cGAS Methylation Modification as a Tumor Diagnosis and Prognosis Analysis Marker, and Its Detection Reagent and Application

Abstract

The present invention discloses the methylation modification of cGAS, which can be used as a marker for tumor diagnosis and prognosis analysis, specifically the methylation modification of human cGAS K362 or murine cGAS K350, and also discloses the detection reagents and applications of the above-mentioned cGAS methylation modification. The above-mentioned methylation modifications of human cGAS K362 and murine cGAS K350 can be used as markers for tumor diagnosis and prognosis analysis, especially playing an important role in tumor immune escape in colorectal cancer and melanoma; the present invention also prepares antibodies that specifically recognize the above-mentioned cGAS methylation modification. Verification shows that the expression level of cGAS methylation modification in tumor tissues is significantly higher than that in normal tissues, and is significantly negatively correlated with the prognosis of tumor patients; inhibiting cGAS methylation modification combined with immune checkpoint inhibitors can inhibit the occurrence and development of tumors. The cGAS methylation modification described in the present invention can be used as a marker for tumor diagnosis and prognosis analysis, and can clearly and accurately characterize tumor immune escape and poor prognosis, providing an important reference basis for clinical diagnosis and treatment plans.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor markers, and in particular to cGAS methylation modification that can be used as a marker for tumor diagnosis and prognosis analysis, and its detection reagent and application. Specifically, the cGAS methylation modification includes human cGAS K362 methylation modification and murine cGAS K350 methylation modification. Background Art

[0002] Tumors, as major diseases, seriously threaten human life and health. With the rise of immune checkpoint monoclonal antibody therapy and CAR-T cell therapy, great achievements have been made in tumor immunotherapy. Currently, the immune checkpoint molecules that have been studied more deeply in clinical research include: cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), programmed death receptor 1 (PD-1) and its ligand (PD-L1). The use of immune checkpoint blockers, PD-1 and PD-L1 antibodies, to block the PD-1 / PD-L signaling pathway has shown good curative effects in clinical trials and has become the standard treatment method for various malignant tumors including hematological tumors and solid tumors such as non-small cell lung cancer. However, although immune checkpoint monoclonal antibody therapy has achieved gratifying results in clinical anti-tumor applications, there are still significant differences in the response rates among different tumors. Currently, the objective remission rate of known PD-1 antibodies in Hodgkin lymphoma can reach 66% - 95%, while in tumors such as malignant melanoma, it is only 10% - 30%. Some patients may also experience disease superprogression after receiving monoclonal antibody therapy. Therefore, in clinical applications, finding biomarkers with high sensitivity and high specificity and new targets for tumor immunotherapy has important clinical value for tumor treatment.

[0003] The cGAS-STING-TBK1 signaling pathway plays an important role in the body's recognition of cytoplasmic DNA and initiation of type I interferon response-mediated anti-tumor effects. During radiotherapy and chemotherapy, the DNA recognition receptor of dendritic cells (DC cells), cyclic GMP-AMP synthase (cGAS), recognizes DNA fragments after tumor cell death and catalyzes the generation of 2',3'-cyclic GMP-AMP (cGAMP). The second messenger cGAMP activates the downstream STING-TBK1 signaling pathway and interferon regulatory factor 3 (IRF3). Thereafter, phosphorylated IRF3 translocates to the nucleus, promoting the generation of type I interferon and antigen presentation, and enhancing the ability of T cells to activate and kill tumor cells. Therefore, in-depth study of the regulation of interferon signaling initiated by the cGAS-STING-TBK1 pathway and analysis of its effects on the specific infiltration and infiltration degree of immune cells in tumor tissues have important guiding significance for anti-tumor immunotherapy.

[0004] However, in the current prior art, the mechanism by which cGAS methylation modification affects cGAS activity has rarely been reported, and the application of cGAS methylation modification in anti-tumor immunotherapy and tumor prognosis analysis has not been reported either. Summary of the Invention

[0005] In order to overcome at least one problem existing in the prior art, based on the fact that the cGAS-mediated DNA sensing signaling pathway promotes the generation of type I interferon, promotes antigen presentation, enhances T cell activation, and plays an important role in anti-tumor immunity, and the inhibition of cGAS activity leads to tumor immune escape and reduced sensitivity to immune checkpoint blocker therapy, the present invention for the first time discovers that cGAS methylation modification (specifically human cGAS K362 methylation modification or murine cGAS K350 methylation modification) inhibits cGAS activity, is highly expressed in tumor tissues, and at the same time shows a negative correlation with the prognosis of patients; the present invention provides a tumor marker for simultaneously characterizing tumor diagnosis and patient prognosis analysis, especially clearly characterizing tumor immune escape and poor prognosis, and obtains a modified antibody for specifically detecting the marker, forming a detection method with good development prospects, providing an important reference basis for clinical diagnosis and treatment plans.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention is to provide a marker for tumor diagnosis and / or prognosis analysis, and the marker is cGAS methylation modification.

[0008] Further, the cGAS methylation modification is human cGAS K362 methylation modification or murine cGAS K350 methylation modification.

[0009] Further, the methylation modification is monomethylation modification.

[0010] Further, the tumors include hematological tumors and solid tumors, etc., such as intestinal cancer and melanoma; specifically, it can be colorectal cancer and melanoma; more specifically, it can be colorectal cancer.

[0011] In a specific embodiment, the above cGAS methylation modification (specifically, it can be cGAS K362 methylation modification) is significantly highly expressed in colorectal cancer tissues, and at the same time shows a significant negative correlation with the prognosis of tumor patients.

[0012] The second aspect of the present invention is to provide an application of a marker for tumor diagnosis and / or prognosis analysis as described in any one of the first aspects of the present invention, and the application is selected from at least one of the following applications: application in preparing a detection reagent or kit for the marker, application as a tumor drug action target, application in screening or preparing a drug for treating tumors and / or improving the prognosis of tumor patients, application in evaluating tumor prognosis, application in characterizing tumor immune escape, and application in formulating a tumor treatment plan.

[0013] The third aspect of the present invention is to provide a drug for treating tumors and / or improving the prognosis of tumor patients, which contains a cGAS methylation inhibitor. The above inhibitor can be any suitable inhibitor in the art that can inhibit the level of methylation modification.

[0014] Furthermore, the cGAS methylation inhibitor is an inhibitor for inhibiting the methylation modification level of human cGAS K362 or an inhibitor for inhibiting the methylation modification level of murine cGAS K350.

[0015] Furthermore, the cGAS methylation inhibitor is Chaetocin.

[0016] Furthermore, the drug for treating tumors and / or improving the prognosis of tumor patients further contains an immune checkpoint blocker. The above immune checkpoint blocker can be any suitable drug in the art, such as CTLA-4 antibody, PD-1 antibody, and PD-L1 antibody, etc.

[0017] Furthermore, the immune checkpoint blocker is a PD-1 antibody.

[0018] In a specific embodiment, the above cGAS methylation inhibitor inhibits the methylation modification level of murine cGAS K350, promotes the cGAS-responsive DNA sensing signaling pathway, and its combination with an immune checkpoint blocker inhibits the occurrence and development of melanoma and colorectal cancer, and significantly inhibits tumor growth.

[0019] The fourth aspect of the present invention is a detection reagent for a marker for tumor diagnosis and / or prognosis analysis as described in any one of the first aspects of the present invention, which contains a reagent for specifically recognizing the cGAS methylation modification in tumor tissues and tumor cells.

[0020] Furthermore, the reagent is an antibody specifically recognizing the methylation modification of human cGAS K362 or an antibody specifically recognizing the methylation modification of murine cGAS K350.

[0021] Furthermore, the method for preparing the antibody includes: conjugating the antigen polypeptide with the carrier protein keyhole limpet hemocyanin to form an antigen and immunizing a host animal, and obtaining a specifically recognizing antibody through a specific affinity purification method. The above antigen polypeptide is a polypeptide sequence based on the cGAS methylation modification site, and the operation methods of each step in the antibody preparation method can all adopt conventional technical means in the art.

[0022] Furthermore, the antibody is a polyclonal antibody.

[0023] Furthermore, the antigen polypeptide of the human cGAS K362 methylation modification antibody is: CQLRLKPFYLVPK(me)HAKE(SEQ ID NO.1); the antigen polypeptide of the mouse cGAS K350 methylation modification antibody is CTNLRREPFYLVPK(me)NAKD(SEQ ID NO.2).

[0024] Furthermore, the detection reagent is a kit containing an antibody specifically recognizing human cGAS K362 methylation modification or an antibody specifically recognizing mouse cGAS K350 methylation modification.

[0025] The fifth aspect of the present invention is to provide an application of any of the detection reagents described in the fourth aspect of the present invention, and the application is selected from one of the following applications: application in preparing a kit for detecting tumors, application in preparing a kit for tumor prognosis analysis, application in preparing a kit for tumor immunotherapy regimens / application in determining a tumor immunotherapy regimen, application in evaluating the role of cGAS methylation in the tumor immune escape mechanism (application in verifying that cGAS methylation promotes colorectal cancer immune escape). The above tumors include: colorectal cancer and melanoma.

[0026] The sixth aspect of the present invention is to provide a method for detecting the cGAS methylation modification level in cells for non-diagnostic purposes, which mixes any of the detection reagents described in the fourth aspect of the present invention with a sample to be tested to detect the cGAS methylation modification level.

[0027] Compared with the prior art, the present invention adopts the above technical solutions and has the following beneficial effects:

[0028] The present invention has studied the mechanism of the effect of cGAS methylation modification on its activity, and for the first time proposed that cGAS methylation modification (specifically human cGAS K362 methylation modification or mouse cGAS K350 methylation modification) can be used as a marker for tumor diagnosis and prognosis analysis, especially clearly characterizing tumor immune escape and poor prognosis, providing an important reference basis for clinical diagnosis and treatment regimens.

[0029] The present invention discovers that the sites of cGAS monomethylation modification are K362 in humans and K350 in mice, and prepares antibodies for the methylation modification of human K362 and mouse K350. Through experimental verification, the present invention finds that the cGAS K362 methylation modification is highly specifically expressed in colorectal cancer tissues, but there is no such highly specific expression phenomenon in normal tissues, and the K362 methylation modification is significantly negatively correlated with the prognosis of tumor patients. Therefore, cGAS K362 methylation can be used as a marker for tumor diagnosis and prognosis analysis. In addition, experimental verification shows that in a mouse tumor model, inhibiting cGAS methylation modification combined with immune checkpoint blockers can significantly inhibit tumor growth. Therefore, cGAS methylation can clearly characterize tumor immune escape and poor prognosis, providing an important reference basis for clinical diagnosis and treatment plans. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The schematic embodiments of the present invention and their descriptions are only used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0031] Figure 1 Schematic diagram of the mass spectrometry identification results after immunoprecipitating cGAS in an embodiment of the present invention;

[0032] Figure 2 Schematic diagram of the analysis results of the conservation of cGAS methylation modification sites in different species in an embodiment of the present invention;

[0033] Figure 3 Schematic diagram of the specific detection results of the cGAS human K362 methylation and mouse K350 methylation modification antibodies in an embodiment of the present invention;

[0034] Figure 4 Schematic diagram of the analysis results of the correlation between the expression of cGAS K362 methylation modification in tumor tissues and the prognosis of tumor patients in an embodiment of the present invention;

[0035] Figure 5 Schematic diagram of the results of the effect of inhibiting cGAS methylation modification combined with immune checkpoint blockers on mouse tumor growth in an embodiment of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0036] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention. For the experimental methods without specific conditions noted in the following embodiments, they are generally determined according to national standards. The experimental materials not indicated the source in the following embodiments are all commercially available raw materials. The equipment used in each step in the following embodiments are all conventional equipment. If there is no corresponding national standard, it shall be carried out according to the general international standards, conventional conditions, or the conditions recommended by the manufacturer. Unless otherwise specified, all parts are in weight parts and all percentages are in mass percentages. Unless otherwise defined or explained, all the professional and scientific terms used in the present invention have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the recorded content can be applied to the method of the present invention.

[0037] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other. The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but it is not limited to the present invention.

[0038] The sources of the experimental materials used in the following embodiments are as follows: anti-pSTING(Ser366)(E9A9K), anti-pSTING(Ser365)(8F4W), anti-pTBK1 / NAK(Ser172)(D52C2), anti-pSTING(Ser365)(D8F4W), anti-human cGAS(D1D3G) Rabbit mAb, anti-mouse cGAS(D3O8O), anti-STING(D2P2F), anti-TBK1(D1B4) are from CST Antibody Company, Anti-FLAG(20543-1-AP), anti-MYC(16286-1-AP), HA(51064-2-AP) are from Proteintech Antibody Company, Anti-HA-tag mouse antibody agarose beads(M20013L) and anti-MYC-tag mouse antibody agarose beads(M20012M) are from Abmart Company. Anti-FLAG-tag mouse antibody agarose beads(A2220) are from Sigma Company.

[0039] Example 1 - Mass Spectrometric Identification of cGAS Methylation Sites

[0040] In this example, a mass spectrometry method was used to determine the cGAS methylation sites, and its implementation methods and steps included:

[0041] Immunoprecipitate overexpressed cGAS in 293T cells. The specific steps were as follows: 1. Transfect 5 μg of FLAG-cGAS plasmid in a 10 cm dish. After 48 hours of transfection, collect the cell pellet; 2. Lyse the cells with cell lysis buffer (20 mM HEPES (pH 7.8), 400 mM KCl, 5% Glycerol, 5 mM EDTA, 0.4% NP40, phosphatase and protease inhibitors), centrifuge at 12,000 rpm at 4°C for 25 minutes, and collect the supernatant; 3. Immunoprecipitate overexpressed FLAG-cGAS with M2 beads, incubate at 4°C for 4 hours, wash three times with cell lysis buffer, and wash three times with PBS. The immunoprecipitated samples were directly analyzed by mass spectrometry (LC-MS / MS, red represents b ions, and blue represents y ions). The mass spectrometric identification results of human cGAS are as Figure 1 shown.

[0042] Figure 1 The results showed the cGAS methylation sites and the signal peaks of methylation modification, and monomethylation modification occurred at lysine (K) 362 of human cGAS (i.e., cGAS 362K methylation modification).

[0043] In this example, the sequences of the cGAS K362 site in different species were also analyzed. The specific analysis method included: analyzing the conservation of the human cGAS K362 site in different species through the NCBI-BLAST online database, and it was found that K362 was very conserved in mammals such as humans, monkeys, mice, and rabbits. The above sequence analysis results are as Figure 2 shown, indicating that the K362 site was very conserved in mammals, and the monomethylation modification site in murine cGAS was located at K350 (i.e., cGAS 350K methylation modification).

[0044] Example 2 - Preparation of cGAS Methylation Modification Antibodies and Verification of Their Specificity and Titer

[0045] Based on the cGAS 362K methylation modification and cGAS 350K methylation modification identified in Example 1, this example was used to prepare human K362 methylation modification and murine K350 methylation modification antibodies and verify their specificity and titer.

[0046] (1) Preparation of cGAS Methylation Modification Antibodies

[0047] The antigen polypeptide sequences of the above-mentioned K362 methylation modification antibodies are shown as follows:

[0048] K362-monomethylated peptide (K362-me): CQLRLKPFYLVPK(me)HAKE.

[0049] The antigen polypeptide sequence of the above-mentioned K350 methylation modification antibody is shown as follows:

[0050] K350-monomethylated peptide (K350-me): CTNLRREPFYLVPK(me)NAKD.

[0051] In this example, rabbit polyclonal antibodies were prepared. The specific steps included: using New Zealand white rabbits as hosts, synthesizing the above antigen polypeptides and coupling them with the carrier protein keyhole limpet hemocyanin (KLH) to form antigens to immunize the host animals. The antibody serum was obtained by specific affinity purification to obtain antibodies that specifically recognize human K362 methylation modification or mouse K350 methylation modification.

[0052] (2) Verification of the specificity and titer of the cGAS methylation modification antibody

[0053] In this example, the specificity and titer of the cGAS methylation modification antibody obtained were detected at the Dot blot, Western blot, and immunohistochemistry levels. The Dot blot method was to fix the polypeptide on the nitrocellulose membrane and then detect the specificity of the cGAS methylation antibody by immunoblotting. The Western blot detection method included: 1. Using the cell lysate with endogenous cGAS knockout to detect the specificity of the cGAS methylation antibody in recognizing endogenous cGAS; 2. Using the methylation site mutant (K mutated to R, lacking the methylation modification ability) to detect the specificity of the cGAS methylation antibody in recognizing the site. The immunohistochemistry method was used to detect the effect of the cGAS methylation antibody in tissue-level detection. The results of the above detections are as Figure 3 shown.

[0054] In the above detection method, the method for constructing the cGAS gene knockout cell line is as follows: 1. The cGAS gene knockout cell line is obtained by the CRISPR-Cas9 gene knockout technology. The B16F10 cell line is used, and 1 sgRNA is selected. The specific sequence is TCTCGTACCCAAGAATGCAA. The sgRNA is constructed onto the GFP-458 vector and transfected into the target cells through plasmid transfection. The positive cells expressing GFP are sorted by flow cytometry and plated into a 96-well plate, with one cell in each well. When the cells grow into monoclonal colonies, the cGAS gene knockout efficiency is detected by the Western blot method to obtain the cGAS gene knockout cell line. 2. The conventional siRNA interference technology is used. By transfecting the siRNA of cGAS into HT-29 cells, the stability of cGAS mRNA is interfered, resulting in a decrease in the cGAS expression level.

[0055] The construction methods of the human K362R mutant and the mouse K350R mutant: They are constructed on the pcDNA 3.1-HA-human cGAS and pcDNA 3.1-cmyc-mouse cGAS wild-type vectors by using the subcloning construction method for conventional mutants. Given that the cGAS expression in the HCT116 cell line is very low, the tumor tissues with low cGAS expression are the tumor tissues formed by HCT116 cells; the tumor tissues with high cGAS expression use clinical colorectal cancer patient samples.

[0056] As can be seen from Figure 3 Part A, the Dot blot method for detecting K362 methylation modification in vitro only recognizes methylated polypeptides and does not recognize polypeptide antigens without methylation modification. As can be seen from Figure 3 Part B, the Dot blot method for detecting K350 methylation modification in vitro only recognizes methylated polypeptides and does not recognize polypeptide antigens without methylation modification. As can be seen from Figure 3 Parts C and D, the cGAS gene knockdown cell line and the K362R mutant are respectively selected to detect the specificity of the K362 methylation modification antibody. The results show that it can specifically recognize human cGAS.

[0057] As can be seen from Figure 3 Parts E and F, the cGAS gene knockdown cell line and the K350R mutant are respectively selected to detect the specificity of the K350 methylation modification antibody. The results show that it can specifically recognize mouse cGAS. As can be seen from Figure 3 Part G, the immunohistochemistry method is used to detect the specificity of the K362 methylation modification antibody in tumor tissues with low and high cGAS expression. The results show that it can specifically recognize endogenous cGAS.

[0058] Analysis of the Correlation between the Expression of cGAS K362 Methylation Modification in Tumor Tissues and the Prognosis of Tumor Patients in Example 3

[0059] In this example, Western blot and immunohistochemistry were used to detect the expression levels of cGAS K362 methylation modification in tumor tissues and normal tissues, and to analyze the correlation between the cGAS K362 methylation modification level and the prognosis of colorectal cancer patients. In this example, 320 colorectal cancer tissue microarrays were used to detect the differences in the cGAS K362 methylation modification levels between tumor tissues and normal tissues, and the detection results are as Figure 4 shown.

[0060] The steps of the Western blot method include: 1. After the target cells are lysed sufficiently with cell lysis buffer (50 mM Tris-HCl (pH 7-9), 300 mM NaCl, 1% Triton X-100, supplemented with protease inhibitor and phosphatase inhibitor cocktails), centrifuge at 12,000 rpm at 4°C for 25 minutes; 2. Take the cell supernatant for downstream experiments. After obtaining the samples, run SDS-PAGE electrophoresis; 3. Transfer the PAGE samples to a PVDF membrane (Millipore) for subsequent immunoblotting experiments. The specific primary antibodies used are as Figure 4 shown in part A of

[0061] Immunohistochemistry and expression level analysis method: 1. Tissue specimens, including heart tissues pretreated by cardiac perfusion, were fixed overnight in 10% neutral buffered formalin, then dehydrated in increasing concentrations of isopropanol, and then cleared of alcohol with xylene; 2. The specimens were embedded in paraffin blocks for tissue sectioning. Standard staining was performed on 3-μm-thick sections of each sample block with hematoxylin and eosin (H&E); 3. For immunohistochemistry, tissue sections were deparaffinized and incubated in citrate buffer at 95°C for 40 minutes to retrieve antigens, and then incubated overnight at 4°C with primary antibodies including anti-cGAS K362me (1:100 dilution); 4. After three washes, tissue sections were incubated with biotinylated anti-rabbit IgG (1:100 dilution) at RT for 1 hour, then washed three times, and then streptavidin-horseradish peroxidase conjugate was added and the slides were incubated for 45 minutes; 5. After washing three times with PBS, DAB solution was added, and the slides were counterstained with hematoxylin. Negative control groups were treated in the same way but without adding primary antibodies; 6. In this experiment, tissue microarray (TMA) was used to study 320 patients with colorectal cancer; 7. Immunohistochemical staining was evaluated by independent pathologists. The staining extent was scored from 0 to 3, corresponding to the percentage of immunoreactive tumor cells (0%-10%, 11%-25%, 26%-75%, and 76%-100% respectively) and staining intensity (negative, score = 0; weak, score = 1; strong, score = 2; very strong, score = 3). By multiplying the staining extent score by the intensity score, a score of 0-3 was calculated to obtain the low (0-1) level or high (2-3) level value for each sample.

[0062] Figure 4 Parts A and B of show that the expression level of cGAS K362 methylation modification in tumor tissues is significantly higher than that in normal tissues; Figure 4 Part C of shows that cGAS K362 methylation modification is significantly negatively correlated with the prognosis of tumor patients, indicating that the above cGAS K362 methylation modification can be used as a tumor marker for the diagnosis and prognosis analysis of colorectal cancer.

[0063] Example 4 - Effect of inhibiting cGAS methylation modification combined with immune checkpoint blocker treatment on tumor growth in mice

[0064] This example studied the effects of cGAS 350K methylation modification on cGAS activity, tumor immune escape, and its effect on tumor growth. A subcutaneous tumor-bearing model of colorectal cancer and melanoma was used to detect tumor growth. An inhibitor, Chaetocin (a specific inhibitor of histone methyltransferase (HMT) SU(VAR)3-9), was used to inhibit the level of cGAS methylation modification, and combined treatment with Chaetocin and anti-PD-1 antibody was used to compare the differences in tumor growth.

[0065] The above experimental steps include: 1. Subcutaneously inject 0.5 - 1×10 6 B16F10 or 1×10 6 MC38 cells into C57BL / 6 mice aged 7 - 8 weeks. The tumor volume was calculated as tumor volume = length × width × width / 2; 2. When the tumor volume reached about 75 mm 3 , intervention treatment was given; 3. The mice were intraperitoneally injected with DMSO (10%) in PBS or chaetocin (25 mg / kg) every three days for three courses; 4. The mice were intraperitoneally injected with anti-mouse-PD-1 (BP0146, CD279) or rat IgG2a isotype mAbs (BP0089) (Bioxcell), 100 μg per mouse (MC38) or 200 μg (B16F10) every three days for three courses; 5. For combined treatment, the experimental groups were as shown in Figure 5 Parts C and D. The detection results are as shown in Figure 5 .

[0066] As can be seen from Figure 5 Parts A and B, cGAS methylation inhibits cGAS activity. The inhibitor Chaetocin inhibits the endogenous cGAS K350 methylation modification level in murine cell lines B16F10 and MC38, promotes the cGAS response to the DNA sensing signaling pathway, and promotes tumor immune escape. As can be seen from Figure 5 Parts C and D, a mouse subcutaneous tumor-bearing model was used to detect the effect of combined treatment with the inhibitor Chaetocin and immune checkpoint blocker on tumor growth. The results showed that combined treatment with inhibition of cGAS methylation (Chaetocin) and immune checkpoint blocker (anti-PD-1 antibody) significantly inhibited tumor growth.

[0067] As can be seen from the above embodiments, the present invention obtains the lysine at position 362 of the human cGAS monomethylation modification site and the lysine at position 350 of the murine cGAS monomethylation modification site through mass spectrometry analysis, and obtains a K362 methylation modification specific antibody and a K350 methylation modification specific antibody through the preparation of polyclonal antibodies. By detecting the cGAS methylation modification level in colorectal cancer tissues, it is found that the expression level of cGAS K362 methylation modification in tumor tissues is significantly higher than that in normal tissues, and it is significantly negatively correlated with the prognosis of tumor patients. The above cGAS methylation modification can be used as a tumor marker for tumor diagnosis and prognosis analysis. Moreover, the present invention uses a subcutaneous mouse tumor-bearing model to detect the effect of inhibiting cGAS methylation on the combined treatment of tumors with immune checkpoint blockers, and finds that Chaetocin inhibits cGAS methylation combined with PD-1 antibody significantly inhibits the growth of mouse tumors, which provides an important reference basis for clinical diagnosis and treatment plans.

[0068] The above are only the preferred embodiments of the present invention, and do not limit the implementation manners and protection scope of the present invention. For those skilled in the art, it should be able to realize that all the equivalent substitutions and obvious changes made by using the description and illustrations of the present invention should be included in the protection scope of the present invention. Sequence Listing <110> Shanghai Tenth People's Hospital <120> cGAS Methylation Modification, Detection Reagent and Application Thereof as Tumor Diagnosis and Prognosis Analysis Markers <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 17 <212> PRT <213> Antigen Polypeptide of cGAS K362 Methylation Modification Antibody (Artificial Sequence) <220> <221> METHYLATION <222> (13)..(13) <400> 1 Cys Gln Leu Arg Leu Lys Pro Phe Tyr Leu Val Pro Lys His Ala Lys 1 5 10 15 Glu <210> 2 <211> 18 <212> PRT <213> Antigen polypeptide of cGAS K350 methylation modification antibody (Artificial Sequence) <220> <221> METHYLATION <222> (14)..(14) <400> 2 Cys Thr Asn Leu Arg Arg Glu Pro Phe Tyr Leu Val Pro Lys Asn Ala 1 5 10 15 Lys Asp

Claims

1. A marker for tumor diagnosis and / or prognosis analysis, characterized in that, The biomarker is human cGASK362 methylation modification or murine cGAS K350 methylation modification.

2. The biomarker for tumor diagnosis and / or prognosis analysis according to claim 1, wherein The tumors include colorectal cancer and melanoma.

3. Use of a marker for tumor diagnosis and / or prognosis analysis as described in claim 1 or 2, characterized in that, The application is selected from at least one of the following applications: application in the preparation of a detection reagent or kit for the biomarker, application in the preparation of a drug targeting a tumor drug target, application in screening or preparing a drug for treating tumors and / or improving the prognosis of tumor patients, application in the preparation of a drug for evaluating tumor prognosis, application in the preparation of a drug for characterizing tumor immune escape, wherein the tumor is selected from at least one of colorectal cancer or melanoma.

4. The application according to claim 3, wherein The drug for treating tumors and / or improving the prognosis of tumor patients contains a cGAS methylation inhibitor; wherein, the cGAS methylation inhibitor is an inhibitor for inhibiting the methylation modification level of human cGAS K362 or for inhibiting the methylation modification level of murine cGAS K350.

5. The application according to claim 4, characterized in that, The cGAS methylation inhibitor is Chaetocin.

6. The application according to claim 4 or 5, characterized in that The drug for treating tumors and / or improving the prognosis of tumor patients further contains an immune checkpoint blocker.

7. The application according to claim 6, characterized in that, The immune checkpoint blocker includes a PD-1 antibody.

8. A detection reagent for a marker for tumor diagnosis and / or prognosis analysis as described in claim 1 or 2, characterized in that, The detection reagent contains a reagent for specifically recognizing cGAS methylation modification in tumor tissues and tumor cells; wherein, the reagent is an antibody specifically recognizing human cGAS K362 methylation modification or an antibody specifically recognizing murine cGASK350 methylation modification.

9. The detection reagent according to claim 8, wherein The method for preparing the antibody includes: coupling an antigen polypeptide with a carrier protein keyhole limpet hemocyanin to form an antigen and immunizing a host animal, and obtaining a specifically recognizing antibody through a specific affinity purification method.

10. The detection reagent according to claim 9, characterized in that, The amino acid sequence of the antigen polypeptide of the antibody specifically recognizing human cGAS K362 methylation modification is as shown in SEQ ID NO.1; the amino acid sequence of the antigen polypeptide of the antibody specifically recognizing murine cGAS K350 methylation modification is as shown in SEQ ID NO.2.

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