Use of chk alpha-based non-metabolic functions as targets for cancer treatment, diagnosis and prognosis prediction

Abstract

The present application belongs to the technical field of tumor medicine, and relates to application of CHK alpha-based non-metabolic function as a target for cancer treatment, diagnosis and prognosis prediction. The present application analyzes the mechanism of choline kinase alpha (CHK alpha) in tumorigenesis and development of human cancer, utilizes the non-metabolic function of CHK alpha in the regulation of phosphatidylcholine synthesis, sugar metabolism (glycolysis), glutathione synthesis, lipid droplet lipolysis, beta-oxidation and the role in tumor growth, and provides a new action target for cancer treatment, diagnosis and prognosis prediction.

Description

[0001] This application is a divisional application. The original application has the application number 202110602766.7 and the application date is May 31, 2021. The invention is entitled "Application of CHKα-based non-metabolic function as a target for cancer treatment, diagnosis and prognosis prediction". Technical Field

[0002] This invention belongs to the technical field of oncology medicine and relates to the application of non-metabolic functions of CHKα as targets for cancer treatment, diagnosis and prognosis prediction. Background Technology

[0003] Choline kinase catalyzes the phosphorylation of free choline to phosphorocholine, which is further converted to cytidine diphosphate choline by phosphorocholine cytosyltransferase, and then to phosphatidylcholine by choline phosphotransferase. Choline kinase α promotes tumor cell proliferation and survival, is highly expressed in 40%–60% of human tumors, and is positively correlated with poor prognosis in multiple tumors, playing a role in tumorigenesis and development. However, the specific mechanism by which choline kinase α (CHKα) enhances tumor progression remains unclear.

[0004] Metabolic reprogramming is a characteristic of tumor cells. The non-metabolic functions of numerous metabolic enzymes in tumor cells are receiving increasing attention and play a crucial role in tumor development and progression. Examples include the protein kinase functions of pyruvate kinase (PKM), phosphoglycerate kinase (PGK), and phosphoenolpyruvate carboxykinase (PCK).

[0005] Increased reactive oxygen species and oxidative stress in tumor cells can counteract oxidative stress and promote tumor progression through glutathione synthesis, while phosphorylation of cystathionine β synthase (CBS) can promote glutathione synthesis.

[0006] Lipid droplets are surrounded by a polar, amphiphilic monolayer of phospholipids, and their structural protein is the lipid droplet coating protein (PLIN). Lipid droplets regulate the hydrolysis of neutral lipids, such as triglycerides, sterol esters, and retinyl esters, which are used for energy production from fatty acid oxidation, membrane biogenesis, and protein modification, thus playing a crucial role in the growth of tumor cells. Summary of the Invention

[0007] The purpose of this invention is to analyze the mechanism of choline kinase α (CHKα) in tumorigenesis and development in human cancer, and to propose the application of CHKα-regulated non-metabolic functions as targets for cancer treatment, diagnosis and prognosis prediction by utilizing the role of CHKα in the regulation of phosphatidylcholine synthesis, glucose metabolism (glycolysis), glutathione synthesis, lipid droplet lipolysis, β-oxidation and tumor growth.

[0008] This invention is achieved using the following technical solution:

[0009] This invention provides an application of the non-metabolic function of CHKα as a target for cancer treatment and diagnosis, the application including:

[0010] 1) To identify the presence of elevated phosphorylation levels at CBS Y484, CHKα S279, CHKα K247, PLIN2 Y232, PLIN3 Y251, enolase 1 (ENO1) Y44, and intramolecular disulfide bonds between C303 and C307 of CHKα, compared to reference levels in the patient's cancer cells or blood.

[0011] (2) Blocking one or more of the following states by inhibition methods: activation of CHKα protein kinase, phosphorylation at CBS Y484, phosphorylation at CHKαS279, acetylation at CHKαK247, phosphorylation at PLIN2 Y232, phosphorylation at PLIN3 Y251, phosphorylation level of enolase 1 (ENO1) Y44, and formation of intramolecular disulfide bonds between C303 and C307 of CHKα; and / or

[0012] (3) Predicting the patient's favorable response to treatment methods;

[0013] The reference level is derived from levels in non-cancerous or early-stage cancerous cells, or from the patient's blood.

[0014] Preferably, the cancer is oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory system cancer, genitourinary system cancer, gastrointestinal cancer, central or peripheral nervous system cancer, endocrine or neuroendocrine system cancer or hematopoietic system cancer, glioma, sarcoma, epithelial cancer, lymphoma, melanoma, fibroma, meningioma, brain cancer, kidney cancer, biliary tract cancer, pheochromocytoma, islet cell carcinoma, Levi-Flaumeni tumor, thyroid cancer, parathyroid cancer, pituitary adenoma, adrenal adenoma, or osteosarcoma.

[0015] Tumors, neuroendocrine system tumors, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer.

[0016] Preferably, the determination method includes using phosphorylation-specific antibodies to perform ELISA, immunoassay, radioimmunoassay, immunohistochemistry, immunoradioassay, fluorescence immunoassay, gel electrophoresis, Western blotting, in situ hybridization, flow cytometry, or microscopy.

[0017] Preferably, the inhibition method includes using a CHKα inhibitor, a CBS Y484 inhibitor, a CHKαS279 inhibitor, a CHKαK247 inhibitor, a PLIN2 Y232 inhibitor, a PLIN3 Y251 inhibitor, or any other method that inhibits CHKα protein kinase activity, or any other method that inhibits phosphorylation at CBS Y484, phosphorylation at CHKαS279, acetylation at CHKαK247, phosphorylation at PLIN2 Y232, phosphorylation at PLIN3 Y251, phosphorylation level of enolase 1 (ENO1) Y44, or the formation of an intramolecular disulfide bond between C303 and C307 of CHKα.

[0018] Preferably, the CHKα inhibitor comprises a small molecule inhibitor targeting CHKα protein kinase activity. The CBS Y484, CHKαS279, CHKαK247, PLIN2 Y232, and PLIN3 Y251 inhibitors selectively target phosphorylation at CBS Y484, phosphorylation at CHKαS279, acetylation at CHKαK247, phosphorylation at PLIN2 Y232, phosphorylation at PLIN3 Y251, the phosphorylation level of enolase 1 (ENO1) Y44, and the formation between C303 and C307 of CHKα.

[0019] Intramolecular disulfide bonds in peptides, small molecule inhibitors, or complementary inhibitory polynucleotides.

[0020] Preferably, the beneficial responses include reduced tumor size or burden, tumor growth arrest, reduced tumor-related pain, reduced cancer-related pathological conditions, reduced cancer-related symptoms, cancer progression-free status, prolonged disease-free interval, prolonged progression time, induced remission, reduced metastasis, prolonged patient survival, or increased sensitivity of the tumor to anticancer therapy.

[0021] This invention also provides the application of CHKα-based protein kinase activity as a target for cancer prognosis prediction, the application including:

[0022] (1) Determine whether the patient’s cancer cells or blood contain: elevated phosphorylation levels at CBSY484, CHKαS279, CHKαK247, PLIN2 Y232, PLIN3 Y251, and various combined expression levels of these five sites compared to reference levels.

[0023] (2) If cancer cells or the patient’s blood contain elevated levels of any of (1), the patient is predicted to have invasive cancer.

[0024] (3) If cancer cells or the patient’s blood contain elevated levels of any of (1), the patient is predicted to have an aggressive cancer in the advanced stage.

[0025] (4) If cancer cells or the patient’s blood contain elevated levels of any of (1), the patient is predicted to have a poor prognosis.

[0026] The reference level is derived from levels in non-cancerous or early-stage cancerous cells, or from the patient's blood.

[0027] Furthermore, if the patient has invasive cancer, inhibitory anticancer therapy is performed by blocking one or more of the following mechanisms: activation of CHKα protein kinase, phosphorylation at CBS Y484, phosphorylation at CHKαS279, acetylation at CHKαK247, phosphorylation at PLIN2 Y232, phosphorylation at PLIN3 Y251, phosphorylation level of enolase 1 (ENO1) Y44, and formation of intramolecular disulfide bonds between C303 and C307 of CHKα.

[0028] The beneficial effects of this invention are as follows: This invention utilizes any upstream signal that can activate oxidative stress signals, leading to the formation of an intramolecular disulfide bond between C303 and C307 of CHKα, thereby phosphorylating the CBS Y484 site, enhancing the binding of CBS to pyridoxal phosphate, and promoting de novo glutathione synthesis, cell proliferation, and tumor growth in tumor cells. This invention also utilizes any upstream signal that can bind CHKα to lipid droplets (including phosphorylation of CHKα at S279 and acetylation of CHKα at K247 caused by glucose deprivation), leading to a conformational change in the catalytic domain of CHKα, resulting in protein kinase activity and phosphorylation of the PLIN2 Y232 and PLIN3 Y251 sites. The phosphorylated PLIN2 / 3 dissociates from the lipid droplets and is degraded by Hsc70-mediated autophagy, thereby promoting lipid droplet lipolysis, β-oxidation, and tumor growth. The importance of the newly identified choline kinase CHKα protein kinase activity in de novo glutathione synthesis, lipolysis, β-oxidation, and tumor growth was emphasized. Importantly, the expression levels of CBS Y484 phosphorylation, CHKα S279 phosphorylation, CHKα K247 acetylation, PLIN2 Y232 phosphorylation, and PLIN3 Y251 phosphorylation were significantly upregulated in tumor tissues, with significant positive correlations between these expression levels, and were associated with tumor progression and poor prognosis. This invention utilizes any signal that enables CHKα to bind to ENO1 and phosphorylate enolase 1 (ENO1) Y44 (including the binding site mutations CHKαF199N / P200N and ENO1 Y44F) to activate, thereby blocking TRIM25 binding and polyubiquitination of CHKα (K195 site) and proteasome degradation of CHKα, thus promoting choline phospholipid metabolism and tumor growth. Importantly, the expression level of ENO1 phosphorylation at the Y44 site was significantly upregulated in tumor tissues and was negatively correlated with longer survival in cancer patients. Attached Figure Description

[0029] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof.

[0030] In the attached diagram:

[0031] Figure 1 For example 1, biotin-pulp assays were performed on LN229 cells after H2O2 treatment, followed by immunoblotting analysis using tubulin as a marker; where WLC was whole cell lysate; Cyto was cytoplasmic extract; and Nuc was nuclear extract.

[0032] Figure 2 This is a three-dimensional structure of the human CHKα protein from Example 1.

[0033] Figure 3 This is the biotin pull-down experiment of Example 1; where WLC is whole cell lysate.

[0034] Figure 4 The image shows the immunoblot analysis of Flag and His after H2O2 treatment in the Ni-NTA pull-down experiment of Example 2, using tubulin as a marker.

[0035] Figure 5 For the in vitro kinase assay experiment in Example 2, after H2O2 treatment, CHKα, CBS, 32 Immunoblot analysis of P-CBS and CBS pY484.

[0036] Figure 6 The pattern used to fill the bar charts for the in vitro kinase assay (left side) and CBS activity assay (right side) of Example 3 is as follows.

[0037] Figure 7 The pattern used to fill the bar charts in Example 4 is a replacement for the ROS level detection experiment (left side) and GSH / GSSG ratio detection experiment (right side).

[0038] Figure 8 For the tumor size detection experiment in Example 5, ***P<0.001 (two-tailed Student's t-test). C1, clone 1; C2, clone 2. Change the pattern used to fill the bar chart.

[0039] Figure 9 Survival curves for CBS Y484 phosphorylation levels in glioma patients of Example 6.

[0040] Figure 10 After glucose-deficient treatment of tumor cells in Example 7, the expression of CHKα and CHKβ was detected by immunoblotting analysis, using tubulin as a marker.

[0041] Figure 11 Immunofluorescence analysis was performed on the colocalization of CHKα, ATGL, Beclin1, and lipid droplets in tumor cells after glucose deprivation treatment in Example 8.

[0042] Figure 12 For the immunoblotting analysis of CHKα binding to lipid droplets in Example 9, tubulin was used as a marker.

[0043] Figure 13 For the immunoblotting analysis of CHKα binding to lipid droplets in Example 10, tubulin was used as a marker.

[0044] Figure 14For the immunoprecipitation and immunoblotting analysis of Example 11, microtubules were used as markers to determine the binding of CHKα to PLIN2 and PLIN3.

[0045] Figure 15 For the immunoprecipitation and immunoblotting analysis of CHKα binding to PLIN2 / 3 in Example 12, microtubules were used as a marker.

[0046] Figure 16 Pull-down analysis and Western blot analysis were performed on the Ni-NTA agarose beads from Example 13 to analyze the CHKα phosphorylation sites of PLIN2Y232 and PLIN3Y251. Tubulin was used as a marker.

[0047] Figure 17 The molecular dynamics simulation analysis of CHKα in Example 14.

[0048] Figure 18 For the immunoprecipitation and immunoblotting analysis of PLIN2 / 3 and Hsc70 in Example 15, tubulin was used as a marker.

[0049] Figure 19 The immunofluorescence analysis of PLIN2 / 3 and lipid droplets in Example 16 shows the colocalization of these two components.

[0050] Figure 20 The immunofluorescence analysis of Example 16 shows the colocalization of ATGL, Beclin1, LC3B with lipid droplets.

[0051] Figure 21 This is a statistical graph of tumor cell proliferation detected under 2-DG treatment conditions in Example 17.

[0052] Figure 22 The results show the size of a mouse glioma as measured in Example 18.

[0053] Figure 23 The results show the accumulation of lipid droplets in a mouse glioma tissue sample from Example 18.

[0054] Figure 24 Immunohistochemical assays were performed on 100 human glioma samples using the ACC S79 phosphorylated antibody, CHKαS279 phosphorylated antibody, CHKαK247 acetylated antibody, PLIN2 Y232 phosphorylated antibody, and PLIN3 Y251 phosphorylated antibody from Example 19.

[0055] Figure 25 The correlation of the expression levels of the tumor markers shown in Example 19 in 60 human glioma samples was detected.

[0056] Figure 26Survival curves for ACC S79 phosphorylation level, CHKαS279 phosphorylation level, CHKαK247 acetylation level, PLIN2 Y232 phosphorylation level, and PLIN3 Y251 phosphorylation level in glioma patients of Example 19.

[0057] Figure 27 The GST pull-down assay and immunoblotting analysis of Example 20 were used to study the binding of ENO1 and CHKα.

[0058] Figure 28 Immunoprecipitation and immunoblotting analysis of CHKα were performed in Example 21 to determine its ubiquitination status.

[0059] Figure 29 The immunoprecipitation and immunoblotting analysis of Example 22 were used to determine the binding of TRIM25 and CHKα.

[0060] Figure 30 The immunoprecipitation and immunoblotting analysis of CHKα ubiquitination sites in Example 22.

[0061] Figure 31 The immunoprecipitation and immunoblotting analysis of Example 23 were used to determine the binding of TRIM25 and CHKα.

[0062] Figure 32 The intracellular phosphatidylcholine production was detected in Example 24.

[0063] Figure 33 This is an in vitro kinase experiment for Example 25.

[0064] Figure 34 This is for the detection of lactic acid production in Example 26. Detailed Implementation

[0065] To make the objectives and technical solutions of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions; unless otherwise specified, the reagents and materials are commercially available.

[0066] I. Testing Method:

[0067] 1. Detection of phosphorylated protein levels:

[0068] The amount of antibody bound is determined by contacting a sample with an antibody that specifically binds to a phosphorylated peptide, for example by detecting or measuring the path of the complex between the antibody and the peptide. The antibody can be labeled (radioactive, fluorescent, etc.) to facilitate the detection of the complex.

[0069] The peptide level detection system used in this invention includes: autoradiographic immunoassay (RIA), immunofluorescence, gel electrophoresis, and Western blotting.

[0070] The antibody used (added): tubulin (sc-8035) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA);

[0071] Anti-CHKα, ENO1, LC3B, ATGL, Beclin 1, ACC pS79, CHK antibodies were purchased from CellSignaling Technology (Danvers, MA); Rabbit antibodies that recognize CHK, CBS(pY484), CBS, CHKαpS279, CHKαAcK247, PLIN2 pY232, PLIN3 pY251 was purchased from Signalway Biotechnology (Pearland, TX); Mouse monoclonal anti-Flag (F1804), rabbit anti-Flag (F7425), anti-His (SAB1305538) antibodies were purchased from Sigma-Aldrich (St. Louis, MO).

[0072] 2. Acquisition and analysis of mouse tumor samples:

[0073] Huh7 cells (1×10⁶) were injected intracranially into athymic nude mice (n=7 / group). Mice were euthanized 28 days after injection, and HCC tumor growth was examined.

[0074] 3. Immunohistochemical (IHC) staining detection of proteins in cells (tissue microarray construction):

[0075] Formalin-fixed paraffin-embedded tissue was obtained by surgical excision and stained with Mayer’s hematoxylin and eosin (H&E; Biogenex Laboratories, San Ramon, California).

[0076] Tissue microarray (TMA) processing was performed on tumor samples and normal tissue samples from cancer cases. An automated tissue array instrument was used. Alphelys (Plaisir, France) extracted cancerous tissue (2 mm in diameter, selected by the pathologist) from each specimen and fixed it in paraffin blocks. After quality control, the TMA blocks were sectioned for immunohistochemical analysis.

[0077] The secondary antibody, anti-Rabbit IgG heavy chain (HRP) (ab99702), was purchased from Abcam (Cambridge, MA).

[0078] 4. Pearson correlation test

[0079] This invention uses the Pearson correlation test to determine the correlation between protein expression in human glioma specimens.

[0080] Immunohistochemical analysis was performed according to previous publications (see Nucleus-TranslocatedACSS2 Promotes Gene Transcription for Lysosomal Biogenesis and Autophagy. Molecular cell. 2017; 66(5):684-97e9). After deparaffining, rehydration, and antigen retrieval, TMA slides were incubated overnight at 4°C with primary antibody rabbit anti-human AKT pS473 (dilution 1:200), primary antibody rabbit anti-human phosphorylated PCK1 pS90 (dilution 1:200), primary antibody rabbit anti-human INSIG1 pS207 and INSIG2 pS151 (dilution 1:500), primary antibody rabbit anti-human SREBP1 (dilution 1:100), or nonspecific IgG (as negative control). The slides were then incubated with anti-rabbit secondary antibody (ready-to-use solution; Cell Signaling Technology; #8114), followed by dichromatographic diaminobenzidine (DAB) staining (Cell Signaling Technology) and hematoxylin staining, and fixed on xylene. The tissue slides were quantitatively scored under a microscope based on the percentage of positive cells and staining intensity. The present invention assigns the following proportional scores: 0, 0% of cells are positive; 1, 0% to 1%; 2, 2% to 10%; 3, 11% to 30%; 4, 31% to 70%; and 5, from 71% to 100%. Staining intensity was also rated on a scale of 0 to 3:0 (negative): 1, weak; 2, moderate; 3, strong. As previously described in the literature, the proportional and intensity scores were then summed to obtain a total score (range 0–8). The reproducibility of the scoring system was independently verified by two pathologists without clinical knowledge.

[0081] 5. Overall survival rate of patients (Kaplan-Meier plot)

[0082] Data analysis was performed using SPSS version 20.0 software (SPSS Inc., Chicago, Illinois, USA). Independent samples t-tests were used to compare biomarker expression levels in tumor and normal tissues. One-way ANOVA (post hoc Bonferroni test) was used for multiple comparisons and least significant differences tests to assess the correlation between biomarker expression levels and patient clinicopathological characteristics. Pearson correlation coefficients were used to analyze the correlation between biomarker expression levels. Overall survival (OS) was defined as the duration from diagnosis to death or the last follow-up. K-means cluster analysis was used to categorize the expression levels of relevant biomarkers, Kaplan-Meier survival curves were plotted, log-rank tests were used to compare survival rates, and Cox regression models with two-way Wald tests were used to calculate hazard ratios (HR) and 95% confidence intervals (CIs) for survival analysis. Data reviewed were used for patients who were alive at the last follow-up or who were lost at follow-up. Variables with a p-value less than 0.05 were included in multivariate analyses. P < 0.05 was considered statistically significant. All statistical tests are two-sided.

[0083] II. The materials used in the following embodiments are as follows:

[0084] 1. Cell types:

[0085] Huh7 cells (human liver cancer cell line Huh7 cells) and LN229 cells (human glioma cells) were derived from ATCC.

[0086] 2. The athymic nude mice are BALB / c athymic nude mice;

[0087] 3. Patient samples:

[0088] Tissue samples of surgically resected, formalin-fixed, and paraffin-embedded NSCLC were retrospectively collected from the biobank of the Zhejiang University Translational Research Institute (Hangzhou, China). One hundred patients pathologically diagnosed with glioma who had not undergone surgical treatment were selected as an independent cohort. Clinical data were obtained by reviewing patient medical histories. Pathological staging was assessed according to the 8th edition of the American Joint Committee on Cancer / International Federation for Cancer Control TNM Classification System.

[0089] 4. The shRNA sequence used for gene knockdown is as follows:

[0090] CHKα:TGATACTAAAGACGGTATTAA

[0091] PLIN2: CAGAAGCTAGAGCCGCAAATT

[0092] PLIN3: CTGGACCACATGGTGGAATAT

[0093] ATGL: CCTGCCACTCTATGAGCTTAA

[0094] Beclin 1:GCTTGGGTGTCCTCACAATTT

[0095] ENO1: CGCATTGGAGCAGAGGTTTAC

[0096] TRIM25:CCGGAACAGTTAGTGGATTTA

[0097] Example 1: CHKα is a direct oxidative stress sensor

[0098] 1. Intramolecular disulfide bond formation in CHKα molecules is a direct event in cellular response to oxidative stress.

[0099] LN229 cells were treated with 50 μM H2O2. Oxidized proteins were labeled with biotin-maleimide and purified using streptavidin-agarose beads. Figure 1 As shown, disulfide bonds are formed within the CHKα molecule when treated with H2O2 for 2 minutes.

[0100] 2. Intramolecular disulfide bonds of CHKα are formed between C3O3 and C3O7.

[0101] (1) As Figure 2 As shown, the human CHKα structure (PDB: 2CKO) reveals the spatial positions of C303 and C307. The regions surrounding C303 and C307 are outlined and magnified. Intramolecular disulfide bonds are formed between the sulfur atoms of C303 and C307.

[0102] (2) Expression of HA-labeled wild-type CHKα, CHKαC303A, or CHKαC307A in LN229 cells. Cells were treated with the reducing agent N-acetylcysteine ​​(NAC) for 30 min, followed by treatment with 50 μM H2O2 for 10 min. Oxidized proteins were labeled with biotin-maleimide and purified using streptavidin-agarose beads. WLC, whole-cell lysate. Figure 3 As shown, in LN229 cells, CHKα's C303 or C307 (only separated by a distance of 0.05 in the CHKα structure) The mutation eliminates the formation of disulfide bonds and allows CHKα to migrate more quickly.

[0103] Example 2: Oxidative stress leads to CHKα binding and phosphorylation of CBS

[0104] 1. After H2O2 treatment, CHKα combines with CBS.

[0105] LN229 cells expressing the Flag-tagged CHKα and the His-tagged CBS were treated with NAC for 10 min, followed by treatment with 50 μM H2O2 for 10 min. Ni-NTA assays showed that H2O2 treatment induced CHKα binding to CBS, and this binding was blocked by NAC treatment. Figure 4 )

[0106] 2. Oxidative stress causes CHKα to phosphorylate the Y484 site of CBS.

[0107] Purified wild-type Flag-CHKα was immobilized on magnetic beads, treated with H2O2 for 10 minutes, washed with PBS, and then incubated with purified wild-type His-CBS or His-CBS Y484F in the presence of 32P-ATP for in vitro kinase assay. Results are as follows: Figure 5 H2O2 treatment causes phosphorylation of CBS, but the Y484F mutation in CBS blocks this phosphorylation.

[0108] Example 3: CHKα-mediated CBS phosphorylation activates CBS.

[0109] Purified wild-type Flag-CHKα or its mutant protein was immobilized on magnetic beads, treated with 50 μM H2O2 for 10 min, then treated with 10 mM DTT for 30 min, washed with PBS, and then incubated with purified wild-type His-CBS or His-CBSY484F in the presence of ATP. In vitro kinase assays (left) and CBS activity assays (right) were performed. Results are as follows: Figure 6 Phosphorylation of CBS at Y484 enhances CBS activity.

[0110] Example 4: CHKα-mediated CBS phosphorylation enhances de novo glutathione synthesis.

[0111] LN229 wild-type cells and LN229 cells with CHKαC307A, CHKαS298A / P299A, or CBS Y484F mutations were treated with 0.25 mM paraquat for 6 hours. Intracellular ROS levels (left) and the GSH / GSSG ratio (right) were then measured. Figure 7 As shown, CHKα-mediated phosphorylation of CBS at the Y484 site activates CBS, increasing de novo glutathione synthesis (a direct response of tumor cells to counteract oxidative stress).

[0112] Example 5: CHKα-regulated glutathione synthesis promotes tumor growth.

[0113] LN229 wild-type cells, as well as CHKαC307A, CHKαS298A / P299A, or CBS Y484F mutant LN229 cells, were intracranially injected into athymic nude mice (n=14 per group). One week later, liposomal doxorubicin (20 mg / kg) was injected intracranially every three days (for a total of four times) (n=7 per group). Tumor size was assessed one month later. Figure 8 As shown, doxorubicin inhibits tumor growth, while the expression of CHKαC307A, CHKαS298A / P299A, or CBS Y484F mutations reduces tumor growth and enhances the tumor-inhibiting effect of doxorubicin.

[0114] Example 6: CBS Y484 phosphorylation level is negatively correlated with survival in patients with glioma.

[0115] The phosphorylation level of CBS Y484 in glioma samples was divided into high and low expression, and a Kaplan-Meier plot of overall patient survival was plotted. Figure 9 As shown, the phosphorylation level of CBS Y484 site is negatively correlated with the survival of patients with glioma.

[0116] Example 7: CHKα binds to lipid droplets and is required for lipid droplet lipolysis.

[0117] Huh7, U87, GP06, and GP08 cells were treated with glucose deficiency for 1 hour, and the expression of CHKα in whole-cell lysate, cytoplasm, and lipid droplets was detected. Figure 10 As shown, glucose deficiency treatment causes a small fraction of cytoplasmic CHKα, rather than CHKβ, to bind to lipid droplets.

[0118] Example 8: CHKα is required for lipid droplet lipolysis.

[0119] Huh7 cells with or without CHKα or CHKβ knockdown were treated with glucose deficiency for 1 hour. Immunofluorescence was then performed on the cells using BODIPY, DAPI, or antibodies recognizing Beclin 1 or ATGL. Figure 11 As shown, only CHKα knockdown inhibited the colocalization of ATGL and autophagy protein Beclin1 with lipid droplets induced by glucose deficiency. This result demonstrates that CHKα is required for lipid droplet lipolysis under glucose-deficient conditions.

[0120] Example 9: AMPK-mediated phosphorylation of CHKα at S279 site to CHKα-binding lipid droplets under glucose-deficient conditions.

[0121] Huh7 cells were treated with 5 μM Compound C (AMPK inhibitor) for 30 minutes followed by glucose deprivation for 1 hour, or with 0.5 mA769662 (AMPK activator) for 30 minutes. Lipid droplets were purified and analyzed using Western blotting. Figure 12 As shown, Compound C treatment blocked the binding of CHKα to lipid droplets induced by glucose deficiency, while A769662 treatment still promoted this binding even under glucose-free conditions.

[0122] Example 10: Phosphorylation of CHKα at S279 leads to KAT5-mediated acetylation of CHKα at K247 and recruitment of CHKα to lipid droplets.

[0123] Huh7 cells expressing wild-type Flag-CHKα or the Flag-CHKαK247R mutation were subjected to glucose deprivation for 1 hour, lipid droplets were purified, and immunoprecipitation analysis was performed using Flag antibody. Figure 13 As shown, the K247R mutant CHKα (with no effect on phosphorylation at CHKαS279) does not translocate to lipid droplets.

[0124] Example 11: CHKα combined with PLIN2 / 3

[0125] Through immunoprecipitation and immunoblotting analysis, such as Figure 14 As shown, in Huh7 cells treated with glucose deficiency for 1 hour, CHKα binds to PLIN2 and PLIN3.

[0126] Example 12: Monomer CHKα bound to PLIN2 / 3

[0127] Huh7 cells expressing Flag-PLIN2, Flag-PLIN3, wild-type His-CHKα, or CHKα mutations were treated with glucose deprivation for 1 hour to obtain whole-cell lysates. A drop-down analysis was performed using Flag antibody agarose beads and Ni-NTA agarose beads. Figure 15 As shown, CHKα transforms from a dimer to a monomer, enabling it to bind to PLIN2 and PLIN3.

[0128] Example 13: CHKα phosphorylation of PLIN2 Y232 and PLIN3 Y251 sites

[0129] Huh7 cells expressing wild-type His-PLIN2, His-PLIN2 Y232F mutation, wild-type His-PLIN3, and His-PLIN3 Y251F mutation were treated with hypoxia for 1 hour, and then analyzed using Ni-NTA agarose beads. Figure 16 As shown, glucose deficiency treatment leads to phosphorylation at PLIN2 Y232 and PLIN3 Y251 sites, which is blocked by the PLIN2 Y232F mutation and the PLIN3 Y251F mutation.

[0130] Example 14: CHKα monomerization mediated by acetylation at the K247 site altered the structure of its catalytic domain and phosphorylated the PLIN2 Y232 and PLIN3 Y251 sites.

[0131] Molecular dynamics simulation analysis of CHKα dimer and monomer yielded the following results: Figure 17 As shown, the top image displays the overall profile of the monomeric CHKα docking with either the PLIN2 Y232 (LHSRAYQQALS) peptide or the PLIN3 Y251 (LRQHAYEHSLG) peptide. The bottom image is a magnified view. The distance between the γ-phosphate group of ATP and the OH group of PLIN2 Y232 or PLIN3 Y251 was measured. Computerized docking analysis revealed that only the monomeric, expanded choline-binding pocket of CHKα allows the catalytic domain of either the PLIN2 Y232 or PLIN3 Y251 peptide to interact with CHKα; and PLIN2 Y232... Or PLIN3 Y251 The OH group of ATP is close enough to the γ-phosphate group of ATP to allow for the transfer of phosphate groups.

[0132] Example 15: Under glucose-deficient conditions, CHKα phosphorylation of PLIN2 / 3 promotes the binding of PLIN2 / 3 to Hsc70.

[0133] Huh7 cells expressing HA-Hsc70, wild-type Flag-PLIN2, wild-type Flag-PLIN3, PLIN2 Y232F mutation, or PLIN3 Y251F mutation were treated with BSA-bound oleic acid for 12 hours and then subjected to glucose deprivation for 1 hour, followed by immunoprecipitation analysis using Flag agarose beads. Results are as follows: Figure 18 As shown, the PLIN2 Y232F mutation or the PLIN3 Y251F mutation blocks the interaction between PLIN2 / 3 and Hsc70.

[0134] Example 16: Under glucose-deficient conditions, CHKα-mediated PLIN2 / 3 phosphorylation leads to the recruitment of ATGL and autophagosomes to lipid droplets for lipolysis.

[0135] U87 wild-type cells and U87 cells with CHKαS279A, CHKαK247R, PLIN2 Y232F, or PLIN3 Y251F mutations were treated with BSA-bound oleic acid for 12 hours, followed by glucose deprivation for 2 hours. Cells were then stained with BODIPY, DAPI, PLIN2 antibody, and PLIN3 antibody, and immunofluorescence analysis was performed. Results are as follows: Figure 19 and Figure 20The CHKαS279A mutation, CHKαK247R mutation, PLIN2 Y232F mutation, or PLIN3 Y251F mutation blocked glucose deficiency-induced dissociation of PLIN2 / 3 from lipid droplets. Figure 19 ), and ATGL, Beclin1, and LC3B dissociate from lipid droplets ( Figure 20 ).

[0136] Example 17: CHKα-mediated lipolysis promotes tumor cell survival

[0137] Huh7 cells expressing CHKα shRNA, PLIN2 shRNA, PLIN3 shRNA, reconstructed wild-type Flag-rCHKα, reconstructed wild-type Flag-rPLIN2, reconstructed wild-type HA-PLIN3, or mutations as shown in the figure, were treated with 25 mM 2-DG for 48 hours, followed by cell counting. Figure 21 As shown, under 2-DG treatment, mutations expressing CHKα or PLIN2 / 3 further reduced cell proliferation.

[0138] Example 18: CHKα-mediated lipolysis promotes brain tumor growth

[0139] U87 cells, as well as U87 cells expressing CHKαS279A mutation, CHKαK247R mutation, PLIN2 Y232F / PLIN3 Y251F mutation (PLIN2 / 3Mut), ATGL shRNA, or Beclin1 shRNA, were administered intracranially to athymic nude mice. Two weeks later, 0.2 ml of 2-DG (500 mg / kg) was injected intraperitoneally daily for 14 days. Tumor size was then assessed (e.g., ...). Figure 22 The left side shows the HE staining results, representing a representative tumor xenograft, while the right side shows the tumor volume measurement. Frozen sections of tumor tissue were prepared and then stained with red oil O. The percentage of areas stained with red oil O was calculated (e.g.,...). Figure 23 The results showed that expression of these mutations, or ATGL shRNA or Beclin1 shRNA, inhibited tumor growth and increased lipid droplet accumulation.

[0140] Example 19: CHKα-mediated lipolysis plays a key role in the malignant clinical manifestations of gliomas.

[0141] Use as Figure 24 Immunohistochemistry was performed on 100 glioma samples using the antibody shown. The results showed positive correlations among the phosphorylation levels of ACC S79, CHKαS279, CHKαK247, PLIN2 Y232, and PLIN3 Y251 (e.g., ...). Figure 25In a sample of 60 patients, the phosphorylation levels of ACC S79, CHKαS279, CHKαK247 acetylation, PLIN2Y232, and PLIN3Y251 were classified as high or low expression. Kaplan-Meier plots of overall survival were then constructed, and the results are as follows: Figure 26 As shown, the phosphorylation levels of CHKαS279, CHKαK247, PLIN2Y232, and PLIN3Y251 were positively correlated with poor prognosis in patients with glioma.

[0142] Example 20: Combination of ENO1 and CHKα

[0143] After co-incubating purified GST-CHKα with purified ENO1, the results were obtained by GST pull-down assay as follows: Figure 27 There is a binding between NO1 and CHKα.

[0144] Example 21: ENO1 inhibits CHKα ubiquitination

[0145] Flag-CHKα, His-Ub, and ENO1 shRNAs were expressed in U87 cells, and analyzed by immunoprecipitation and Western blotting, such as... Figure 28 As shown, knocking down ENO1 enhances CHKα ubiquitination.

[0146] Example 22: TRIM25 binds to and ubiquitinates the K195 site of CHKα.

[0147] Mutations of Flag-CHKα, HA-TRIM25, CHKαK195R, CHKαK273R, CHKαK276R, and CHKαK325R were expressed in U87 cells. Immunoprecipitation and Western blot analysis were performed, such as... Figure 29 TRIM25 binds to CHKα; such as Figure 30 The CHKαK195R mutation reduces its ubiquitination level.

[0148] Example 23: The binding of ENO1 to CHKα reduces the binding of TRIM25 to CHKα.

[0149] HA-TRIM25, Flag-CHKα, Flag-CHKαF199N, and Flag-CHKαP200N were expressed in U87 cells. Immunoprecipitation and Western blot analysis were performed, such as... Figure 31 As shown, mutations in the binding sites of ENO1 and CHKα increase the binding of TRIM25 to CHKα.

[0150] Example 24: Upregulation of CHKα by ENO1 promotes phosphatidylcholine production

[0151] ENO1 shRNA and TRIM25 shRNA were expressed in U87 cells, and the levels of phosphatidylcholine in the cells were detected. Figure 32 As shown, knocking down ENO1 reduced the production of phosphatidylcholine, while knocking down TRIM25 enhanced the production of phosphatidylcholine; knocking down ENO1 on the basis of knocking down TRIM25 weakened the production of phosphatidylcholine.

[0152] Example 25: CHKα phosphorylation of ENO1 Y44 site

[0153] Through in vitro kinase assays, immunoprecipitation, and Western blotting, such as... Figure 33 As shown, CHKα phosphorylates ENO1 at site Y44.

[0154] Example 26: CHKα-mediated ENO1 phosphorylation enhances tumor cell glycolysis.

[0155] like Figure 34 As shown, knocking out CHKα or knocking down ENO1 both reduced lactate production in LN229 tumor cells, thus reducing glycolysis.

[0156] Of course, the above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of reagents for detecting the phosphorylation level of CHKα S279 site and / or the acetylation level of CHKα K247 site in the preparation of prognostic prediction products for glioma.

2. The application according to claim 1, characterized in that, When the phosphorylation level of CHKα S279 site and / or the acetylation level of CHKα K247 site are elevated compared with the reference level, it indicates that the patient has a poor prognosis. The reference level is derived from non-glioma cells or early-stage glioma cells.

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