RRM1 T52 phosphorylation antibody and its application in glioma.

Abstract

The present application relates to a kind of RRM1T52 phosphorylation antibody and its application in brain glioma, belong to biological medicine technical field.The present application first proposes that RRM1T52 can be used as new index for predicting diagnosis glioblastoma, provides new judging basis for the progress of current clinical glioblastoma patient after using temozolomide treatment, perfects the early diagnosis of present stage to glioblastoma.The present application finds biomarker for glioblastoma drug resistance based on the mechanism of glioblastoma drug resistance, and early diagnosis of glioblastoma can be realized by detecting biomarker expression level, changes the limitation and credibility of previous detection means and index.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an RRM1 T52 phosphorylated antibody and its application in glioma. Background Technology

[0002] To date, cancer has become a major threat to the health and lives of the Chinese people, severely impacting their quality of life. According to the latest national cancer statistics released by the National Cancer Center in 2022, detailing the cancer burden in China in 2016, the crude incidence and mortality rates of cancer in my country continued to rise from 2000 to 2016. Brain tumors and other nervous system cancers account for 1%-4% of new cancer cases and 3% of cancer deaths. Glioblastoma (GBM) is the most common primary malignant brain tumor in adults and also the deadliest cancer of the central nervous system. Its high malignancy results in the lowest overall survival rate, with a 5-year survival rate of only 0.05%-4.7% after diagnosis.

[0003] Patients diagnosed with glioblastoma typically have a poor prognosis, and their quality of life deteriorates as the disease progresses. Current diagnostic methods for glioblastoma primarily rely on: 1. Clinical manifestations: memory impairment, seizures, and headaches; 2. Imaging features: including invasive, heterogeneous, ring-enhancing lesions with central necrosis and surrounding tumor edema; 3. Pathological diagnosis. However, these methods are insufficient for early detection and diagnosis of glioblastoma. Often, the tumor is only diagnosed after it has progressed to a certain stage and the patient has developed severe symptoms, at which point the optimal treatment window has often been missed, leading to irreversible consequences.

[0004] The high heterogeneity of glioblastoma leads to a lack of clearly effective treatment methods. Furthermore, due to the complex mechanisms of its development and progression, research remains insufficient. Currently, key challenges in its treatment focus on drug resistance in tumor cells and high recurrence rates. The standard clinical treatment is surgical resection followed by chemoradiotherapy. Temozolomide (TMZ) has been shown to be the only chemotherapy drug capable of improving the survival of GBM patients; however, even after a standard course of treatment, the median survival remains only 12-15 months. Moreover, the mechanism of GBM cell resistance to TMZ is still unclear, significantly delaying the development of new diagnostic and therapeutic methods for gliomas. Only through a more comprehensive and systematic understanding of the mechanisms of chemoradiotherapy resistance in gliomas and the identification of biomarkers for glioblastoma drug resistance can we overcome the current bottlenecks in glioblastoma diagnosis and truly achieve early diagnosis of glioblastoma from the perspective of drug resistance.

[0005] TMZ is a small lipophilic molecule of only 194 Da and an orally administered imidazole tetrazine monofunctional DNA alkylating agent. It is rapidly and completely absorbed by the body and spontaneously decomposes to form monomethyltriazine 5-(3-methyltriazine-1-yl)-imidazolium-4-carboxamide (MTIC). MTIC then reacts with water to release 5-aminoimidazolium-4-carboxamide (AICA) and a highly reactive methyldiazonium ion. This highly reactive methyldiazonium ion can methylate DNA at the N7 position of the guanine-rich region, and also leads to methylation of the N3 adenine and O6 guanine sites, causing DNA damage in tumor tissue. However, this reaction can be reversed by oxy-6-methylguanine-DNA methyltransferase (MGMT). Therefore, current in-depth research mainly focuses on the regulation of TMZ-derived methyldiazonium ions, but the role of its derivative AICA in TMZ chemotherapy resistance remains unclear.

[0006] TMZ is currently a standard treatment for glioblastoma patients, but its actual efficacy is poor, and intrinsic and acquired TMZ resistance is a major clinical challenge in GBM treatment. TMZ chemoresistance is mainly attributed to the removal of DNA methyl adducts mediated by oxo-6-methylguanine-DNA methyltransferase (MGMT) and the activation of DNA damage repair systems, such as mismatch repair, base excision repair, non-homologous end joining (NHEJ), and homologous recombination (HR) repair. Adequate DNA damage repair requires sufficient deoxyribonucleoside triphosphates (dNTPs), which are produced by the reduction of ribonucleoside diphosphates (NDPs) mediated by ribonucleotide reductase (RNR). Ribonucleotide reductase (RNR) is a key enzyme initiating the de novo biosynthesis of dNTPs; besides the rescue pathway through nucleotide phosphorylation to regenerate nucleotides, this process is the only way to produce deoxyribonucleoside triphosphates. RNR is a heterotetramer composed of the large subunit RRM1 and the small subunit RRM2. It primarily regulates its enzymatic activity and substrate specificity by binding to different small nucleotides through allosteric changes, and all allosteric regulatory sites are located in RRM1, indicating that RRM1 plays a major role in maintaining the homeostasis of the intracellular nucleotide pool. As a key subunit of RNR, RRM1 participates in regulating cell proliferation, cell migration, tumor metastasis, and the synthesis of deoxyribonucleotides for DNA synthesis; however, its potential value in combating TMZ resistance in glioblastoma is poorly understood. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention discloses for the first time RRM1 pT52 as one of the drug resistance markers for glioblastoma and successfully established a detection protocol for this marker. Furthermore, in clinical practice, it can be combined with the existing TMZ resistance marker MGMT for more accurate diagnosis of patients, while also improving current routine diagnostic protocols for glioblastoma and temozolomide resistance. The establishment of the RRM1 pT52 detection method overcomes the limitations and reliability of previous detection methods and markers.

[0008] The first objective of this invention is to provide a molecular marker for detecting glioma, which is the 52nd threonine residue of the RRM1 gene (RRM1 T52 site), and the phosphorylation level at this site indicates the disease status.

[0009] Furthermore, the higher the phosphorylation level at the RRM1 T52 site, the higher the risk of disease and the worse the prognosis.

[0010] Furthermore, the amino acid sequence of the RRM1 gene is shown in SEQ ID NO.1 (Gene ID: 6240).

[0011] A second objective of this invention is to provide the application of the RRM1 T52 site of the RRM1 gene in the preparation of a glioma detection kit for detecting the phosphorylation level of the RRM1 T52 site.

[0012] Furthermore, it is used for the diagnosis of early, intermediate, or late-stage glioblastoma.

[0013] Furthermore, the test kit contains an RRM1 T52 phosphorylated antibody.

[0014] Furthermore, the detection kit contains a first antibody and a second antibody. The first antibody is an RRM1 T52 phosphorylated antibody (RRM1 pT52), and the second antibody is an antibody homologous to the first antibody and carrying a recognition marker. The recognition marker can be an enzyme label such as horseradish peroxidase. Samples can be collected from the patient's venous peripheral blood, and ELISA detection is performed sequentially using the first antibody (RRM1 T52 phosphorylated antibody) and the second antibody (homogeneous to the first antibody and labeled with horseradish peroxidase), greatly reducing the psychological burden and physical harm to the subjects.

[0015] A third objective of this invention is to provide the application of the RRM1 T52 site of the RRM1 gene in the preparation of drugs for the prevention or treatment of glioma.

[0016] Furthermore, the drug is used to reduce the phosphorylation level at the RRM1 T52 site.

[0017] Furthermore, the drug contains an RRM1 T52 phosphorylated antibody.

[0018] A fourth objective of this invention is to provide the application of the RRM1 T52 site of the RRM1 gene in the preparation of anti-drug-resistant tumor drugs or tumor drug resistance detection reagents, wherein the tumor is a glioma.

[0019] Furthermore, the drug resistance is temozolomide resistance.

[0020] Furthermore, drug resistance testing or anti-drug resistance treatment can be performed using RRM1 T52 phosphorylation antibodies.

[0021] The fifth object of the present invention is to provide an RRM1 T52 phosphorylated antibody, which is prepared using the sequence shown in SEQ ID NO.2 as the antigen.

[0022] A sixth object of the present invention is to provide the use of the above-mentioned RRM1 T52 phosphorylation antibody in the preparation of diagnostic reagents or therapeutic agents for diseases related to AMPK / RRM1 interaction, wherein the antibody is used to block the interaction between AMPK and RRM1, thereby inhibiting the phosphorylation of threonine at position 52 of RRM1 by AMPK.

[0023] Furthermore, the diseases associated with AMPK / RRM1 interaction include, but are not limited to, brain tumors, head and neck tumors, lung cancer, gastric cancer, liver cancer, prostate cancer, endometrial cancer, hematopoietic and lymphatic system malignancies, cardiovascular diseases, and autoimmune diseases.

[0024] By means of the above-described solution, the present invention has at least the following advantages:

[0025] 1) This invention provides a solid theoretical basis for the early prevention and diagnosis of glioblastoma, and proposes that RRM1 can be used as a new indicator for predicting the diagnosis of glioblastoma. It provides a new basis for judging the progression of glioblastoma patients after treatment with temozolomide in clinical practice, and improves the current early diagnosis and prognosis of glioblastoma.

[0026] 2) This invention proposes a new indicator and method for detecting whether a patient has developed resistance to temozolomide, by directly detecting the RRM1 T52 site as a resistance indicator for temozolomide treatment of glioblastoma.

[0027] 3) This invention establishes an RRM1 pT52 antibody, which can be used in immunohistochemistry and immunoblotting analysis. This antibody achieves qualitative detection of temozolomide resistance by specifically recognizing this site.

[0028] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the following describes the preferred embodiments of the present invention in conjunction with detailed drawings. Attached Figure Description

[0029] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0030] Figure 1 This diagram illustrates the breakdown of TMZ into AICA and methyldiazo cations in the body.

[0031] Figure 2 The results of TMZ treatment increasing the levels of AICA and AICAR in glioma cells.

[0032] Figure 3 Figure showing the results of TMZ treatment of glioma cells, which showed dose- and time-dependent activation of AMPK.

[0033] Figure 4 The results of AICA treatment that can activate AMPK in a dose- and time-dependent manner.

[0034] Figure 5 The effect of methyldiazo cations derived from TMZ on DNA damage.

[0035] Figure 6 The effect of methyldiazo cations derived from TMZ on the activation level of the AMPK signaling pathway.

[0036] Figure 7 The figure shows the results of the reactive oxygen species scavenger NAC partially inhibiting the activation of the AMPK signaling pathway by TMZ.

[0037] Figure 8 This diagram illustrates the structural differences between AICA and AICAR.

[0038] Figure 9 A diagram illustrating the phosphoribosyltransferase reaction between AICA and AICAR mediated by the unique transferase HPRT1.

[0039] Figure 10 In in vitro enzyme activity assays, HPRT1 can catalyze the formation of AICAR from AICA.

[0040] Figure 11 The graph shows the results of chemotherapy sensitivity to AICA or AMPK activator A769662-gliotropic TMZ.

[0041] Figure 12 Figure showing the results of enhanced TMZ chemosensitivity in gliomas after knocking out AMPKα1 and α2.

[0042] Figure 13 To confirm the binding of AMPK to RRM1 via immunoprecipitation.

[0043] Figure 14 Mass spectrometry analysis revealed phosphorylation at the RRM1 T52 site.

[0044] Figure 15 TMZ treatment induces phosphorylation at the RRM1 T52 site, but phosphorylation cannot be achieved after mutation of this site.

[0045] Figure 16 Knockout of AMPK affects TMZ-induced phosphorylation of RRM1 T52.

[0046] Figure 17 HPRT1 knockout reduces TMZ-induced RRM1 T52 phosphorylation.

[0047] Figure 18 Phosphorylation of RRM1 T52 can enhance RNR activity, thereby promoting the binding ability of RNR to ATP.

[0048] Figure 19 Figure showing the results of chemosensitivity of HPRT1 knockout or RRM1-expressing T52A gliomas to TMZ.

[0049] Figure 20 The use of the HPRT1 inhibitor 6-MP resulted in a decrease in AMPK-mediated phosphorylation levels at the RRM1 T52 site.

[0050] Figure 21 To facilitate the treatment of tumor-bearing mice, relevant molecular markers were detected in mouse tumor sections using IHC.

[0051] Figure 22 The ROC curve for TMZ resistance.

[0052] Figure 23 To detect the expression of HPRT1, AMPK pT172, and RRM1 pT52 in 50 clinical samples of relapsed GBM by immunohistochemistry and to analyze the correlation between their expression levels and patient prognosis. Detailed Implementation

[0053] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0054] The terms involved in this invention are explained as follows:

[0055] Glioblastoma (GBM): Glioblastoma is the most malignant glioma among astrocytic tumors. The tumor is located subcortical, mostly growing in various supratentorial hemispheres of the cerebral body. It exhibits infiltrative growth, often invading several lobes and deep structures, and can also spread to the contralateral cerebral hemisphere via the corpus callosum. The frontal lobe is the most common site of occurrence.

[0056] Temozolomide (TMZ): As an anti-tumor drug, it spontaneously and rapidly degrades in the body to produce active metabolites, thereby exerting its anti-tumor effect. It is used as a treatment for adult malignant gliomas and malignant melanomas.

[0057] This product is a dabenzimidazole class of alkylating antitumor drugs, and is an analogue of mitozolamide. It is one of the most effective drugs for the clinical treatment of brain tumors. It can cross the blood-brain barrier, has few toxic side effects, and is well tolerated.

[0058] O-6-methylguanine-DNA methyltransferase (MGMT): A DNA repair enzyme that prevents mutations in cellular DNA by removing the alkyl group at the 6-position of oxygen on guanine and can repair damaged DNA.

[0059] Mismatch repair: refers to a repair mechanism used when a DNA molecule experiences a base mismatch due to the insertion or deletion of bases during replication.

[0060] Base excise repair (BER) is an important repair mechanism for maintaining DNA stability. It can remove DNA damage caused by factors such as deamination or base loss, oxy-free radiation, or methylation of cyclic nitrogen compounds caused by endogenous substances.

[0061] Non-homologous end joining (NHEJ): A special DNA double-strand break repair mechanism in eukaryotic cells that forcibly joins two DNA breaks together without relying on DNA homology in order to avoid the retention of DNA or chromosome breaks and the resulting DNA degradation or impact on vitality.

[0062] Homologous Recombination Repair (HRR) is one of the core repair mechanisms for DNA double-strand damage. It uses homologous DNA templates to repair DNA double-strand breaks and is initiated by excision of the DNA break ends to generate a long single-stranded DNA strand for strand invasion, thereby completing the repair of the double-strand damage site.

[0063] Deoxyribonucleoside triphosphates (dNTPs): a collective term including dATP, dGTP, dTTP, and dCTP, which play a role as raw materials in biological DNA synthesis.

[0064] Ribonucleotide reductase (RNR): Ribonucleotide reductase is widely found in various organisms and is the only enzyme in living organisms that catalyzes the reduction of four ribonucleotides to the corresponding deoxyribonucleotides. This enzyme is also a key and rate-limiting enzyme in DNA synthesis and repair, and plays a regulatory role in cell proliferation and differentiation.

[0065] Example 1

[0066] I. Testing

[0067] The detection method of this invention is Western blotting. Western blotting, or protein immunoblotting, is a technique commonly used in research to isolate and identify proteins. It utilizes SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to separate various proteins contained in a specified sample. The separated proteins are then transferred to a nitrocellulose or PVDF membrane, which is subsequently incubated with a specific antibody against the target protein. During membrane washing, unbound antibodies are washed away, leaving only antibodies bound to the target protein. Finally, the bound antibodies are detected by developing film or fluorescence scanning. Because the antibody binds only to the target protein, generally only a clear band is visible, with the band thickness corresponding to the protein content. By analyzing the location and intensity of specific reactions, the expression information of the target protein in a given cell or tissue homogenate can be obtained. Due to the high resolution of gel electrophoresis and the high specificity and sensitivity of immunoblotting, Western blotting analysis can detect target proteins as low as 1 ng. This method is widely used in molecular biology fields such as molecular biology, biochemistry, and immunogenetics. The specific detection steps are as follows:

[0068] 1. Prepare samples

[0069] 1) Sample collection: Collect peripheral blood from the vein, let it stand on ice for 15 minutes, and centrifuge at 3500 rpm for 15 minutes to obtain serum for testing.

[0070] 2) Make ice and prepare ice boxes;

[0071] Preparation of cell lysis buffer: Determine the required lysis buffer based on the number of cells: 50 μl / well of a six-well plate;

[0072] Lysis buffer formulation: 100 μl Beyotime lysis buffer + 1 μl protease inhibitor mixture + 1 μl PMSF.

[0073] 3) Prepare cells according to experimental requirements: Remove the culture medium, wash 3 times with 1×PBS (to remove serum from the culture medium), and add an appropriate amount of lysis buffer to each well (6-well plate). Quickly scrape cells off with a cell scraper and transfer them to a 1.5ml tube, place on ice for 20min, vortex to mix, and then place on ice for another 10min.

[0074] 4) Centrifuge at 12,000g, 4℃ for 15 min, and collect the supernatant into another 1.5ml tube.

[0075] 5) Take 2.5 μl of sample and dilute it with 22.5 μl of triple-distilled water for use in the BCA method to determine protein concentration.

[0076] 6) Add 5× Loading Buffer (2.5ml Buffer / 10ml protein) to the remaining samples and boil at 95℃ for 10 minutes, shaking once during the process.

[0077] 7) Directly load the sample for gel running or dispense it for long-term storage at -80℃.

[0078] 2. SDS-PAGE polyacrylamide gel electrophoresis

[0079] 1) Prepare the separating gel (5 ml / gel).

[0080] 2) Carefully inject the separating gel, leaving about 2cm of space (below the red border of the gel casting frame) for the concentrating gel. Cover the top with deionized water and let it stand for about 30 minutes.

[0081] 3) Prepare the concentrated gel (2 ml / gel).

[0082] 4) Pour the stacking gel into the top of the separating gel, being careful to avoid air bubbles.

[0083] 5) Insert the comb and wait for the concentrated gel to solidify (there is a clear boundary between the gel and the comb, and the solidification time of the separating gel should be greater than 2 hours). Clean the hole with double-distilled water to remove gel fragments, and then blot dry with filter paper.

[0084] 6) Place the gel into the electrophoresis tank, and add 1× electrophoresis buffer to both the upper and lower tanks (do not reuse more than 3 times).

[0085] 7) Sample loading: Take 5 μl of prestained marker into the marker well and add an appropriate amount of 1× loading buffer to make the total volume the same as the sample well. The sample loading volume is generally 15-25 μl. First, boil it in a heating block at 95℃ for 5-10 minutes, shaking once during the process, then quickly centrifuge and load the sample for gel running; add an equal volume of 1× loading buffer to the wells without sample loading.

[0086] 8) Electrophoresis: Start with a constant voltage of 60-80V. After running through the stacking gel, increase the current to 100-120V. The electrophoresis time is determined according to the size of the target protein and the position of the marker. Generally, the target protein is ready when it reaches two-thirds of the separating gel.

[0087] 3. Membrane transfer

[0088] 1) Cut the gel according to the marker indication and the position of the target band (note to mark the cut corner of the gel). Immerse the eluted gel in the transfer buffer for 15 minutes.

[0089] 2) After marking the PVDF membrane, immerse it in methanol for 1 minute, then immerse it along with 4 sheets of 3mm filter paper and a sponge in transfer buffer for 15 minutes.

[0090] 3) Preparing the "sandwich cookie":

[0091] The order is as follows: fiber mat -- filter paper -- PVDF membrane -- gel -- filter paper -- fiber mat;

[0092] Note: Align each item as you add it, making sure there are no air bubbles.

[0093] 4) Transfer: The transfer time is determined according to 1).

[0094] One side of the PVDF membrane is connected to the positive electrode (red), and the other side of the gel is connected to the negative electrode (black).

[0095] 4. Membrane blocking and antibody incubation

[0096] 1) After the transfer is complete.

[0097] 2) Incubate a 5% skim milk powder solution at room temperature for 2 hours or at 4°C with gentle shaking overnight. Since milk powder is relatively difficult to dissolve, it should be prepared at least 1 hour in advance.

[0098] 3) Wash the membrane three times with 10ml of PBS / T, each time for 5 minutes.

[0099] 4) Add 5 ml of primary antibody dilution buffer (divide the antibody according to the instructions), incubate at room temperature for 2 hours or at 4°C with gentle shaking overnight, recover the primary antibody, add sodium azide (which can inhibit bacterial growth) at 5 μl / ml of primary antibody solution, and store at 4°C (less frequently used antibodies can be stored at -20°C for a long time). It can be reused.

[0100] 5) Wash the membrane three times with 10ml of PBS / T, each time for 5 minutes.

[0101] 6) Add the secondary antibody (usually diluted 1:2000) and shake gently at room temperature for 1 hour.

[0102] 7) Wash the membrane three times with 10ml of PBS / T, each time for 5 minutes.

[0103] 5. Development and fixing (or direct fluorescence scanning after incubation with fluorescently labeled secondary antibody)

[0104] 1) Developing steps: First, lay down plastic wrap, then place another layer of plastic wrap on top of absorbent paper. Pour water, developer (if the color darkens, do not use), and fixer into their respective trays. Mix 2.5ml of ECL-A and 2.5ml of ECL-B, and keep away from light. Take the ECL mixture, film, etc., into the darkroom. Close and lock the door, and pull back the curtain. Pour the ECL mixture into a small box. Slightly pat the film dry with absorbent paper and place it in the ECL mixture, shaking at room temperature for 5 minutes (to ensure the ECL is evenly spread over the film). After patting dry with absorbent paper, place it on plastic wrap, film side down, and place it on a clip. Cut an X-film (note that you can only hold the edge of the X-film) and place it on the film, cutting off one corner as a marker (Note: When handling the film, turn the light to minimum and do not face the light; keep your back to the light). Adjust the exposure time according to the brightness of the strips. Generally, you can expose for 2 minutes first, observe the depth of the strips, and then determine the optimal exposure time.

[0105] 2) Development and fixing: After taking out the X-film and placing it in the developer for a certain period of time (depending on the intensity of the target band and the background), wash it once with water and then place it in the fixer for at least 5 minutes.

[0106] Finally, the target protein band on the PVDF membrane is obtained. By comparing the size with the protein marker, the size and location of the target protein can be determined. Then, by comparing with the control, the result of the change in the expression of the target protein can be obtained.

[0107] II. Diagnostic Analysis of Test Results

[0108] Elevated RRM1 T52 phosphorylation levels compared to the reference level indicate that the subject has invasive cancer, advanced invasive cancer, or a poor prognosis.

[0109] III. The biomarkers of this invention were obtained through the following research:

[0110] 1. Materials and Methods

[0111] Materials used:

[0112] (1) Cell types:

[0113] U87, T98G, and LN18 cells were obtained from ATCC. U251 cells were purchased from Sigma-Aldrich (Shanghai, China).

[0114] (2) The athymic nude mice are BALB / c athymic nude mice.

[0115] (3) Patient samples:

[0116] Tissue samples were obtained from the First Affiliated Hospital of Nanjing Medical University. IHC staining was performed on human glioma tissues to compare protein expression between tumor and normal tissue samples. The staining of tissue sections was quantitatively scored based on the percentage of positive cells and staining intensity.

[0117] 2. Results and Analysis

[0118] Studies have found that when TMZ is used to treat glioma patients, it is metabolized into methyldiazo cations and AICA under physiological conditions. Figure 1 Methyldiazo cations can cause DNA damage, and AICA can be further reacted to form AICAR ( ). Figure 2 ,8).

[0119] AICAR, as an AMP analog, stimulates AMPK expression, which is crucial for many key cellular activities. We observed a dose- and time-dependent activation of AMPK in glioma cells after treatment with TMZ, as evidenced by elevated levels of AMPK pT172 and its substrate ACC1 pS79. Figure 3 Similarly, the same result was obtained after directly using AICA. Figure 4 Although methyldiazo cations derived from TMZ induce ROS production and thus activate the AMPK pathway, the effect is not significant. Figure 5-7 Among them, treatment of cells with MESNA and WR-1065 can react with active methyl groups to eliminate methyl diazo cations from TMZ; treatment of cells with NAC can effectively remove ROS, indicating that TMZ can activate AMPK mainly through the production of AICA and downstream AICAR.

[0120] To investigate the specific mechanism by which AICA transforms into AICAR, based on the structural differences between AICA and AICAR, AICAR has an additional ribose-5 phosphate group compared to AICA. Figure 8 We hypothesized that a specific phosphoribosyltransferase mediated the reaction. To confirm this hypothesis, we knocked out five known phosphoribosyltransferases in GBM cells and found that only the knockout of hypoxanthine phosphoribosyltransferase (HPRT1) led to an increase in intracellular AICA levels, because it could not be converted into AICAR. Figure 9 Furthermore, we confirmed through protein purification and in vitro enzyme activity assays that HPRT1 can effectively mediate the conversion of AICA. Figure 10 This indirectly affects the activation of the AMPK pathway.

[0121] The current prevalence of TMZ resistance in clinical patients primarily stems from tumor cells' ability to repair TMZ-induced DNA damage. Further investigation into the specific DNA damage repair mechanism revealed that pretreatment of GBM cells with AICA or AMPK activators can reduce TMZ-induced apoptosis. Figure 11 (12) This indicates that activation of the AICA and AMPK pathways may be a key factor in DNA damage repair during GBM. To clarify the AICA or AMPK-mediated DNA damage repair mechanism, we used immunoprecipitation tandem mass spectrometry to identify the potential downstream target gene that might be regulated as the catalytic subunit RRM1 of RNR. AMPK can interact with RRM1 and phosphorylate the evolutionarily conserved site RRM1 T52. Figure 13 ,14), but currently there are no studies targeting RRM1T52 phosphorylation, nor are there antibodies that specifically recognize phosphorylation at this site. Therefore, we synthesized the RRM1 pT52 peptide (CQGLYSGVT(P)TVELDCQGLYSGVTTVELD, SEQ ID NO.2) and used it as an antigen to immunize rabbits to prepare antibodies that specifically recognize RRM1 pT52. We found that TMZ significantly activated the AMPK signaling pathway and promoted the phosphorylation level of the RRM1 T52 site ( Figure 15 Knocking out AMPKα1 / 2 or knocking down HPRT1 can significantly inhibit the regulatory effect of TMZ on the phosphorylation level of RRM1 T52 site. Figure 16 ,17). RRM1, as a catalytic subunit of RNR, affects RNR activity. We confirmed through biochemical experiments that AMPK can enhance RNR activity and promote the binding affinity of RNR to ATP ( ). Figure 18 The main physiological function of RNR is to generate sufficient dNTPs for DNA damage repair. Studies have confirmed that RRM1 pT52 promotes the repair of TMZ-induced DNA damage and prolongs GBM cell survival. Figure 19 The combined use of the HPRT1 inhibitor 6-MP significantly inhibited the regulatory effect of TMZ on the phosphorylation level of RRM1 T52 site. Figure 20 ).

[0122] We demonstrated through animal experiments that inhibiting HPRT1-mediated RRM1 pT52 significantly increased the sensitivity of tumor cells to TMZ. Figure 21 This further supports the key role of HPRT1-regulated RRM1 pT52 in TMZ chemotherapy resistance, providing a theoretical basis for its use as a new clinical diagnostic and therapeutic indicator.

[0123] Our ROC curve analysis showed that RRM1 pT52 has high reliability as a clinical diagnostic and therapeutic indicator of TMZ resistance in GBM patients (AUC = 0.805). Figure 22 ).

[0124] Meanwhile, we collected 50 clinical samples of relapsed GBM patients who had undergone TMZ chemotherapy. Immunohistochemical analysis revealed that HPRT1, AMPK pT172, and RRM1 pT52 can serve as clinical diagnostic markers to indicate patient prognosis. Figure 23 ).

[0125] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. Use of an RRM1 T52 phosphorylation antibody for the preparation of a kit for the detection of brain glioma, characterized in that: Prepared using the sequence shown in SEQ ID NO. 2 as an antigen.

2. Use of the RRM1 T52 phosphorylation antibody for the manufacture of a medicament for the prevention or treatment of brain glioma, characterized in that: Prepared using the sequence shown in SEQ ID NO. 2 as an antigen.

3. Use of RRM1 T52 phosphorylation antibody in the preparation of a test reagent for detecting the resistance of brain glioma to temozolomide, characterized in that: Prepared using the sequence shown in SEQ ID NO. 2 as an antigen.

4. An RRMl T52 phosphorylation antibody characterized by: Prepared using the sequence shown in SEQ ID NO. 2 as an antigen.

5. Use of the RRM1 T52 phosphorylation antibody of claim 4 in the preparation of a diagnostic reagent for brain glioma.

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