicians effectively treat glioma patients, having important clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Hereinafter, the embodiments of the present invention will be described in detail with reference to the drawings, where:

[0063] Figure 1 It is a diagram for detecting the correlation between CYP3A5 and GBM progression and stemness; where Figure 1 A in it is a flow chart describing the strategy for identifying the CYP3A5 target; Figure 1Panel B shows representative immunohistochemical images and CYP3A5 expression quantification graphs of low-grade gliomas (LGG, n = 59), high-grade gliomas (HGG, n = 121), and 3 normal brain tissues detected using microarrays; Figure 1 Panel C shows box plots of CYP3A5 expression levels in individual samples of the TCGA glioma cohort stratified by WHO grade; Figure 1 Panels D - F show Kaplan–Meier curves obtained by stratifying GBM samples according to the optimal cut-off value of CYP3A5 expression levels in different datasets; Figure 1 Panel G shows a principal component analysis (PCA) plot of single-cell RNA sequencing data from 65,655 GSCs and 14,207 GBM cells; Figure 1 Panel H shows the expression of CYP3A5, CD133, SOX2, and GFAP in GSCs and their matched adherent DGCs analyzed using Western blot; Figure 1 Panel I shows immunofluorescence analysis of CYP3A5, SOX2, and CD133 in 5 randomly selected microscopic fields of 6 GBM samples;

[0064] Figure 2 These are the result graphs of in vivo and in vitro experiments verifying the inhibitory effect of CYP3A5 knockout on gliomas; among them, Figure 2 Panel A shows the result graph of CYP3A5 expression levels in glioma cells after knocking out CYP3A5; Figure 2 Panel B shows the result graph of the protein levels of SOX2 and CD133 in GSCs after knocking out CYP3A5 detected by immunoblotting experiments; Figure 2 Panel C shows representative images and quantification graphs of neurospheres in GSCs with or without knocking out CYP3A5; Figure 2 Panel D shows the result graph of the frequency of neurosphere formation after knocking out CYP3A5 detected by in vitro limiting dilution assays (ELDAs); Figure 2 Panel E shows the growth of GSCs after knocking out CYP3A5 determined by cell viability assays; Figure 2 Panel F shows representative images and quantification graphs of the proliferation index (EdU+ cells / Hoechst cells) of wild-type and CYP3A5-KO GSCs; Figure 2 Panel G shows the immunoblot results of CYP3A5, SOX2, and CD133 in GSCs with overexpression or without overexpression of CYP3A5; Figure 2 Panel H shows the neurosphere formation ability of GSCs after overexpressing CYP3A5; Figure 2 Panels I - J show representative images and quantification graphs of bioluminescence intensity of intracranial implants of U87MG GSCs with or without CYP3A5 KO in nude mice; Figure 2 Panel K shows the Kaplan-Meier survival curve of nude mice; Figure 2L is a representative figure of H&E staining of tumor sections; Figure 2 M is a representative image and quantitative figure of Ki-67 and SOX2 immunohistochemical staining;

[0065] Figure 3 is a figure showing that CYP3A5 makes tumors sensitive to TMZ; among them Figure 3 A is a box plot of the ROC plotter dataset of CYP3A5 mRNA expression in GBM patients after TMZ treatment (n = 55 responders, n = 35 non-responders); Figure 3 B is a Kaplan-Meier curve of TCGAGBM samples treated with TMZ stratified by the optimal cut-off value of CYP3A5 expression level; Figure 3 C and D are figures showing the results of cell viability assays as the concentration of TMZ increases in GSCs with or without CYP3A5KO; Figure 3 E is an immunoblot of PARP and γH2AX in control and CYP3A5 KO GSCs treated with DMSO or TMZ; Figure 3 F-H are figures showing the results of cell viability assays as the concentration of TMZ increases in GSCs with overexpression or without overexpression of CYP3A5; Figure 3 I is an immunoblot of γH2AX in control and CYP3A5-overexpressing GSCs treated with DMSO or TMZ;

[0066] Figure 4 is a figure showing the therapeutic effect of the CYP3A5 inhibitor Cobicistat (Cobi) combined with TMZ on glioma; among them, Figure 4 A is the structural diagram of Cobicistat (Cobi); Figure 4 B is a figure showing the NAD+ / NADH results of GSCs treated with indicated concentrations of Cobi; Figure 4 C is a figure showing the change in oxygen consumption rate (OCR) of GSCs treated with specified concentrations of Cobi; Figure 4 D is a figure showing the effect of Cobi on cell viability; Figure 4 E is an immunoblot analysis of PARP, γH2AX, CD133, and PUMA in GSCs treated with Cobi and TMZ; Figure 4 F is a figure showing the calculation and visualization of the synergy score of combined use of Cobi and TMZ; Figure 4 G-H are representative images and quantitative figures of apoptosis detected by flow cytometry; Figure 4 I-J are representative images and quantitative figures of bioluminescence intensity of nude mice U87MG GSCs treated by gavage with DMSO, TMZ (5 mg / kg), Cobi (10 mg / kg), or TMZ (5 mg / kg) + Cobi (10 mg / kg);Figure 4 K is the Kaplan-Meier survival curve of nude mice; Figure 4 L is the representative image of H&E staining of tumor sections; Figure 4 M is the representative image and quantitative analysis of Ki-67 immunofluorescence staining; Figure 4 N is the graph of the changes in the activities of AST and ALT in the experimental group of mice; Figure 4 O is the graph of the concentration of Cobi (10 mg / kg, gavage) detected by liquid chromatography-tandem mass spectrometry (LC-MS) in the plasma, brain and xenografts of tumor-bearing mice;

[0067] Figure 5 is the graph of the effect of STAT3 on the transcriptional level of CYP3A5 in GSCs; where Figure 5 A is the graph of the screening results of potential transcription factors of CYP3A5; Figure 5 B is the graph of the expression level of CYP3A5 mRNA analyzed by qPCR after knocking out STAT3; Figure 5 C is the graph of the results of immunoblotting detection of total STAT3, phosphorylated STAT3 (Try 705, p-STAT3) and CYP3A5 after knocking out STAT3; Figure 5 D is the graph of predicting the STAT3 binding site in the CYP3A5 promoter region using JASPAR data; Figure 5 E is the graph of the enrichment of STAT3 binding on the input by ChIP-qPCR using primers for two CYP3A5 promoter regions and a negative control primer; Figure 5 F is the immunoblotting graph of GSCs treated with epidermal growth factor (EGF) at a specified concentration; Figure 5 G is the immunoblotting graph of GSCs treated with EGF at different times; Figure 5 H is the immunoblotting graph of GSCs with overexpression or non-overexpression of EGFRVIII; Figure 5 I is the immunoblotting graph of GSCs treated with TMZ at different times; Figure 5 J is the schematic diagram of the hypothetical signaling cascade. Detailed implementation manners

[0068] In the present invention, the experiments were all completed with at least 3 repetitions, and all statistical analyses were performed using R (version 4.1.3) and GraphPad Prism (version 9.5.0). The two-tailed unpaired Student's t-test and Wilcoxon test were used to compare the differences in two groups of continuous variables. One-way analysis of variance was used to test multiple groups of data. Log-rank statistical analysis was used for survival analysis of the Kaplan-Meier survival curve. Unless otherwise specified, all data are expressed as mean ± standard deviation. A difference was considered significant when p ≤ 0.05.

[0069] The present invention will be further described below in conjunction with specific embodiments, which are only used to explain the present invention and should not be construed as a limitation to the present invention. Those of ordinary skill in the art can understand that: various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and purposes of the present invention, and the scope of the present invention is defined by the claims and their equivalents. The experimental methods used in the following embodiments are all conventional methods unless otherwise specified; the reagents, biological materials, etc. used in the following embodiments can all be obtained from commercial channels unless otherwise specified.

[0070] Example 1 Detection of the correlation between CYP3A5 and GBM progression and stemness

[0071] 1. Experimental methods

[0072] 1) Computer data acquisition

[0073] The Cancer Genome Atlas (TCGA) data was obtained using the TCGAbiolinks R package. In addition, this study also included glioma patient cohorts from GSE (http: / / www.ncbi.nlm.nih.gov / geo / ), including GSE68848, GSE13041, GSE16011, and the Chinese Glioma Genome Atlas (CGGA, http: / / www.cgga.org.cn / ).

[0074] Single-cell sequencing data of glioblastoma cells was downloaded from the Broad Institute single-cell portal website.

[0075] 2) Human glioma specimens

[0076] Informed consent was obtained from patients who underwent glioma resection in the Department of Neurosurgery, the First Medical Center of Chinese PLA General Hospital for the tumors and adjacent non-tumor tissues used in this study. The experimental protocol was approved by the Ethics Committee of the First Medical Center of Chinese PLA General Hospital (Approved by the Ethics Committee of the First Medical Center of Chinese PLA General Hospital; No. S2018-089). The tissue microarray slides contained 122 high-grade and 58 low-grade glioma samples and were operated by Shanghai Well Biotechnology Co., Ltd.

[0077] 3) Cell culture and reagents

[0078] As described above, patient-derived GSCs donated by Beijing Tiantan Hospital were used in this study. U87-MG, LN229, and U251 were purchased from the American Type Culture Collection (ATCC). Patient-derived GSCs, U87MG, LN229, and U251 cultures were cultured as neurospheres in serum-free medium consisting of DMEM / F12, vitamin A-free B27 (Invitrogen), 2 μg / mL heparin (STEMCELL), 20 ng / mL EGF (R&D), and 20 ng / mL bFGF (R&D). Spheres were dissociated with Accutase (Sigma) for in vitro experiments. GSCs were cultured in DMEM / F12 containing 10% fetal bovine serum for 7 days to differentiate into DGCs. The authenticity of all cultures was monitored by short tandem repeat (STR), and mycoplasma detection was routinely performed. Nicotinamide mononucleotide, corbisat, resveratrol, and WP1066 were purchased from MCE. Temozolomide was purchased from Sigma Aldrich.

[0079] 4) Immunoblotting

[0080] Proteins were extracted from cells using radioimmunoprecipitation assay (RIPA) lysis buffer, separated by SDS-PAGE, and then transferred to a polyvinylidene fluoride (PVDF) membrane. After incubation with the primary antibody overnight at 4°C, the membrane was incubated with an enzyme-conjugated secondary antibody.

[0081] 5) Immunoprecipitation

[0082] Equal volumes of 500 μl cell lysates were incubated with the primary antibody by rotation overnight at 4°C. The next day, pre-washed magnetic beads were added to the immune complex and rotated for 30 minutes at room temperature. The magnetic separation rack was washed 3 times, boiled at 100°C for 10 min in loading buffer, and then subjected to western blot analysis.

[0083] 6) Immunofluorescence staining

[0084] Cultures or tissue sections were fixed with 4% formaldehyde for 20 minutes at room temperature and then washed with PBS. Subsequently, they were permeabilized with 0.3% Triton X-100 for 5 minutes at room temperature. Then they were incubated with the primary antibody overnight at 4°C and then with the corresponding secondary antibody for 1 hour at room temperature. Finally, the nuclei were counterstained with DAPI.

[0085] For TSA staining, after antigen retrieval, the samples were blocked with 3% BSA at room temperature for 30 minutes, then reacted with the primary antibody overnight at 4°C, and then reacted with the appropriate secondary antibody at room temperature for 1 hour. Then, they were incubated with the tyramide signal amplification (TSA) plus fluorescein kit at room temperature for 10 minutes. The slides were heated by microwave for antibody elution. Then, the next primary antibody was added according to the above steps. The cell nuclei were counterstained with DAPI. All images were observed with an FV-1000 type laser scanning confocal microscope (Olympus Corporation, Japan).

[0086] 2. Experimental results

[0087] We attempted to identify druggable target genes that might have potential therapeutic significance for glioma patients, and the screening process is as Figure 1 shown in A. A total of 2249 druggable target genes were screened from the drug repurposing center, and four key candidate genes - CKMT2, CYP3A5, CLCN2, and PIEZO1 were identified. Among the four candidate genes, CYP3A5 performed better in differentiating prognostic outcomes using the univariate Cox proportional hazards model, and analysis of the perturbation effects of target depletion in the Cmap database showed that the normalized connectivity score of CYP3A5 was the lowest.

[0088] Then we evaluated the relationship between the expression status of CYP3A5 and the clinical and molecular characteristics of gliomas. As Figure 2 shown in B and C, the expression of CYP3A5 was significantly elevated in glioma patients and increased with the WHO grade of gliomas, and was higher in the highly invasive classical and mesenchymal subtypes. In addition, it was found from various databases that there was a significant association between elevated CYP3A5 expression and poor prognosis in GBM patients ( Figure 1 D - F), and patients with high CYP3A5 expression had a shorter survival time.

[0089] Next, we detected whether CYP3A5 was involved in the occurrence of GBM. Through the analysis of a large amount of transcriptomic data of gliomas in the TCGA and CGGA cohorts and RT-qPCR analysis of 28 frozen glioma samples in our cohort, we found that the expression level of CYP3A5 was positively correlated with most stem cell-related genes. In addition, principal component analysis of single-cell RNA sequencing data of 65,655 GSCs and 14,207 GBM cells found that regions with high CYP3A5 expression also contained cells that significantly expressed cancer stem cell markers such as PROM1, NOTCH1, and BMI1, etc. ( Figure 1G). Additionally, in glioblastoma stem cell-like cells (GSCs) maintained under serum-free conditions, the transcriptional and translational levels of CYP3A5 are higher than those in differentiated glioblastoma adherent cells (DGCs) cultured in serum-supplemented medium ( Figure 1 H). Moreover, co-immunofluorescence (IF) staining of human GBM surgical specimens revealed that CYP3A5 is preferentially expressed in cancer cells expressing the GSC markers SOX2 and CD133 ( Figure 1 I).

[0090] Collectively, these results indicate that CYP3A5 is associated with the progression and stemness of GBM, suggesting that CYP3A5 is highly expressed in glioma patients and is associated with poor prognosis in GBM patients, indicating that CYP3A5 is a potential therapeutic target for GBM.

[0091] Example 2 Therapeutic effect of CYP3A5 on glioma patients

[0092] 1. Experimental methods

[0093] 1) Xenograft study

[0094] Four-week-old female BALB / c nude mice purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. were housed in the pathogen-free barrier animal facility of Tianjin Neurological Institute, Tianjin Medical University General Hospital. All mouse experiments were conducted in accordance with the Animal Ethics and Welfare Committee of Tianjin Medical University (No: IRB2022-DWFL-069). For intracranial tumor xenografts, luciferase lentivirus-infected neurospheres were stereotactically implanted into the right frontal lobe of mice at a depth of 3 mm. The growth of tumors was monitored using an IVIS Lumina imaging station. Mice in each group were sacrificed on the same day after transplantation, and the tumor sizes were compared by H&E staining. Then, the brains were collected, fixed in 4% paraformaldehyde, and subsequently paraffin-embedded and sectioned. For survival analysis, the animals were maintained until neurological signs were observed.

[0095] 2) Construction of knockout, knockdown, and overexpression cells

[0096] To stably express cas9, we first infected glioma cells with the leti-FLAG-Cas9-Bsdr lentivirus and then screened them with 10 μg / ml Blasticidin. Then, CYP3A5 KO cells were constructed by transducing the let-sgRNA-puro lentiviral vector into cells expressing cas9. The stable cell line was transfected with 2 μg / ml puromycin, and the CRISPR-KO efficiency was detected by western blot. siRNAs targeting human STAT3 were transduced into cells using Lipofectamine-3000 (Invitrogen). For CYP3A5 overexpression, the CYP3A5 overexpression plasmid was transfected using Lipofectamine-3000. For EGFRvIII overexpression, cells were transduced with EGFR-vIII lentiviral particles and screened with 2 μg / mL puromycin. Gene knockdown and overexpression efficiencies were detected by qPT-PCR and western blot.

[0097] 3) In vitro limiting dilution assay and tumor sphere formation assay

[0098] Glioma sphere cells were seeded into 96-well plates at a density of 1, 5, 10, 20, 40, 80, and 160 cells per well, and repeated 10 times. After 7 days, cells without spheres (diameter ≥ 50 μm) were calculated and analyzed through the ELDA website (https: / / bioinf.wehi.edu.au / software / elda / ). For the tumor sphere formation assay, 1000 glioma sphere cells per well were implanted into 96-well plates and cultured for 7 days. Neurospheres with a diameter ≥ 50 μm in each well were counted.

[0099] 4) Cell viability assay

[0100] Cells were seeded into 96-well plates, with 2 or 5×10 3 cells per well, and cell proliferation and drug toxicity were observed respectively. According to the manufacturer's instructions, the CCK8 test (Dojindo) was used to detect cell viability. Cell proliferation data were normalized to day 1.

[0101] 5) EdU incorporation assay

[0102] The EdU incorporation experiment was performed using the Click-iT EdU Imaging Kit (Thermo Fisher) according to the manufacturer's protocol. Briefly, GSCs were incubated in 10 μM Edu for 2 hours. Then the cells were fixed and permeabilized, followed by the Click-iT reaction and Hoechst 33342 staining. The proliferation index was calculated by dividing the number of EDU-labeled cells by the number of Hoechst-labeled cells by blinded researchers.

[0103] 2. Experimental Results

[0104] To further explore the role of CYP3A5 in GSC maintenance, we knocked out or overexpressed CYP3A5 in GSCs. We used two non-overlapping SgRNAs to target KO of CYP3A5, resulting in effective deletion of endogenous CYP3A5 expression. After the deletion, we observed a significant decrease in the expression levels of GSC markers CD133 and SOX2 ( Figure 2 A - B). Meanwhile, inhibition of GSC self-renewal was also observed in CYP3A5 KO, and the results of sphere formation assay and in vitro limiting dilution assay supported this ( Figure 2 C - D). In addition, attenuation of the proliferation ability of GSCs after CYP3A5 KO was demonstrated by cell viability assay and EdU incorporation assay ( Figure 2 E - F). Consistent with this, after overexpression of CYP3A5, an increase in the expression of GSC markers CD133 and SOX2 and enhancement of GSC self-renewal were also detected ( Figure 2 G - H).

[0105] Next, by transducing non-overlapping SgRNAs into GSCs expressing cas9 to target knockout (KO) of CYP3A5, the growth of xenograft tumors could be inhibited, and the survival rate of mice was increased, which was demonstrated by bioluminescence imaging (BLI) analysis, hematoxylin and eosin (H&E) staining, and survival analysis ( Figure 2 I - L). Meanwhile, we found that CYP3A5 KO led to a significant decrease in the number of SOX2 and Ki67 tumor cells in GBM xenografts ( Figure 2 M). Taken together, these results indicate that inhibition of CYP3A5 expression can inhibit the proliferation of glioma cells, and at the same time demonstrate that targeting CYP3A5 can play a therapeutic role in glioma patients.

[0106] Example 3 CYP3A5 Sensitizes Tumors to TMZ

[0107] 1. Experimental Methods

[0108] Flow Cytometry Assay for Apoptosis and Cell Cycle

[0109] According to the manufacturer's instructions, apoptosis assay was performed with 7 - AAD and Annexin V (Yeasen, Cat#40310ES60). The proportion of apoptotic cells was detected by flow cytometry (BD Biosciences, USA). Cells were collected and stored in 70% ethanol at 4°C for 12 hours. Subsequently, the fixed cells were rinsed with PBS and treated with RNase I. Finally, the cells were stained with PI for 15 minutes and then subjected to cell cycle analysis using a BD flow cytometer.

[0110] 2. Experimental results

[0111] In GBM patients receiving TMZ treatment, higher levels of CYP3A5 were observed in non-responding patients compared to responding patients in the ROC plotter dataset ( Figure 3 A). Additionally, in GBM patients receiving TMZ treatment, low expression of CYP3A5 was associated with a better prognosis than high expression ( Figure 3 B). Next, we tested in vitro whether CYP3A5 promoted GSCs resistance to TMZ. Cell viability experiments with increasing TMZ doses showed that CYP3A5 KO sensitized GSCs to TMZ ( Figure 3 C-D). Moreover, CYP3A5 KO enhanced TMZ-induced DNA damage and apoptosis, as shown by increased levels of γH2AX and cleaved PARP ( Figure 3 E). Consistently, overexpression of CYP3A5 enhanced GSCs resistance to TMZ, as indicated by increased cell viability and decreased γH2AX levels after TMZ treatment ( Figure 3 F-I).

[0112] Collectively, these results demonstrated that CYP3A5 could predict whether glioma patients were sensitive to TMZ and that inhibitors targeting CYP3A5 could enhance the sensitivity of glioma patients to TMZ.

[0113] Example 4 Therapeutic effect of CYP3A5 inhibitor combined with TMZ on glioma

[0114] 1. Experimental methods

[0115] 1) Animal experiment: Four-week-old female BALB / c nude mice purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. were housed in the pathogen-free barrier animal facility of Tianjin Neurological Institute, Tianjin Medical University General Hospital. For combination therapy, 5 mg / kg TMZ (Sigma) and 10 mg / kg Cobicistat (MCE) were administered by gavage at the specified frequency.

[0116] 2) Synergy calculation method

[0117] The SynergyFinder web interface was used to evaluate the synergy of combined drug use. The HSA model was used to calculate the synergy score, and a score exceeding 10 indicated the presence of a synergistic interaction.

[0118] 3) Determination of NAD + / NADH levels

[0119] According to the manufacturer's instructions, WST-8-based NAD +The intracellular NAD⁺ / NADH ratio was evaluated using a / NADH assay kit (Beyotime). Briefly, 1×10 6 cells were collected, lysed in 200 μl of cold lysis buffer, and then centrifuged at 12,000 g for 10 min at 4 °C. To measure NADH, a portion of the supernatant was incubated at 60 °C for 30 min to selectively degrade NAD + . Subsequently, the ratio of NAD + / NADH was determined using a microplate reader at 450 nm.

[0120] 4) LC-MS analysis

[0121] Tumor-bearing mice were gavaged with cobicistat (10 mg / kg) buffer solution. Plasma, brain, and xenograft tissues were collected before dosing (0 h) and at 0.5, 1, 4, 8, and 12 h after dosing and analyzed using a Waters UPLC system. The cobicistat concentration in the samples was calculated using an internal standard curve and the sample dilution factor, and its level was determined by measuring the area under the curve and the maximum concentration.

[0122] 5) Seahorse extracellular flux assay

[0123] The oxygen consumption rate (OCR) was detected using an XFe24 extracellular flux analyzer (Agilent) according to the manufacturer's instructions. Briefly, cells were plated into Seahorse Bioscience culture plates in Seahorse XF RPMI medium buffer. Then, 2 μM oligomycin, 2 μM FCCP, and 0.5 μM rotenone / antimycin A were sequentially injected to evaluate mitochondrial function. The OCR reading data were normalized according to the cell number.

[0124] 2. Experimental results

[0125] To pharmacologically inhibit CYP3A5, cobicistat (Cobi) is an effective and selective human CYP3A inhibitor ( Figure 4 A). Cobi significantly downregulated the NAD + / NADH ratio in a dose-dependent manner in GSCs ( Figure 4 B). In addition, a decrease in OCR was also observed after treating GSCs with Cobi in a dose-dependent manner ( Figure 4 C). These results indicate that Cobi can replicate the effects of CYP3A5 deficiency on NAD + levels and mitochondrial fitness. In addition, compared with matched DGCs, Cobi showed preferential cytotoxic effects on GSCs ( Figure 4D). Next, we tested the synergistic effect of the combination of TMZ and Cobi on GSCs. As expected, Cobi enhanced TMZ-induced apoptosis and DNA damage, such as elevated levels of cleaved PARP and γH2AX ( Figure 4 E). Notably, Cobi reversed the TMZ-induced upregulation of the stemness marker CD133 and enhanced the TMZ-induced expression of the mitochondrial outer membrane permeabilization protein PUMA ( Figure 4 E). Further analysis of the drug combination dose-response matrix data and flow cytometry apoptosis assays indicated that Cobi and TMZ had a synergistic effect ( Figure 4 F-H).

[0126] Next, we determined the in vivo efficacy of the combination of Cobi and TMZ in treating GBM. Notably, in an established orthotopic GBM xenograft model, the combination of Cobi and TMZ showed superior efficacy in inhibiting tumor growth and prolonging overall survival compared to treatment with Cobi or TMZ alone ( Figure 4 I-L). Immunofluorescence staining of tumor sections showed a significant decrease in the expression of the proliferation marker Ki67 after combination treatment ( Figure 4 M). In addition, based on the assessment of serum aspartate aminotransferase and alanine aminotransferase levels and histological staining of the liver, kidney, or heart, neither single treatment nor the combination of Cobi and TMZ caused any observable signs of toxicity ( Figure 4 N). Drug distribution analysis showed that after entering the plasma, the concentration of Cobi in tumor tissue was higher than that in non-tumor brain tissue ( Figure 4 O). Collectively, these results indicate that Cobi can be delivered to brain tumors, exert a synergistic effect with TMZ, and inhibit the growth of GBM.

[0127] Example 5 In GSCs, STAT3 regulates the transcriptional level of CYP3A5

[0128] 1. Experimental methods

[0129] 1) RNA isolation and real-time quantitative PCR

[0130] Total RNA was extracted using TRIzol reagent and reverse transcribed. Then, qRT-PCR was performed using SYBR Green master mix on a QuantStudio 3 Real-Time PCR system (Thermo Scientific). Normalization was performed with GAPDH.

[0131] 2) Chromatin immunoprecipitation (ChIP) assay

[0132] Using The ChIP assay was performed according to the manufacturer's protocol using the Enzymatic Chromatin IP Kit (#9003, CST). Briefly, 1×10 7 cells were cross-linked with 1% formaldehyde for 10 minutes at room temperature and then quenched with glycine. Subsequently, the cells were collected, chromatin was digested with micrococcal nuclease, and then sonicated to obtain DNA fragments of 150 - 900 bp. For immunoprecipitation, 5 μl of STAT3 antibody (#9139, CST) was added to the diluted chromatin, and the mixture was incubated overnight with rotation at 4°C, and then magnetic beads were added for 2 h at 4°C. After eluting the chromatin from the beads and reverse cross-linking, the chip DNA samples were purified and analyzed by qRT-PCR.

[0133] 2. Experimental results

[0134] To explore the mechanism underlying the elevated expression of CYP3A5 in GSCs, we first queried the KockTF dataset and identified 23 potential transcription factors (TFs) of CYP3A5 ( Figure 5 A). We analyzed these transcription factors (TFs) using the University of California, Santa Cruz (UCSC) Genome Browser and found that the JASPAR binding scores of 2 TFs (ESRRA, STAT3) exceeded 400 ( Figure 5 A). We next tested whether STAT3 promoted CYP3A5 transcription. Notably, knockdown of STAT3 with two independent non-overlapping siRNAs led to a significant decrease in the mRNA and protein expression levels of CYP3A5 ( Figure 5 B - C). Further ChIP-qPCR analysis showed that the binding affinity of STAT3 to the CYP3A5 promoter region in GSCs was enhanced relative to the negative control site ( Figure 5 D - E).

[0135] Since the EGFR signaling pathway and TMZ have been reported to increase the level of p-STAT3, we hypothesized that STAT3 activation induced by the EGFR pathway or TMZ might potentially upregulate the expression of CYP3A5, partly explaining CYP3A5-mediated stemness and chemoresistance. After overnight starvation, treatment of GSCs with epidermal growth factor (EGF) significantly increased the levels of p-STAT3 and CYP3A5 in a time- and dose-dependent manner, accompanied by activated EGFR signaling ( Figure 5 F - G). The EGFR signal can be activated by EGFR variant III (EGFRvIII) in a ligand-independent manner. As expected, overexpression of EGFRvIII in GSCs recapitulated the effects observed with EGF treatment ( Figure 5H). In addition, a period of TMZ treatment also significantly increased the levels of p-STAT3 and CYP3A5 ( Figure 5 I). Taken together, these results suggest that EGFR signaling and TMZ can activate p-STAT3, thereby increasing the expression of CYP3A5 in GSCs ( Figure 5 J).

[0136] The description of the above embodiments is only for understanding the method of the present invention and its core idea. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A pharmaceutical composition for treating glioblastoma, characterized in that, The pharmaceutical composition consists of TMZ and Cobicistat.

2. The pharmaceutical composition according to claim 1, wherein The ratio of Cobicistat to TMZ is 2:

1.

3. The pharmaceutical composition according to claim 1 or 2, characterized in that, The dosage ratio of Cobicistat to TMZ is 10 mg / kg : 5 mg / kg.

4. Use of an inhibitor targeting CYP3A5 in the preparation of a product for preventing and / or treating glioblastoma, characterized in that, The inhibitor targeting CYP3A5 is Cobicistat.

5. Use of the pharmaceutical composition according to any one of claims 1-3 in the preparation of a product for preventing and / or treating glioblastoma.

6. Use of an inhibitor targeting CYP3A5 in the preparation of a drug for sensitizing glioblastoma patients to TMZ, characterized in that, The inhibitor targeting CYP3A5 is Cobicistat.

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