Use of CK2 in analyzing cold and hot properties of lung cancer and total survival of KRAS mutant lung cancer patients and screening lung cancer patients suitable for immunotherapy

Abstract

The application belongs to the technical field of biological medicine, and specifically discloses the use of CK2 in analyzing the cold and hot properties of lung cancer and the total survival of KRAS mutant lung cancer patients and screening lung cancer patients suitable for immunotherapy. It relates to the use of a reagent for detecting KRAS mutation in the preparation of a product for diagnosing whether lung cancer is a hot tumor; the use of a reagent for detecting the expression level of CK2 protein in the preparation of a product for screening KRAS mutant lung cancer patients suitable for immunotherapy; the use of a CK2 protein expression inhibitor in the preparation of a KRAS mutant lung cancer PD-1 / PD-L1 immunotherapy adjuvant drug; and the use of a reagent for detecting the interaction between CK2 and FASN protein in the preparation of a product for analyzing the immunotherapy effect of KRAS mutant lung cancer. The present disclosure can predict the immunotherapy effect of KRAS mutant lung cancer patients, and at the same time, can provide a new target for immunotherapy of KRAS mutant lung cancer.

Description

[0001] The present application is a divisional application of the Chinese invention patent application No. 202311456700.7, entitled "New application of protein kinase CK2 in predicting the efficacy of immunotherapy for KRAS mutant lung cancer", filed on November 3, 2023. The present application incorporates by reference the entire contents of CN117405894A. TECHNICAL FIELD

[0002] The present application belongs to the field of medicine, and specifically relates to the use of protein kinase CK2 in predicting the efficacy of immunotherapy for KRAS mutant lung cancer. BACKGROUND

[0003] The diagnosis and treatment of patients with driver gene positive lung cancer is a hot topic in lung cancer research. KRAS mutation is one of the most common genetic mutations in lung cancer, accounting for about 25-30% in lung adenocarcinoma. As early as 1984, KRAS mutation was discovered in lung cancer, but there have been few breakthroughs in basic research and clinical treatment. The reason is that KRAS protein is small in size and smooth in surface, and its affinity with GTP is extremely high, resulting in the failure of most KRAS targeted drug research and development. Although the latest NCCN guidelines recommend the targeted drug AMG510 for the treatment of KRAS G12C mutation subtype (accounting for 13.8% of all KRAS mutant lung cancer), the effective rate is only 37.1%, and the efficacy is not satisfactory, and there is no breakthrough in targeted drugs for most other KRAS mutant subtypes. Therefore, traditional platinum-containing chemotherapy is still the first choice for the treatment of KRAS mutant lung cancer. Immunotherapy has brought hope to patients with KRAS mutant lung cancer, and its efficacy is significantly better than that of other driver gene mutant lung cancer patients. For example, the ImmunoTarget study, the CheckMate057 study subgroup analysis, and the Mazieres J study confirmed that the effective rate of single-agent immunotherapy for KRAS mutant lung cancer patients can reach 26%, but the problem of immunotherapy resistance still needs to be solved. Therefore, exploring the resistance mechanism of immunotherapy for KRAS mutant lung cancer, finding a sensitization approach, and exploring a new mode of "immunotherapy +", have important theoretical significance and clinical value.

[0004] Currently, there are two main methods to predict the effect of immune checkpoint inhibitor therapy. First, the expression level of PD-L1 protein in tumor tissue is detected by immunohistochemistry to understand its high or low level to predict the treatment effect of patients. PD-L1 is a protein regulated by effector T cells, and its high expression implies the presence of more effector T cells in tumor tissue, which indicates a stronger immune response, thus indicating that patients may have a better response to immunotherapy. However, clinical research results show that PD-L1 high expression only has a significant correlation with the effect of immunotherapy in some specific tumor types, and other tumor types may not have the same correlation. In addition, there are still some patients with no obvious PD-L1 expression who may still benefit from immunotherapy. This shows that the expression level of PD-L1 cannot accurately predict the effect of immunotherapy. On the other hand, there are multiple brands and models of PD-L1 antibodies on the market, and their detection methods, staining levels, and result interpretation methods differ. This difference may interfere with doctors and patients deciding whether to undergo immunotherapy and how to treat to some extent. Therefore, standardizing and unifying the PD-L1 detection method and interpretation standard are very important to ensure the effectiveness of immunotherapy and for patients to obtain the best treatment results. This requires the development of consistent guidelines and standards in clinical practice to improve the accuracy and reliability of immunotherapy.

[0005] Metabolic immune microenvironment is a hot issue in the field of tumor research. Tumor cell metabolic reprogramming affects the function and state of immune checkpoints, further promoting malignant biological behavior and immune escape. Studies have found that palmitic acid, a saturated fatty acid, can activate M2 macrophages and tumor-associated fibroblasts to exert immune suppression function; at the same time, it can enhance the expression of PD-L1 in tumor cells, induce the exhaustion of cytotoxic T cells, and lead to immune escape. Hexokinase-2, a key enzyme of glycolysis, has been reported to up-regulate PD-L1 expression in tumor cells through activation of the NF-κB pathway, leading to immune escape. In addition, methylenetetrahydrofolate dehydrogenase 2, a folate cycle enzyme, promotes the up-regulation of PD-L1 expression in tumor cells by efficiently driving folate circulation, promoting immune escape.

[0006] Protein kinase CK2 is the first protein kinase discovered in humans, which phosphorylates more than 300 substrates and plays an important role in the physiological and pathological metabolic processes of the body. In January 2022, the FDA has approved its specific inhibitor (CX4945) for the clinical treatment of cancer. The relationship between CK2 and cellular lipid metabolism is the focus of current research in the field of CK2. Barbara Guerra et al. reported the potential role of CK2 in lipid metabolism and found that CK2 could phosphorylate MED17 in the liver of mice on a high-fat diet, and MED17 is an important part of the FASN promoter; in addition, CK2 activates the PI3K / AKT / mTOR lipid metabolism pathway by phosphorylating the Ser129 site of AKT and the C-terminal of PTEN. SUMMARY

[0007] To solve the above problems, the present application provides the use of CK2 in analyzing the cold and hot properties of lung cancer and the overall survival of KRAS mutant lung cancer patients and screening lung cancer patients suitable for immunotherapy, mainly to solve the problem that the efficacy of KRAS mutant lung cancer immunotherapy is difficult to evaluate.

[0008] To solve the above problems, the present application adopts the following technical solutions:

[0009] A first aspect of the present application The reagent for detecting the expression of CK2 is used in the preparation of a product for analyzing the effect of immunotherapy of KRAS mutant lung cancer; wherein the application is at least one of the following: 1) the reagent for detecting the expression level of CK2 is used in the preparation of a product for detecting the prognosis effect of immunotherapy of KRAS mutant lung cancer, which is mainly used for evaluating the treatment effect after immunotherapy of KRAS mutant lung cancer; 2) the reagent for detecting the expression level of CK2 is used in the preparation of a product for predicting the prognosis effect of immunotherapy of KRAS mutant lung cancer, which is mainly used for evaluation before immunotherapy of the patient, to predict whether the patient will achieve good effect after immunotherapy.

[0010] Another combination, CK2 can also be combined with FASN as an important molecular marker in the treatment of KRAS mutant lung cancer. The reagent for detecting the interaction between CK2 and FASN is used in the preparation of a product for analyzing the effect of immunotherapy for KRAS mutant lung cancer; wherein the application is at least one of the following: 1) the reagent for detecting the interaction between CK2 and FASN is used in the preparation of a product for detecting the prognosis of immunotherapy for KRAS mutant lung cancer, 2) the reagent for detecting the interaction between CK2 and FASN is used in the preparation of a product for predicting the prognosis of immunotherapy for KRAS mutant lung cancer. The reagent for detecting the interaction between CK2 and FASN can use the existing reagent for detection. The surface plasmon resonance (SPR) technology represented by Biacore can realize detection. The aforementioned reagent for detecting the interaction between CK2 and FASN is a broad concept, which is a collection of essential reagents or alternative reagent schemes used in the detection process (such as the S series CM5 chip involved in the product Biacore T200), and its role is to detect protein interaction. And the degree of interaction between CK2 and FASN can be used to predict the effect of immunotherapy for KRAS mutant lung cancer. The stronger the interaction, the worse the prognosis.

[0011] Another combination, CK2, FASN and DHHC3 joint detection reagent is used in the preparation of a product for analyzing the effect of immunotherapy for KRAS mutant lung cancer; wherein the application is at least one of the following: 1) the reagent for detecting the expression of CK2 is used in the preparation of a product for detecting the prognosis of immunotherapy for KRAS mutant lung cancer, 2) the reagent for detecting the expression of CK2 is used in the preparation of a product for predicting the prognosis of immunotherapy for KRAS mutant lung cancer. The joint detection reagent can be several combinations, or one (such as a chip).

[0012] KRAS mutant lung cancer is lung cancer induced by KRAS mutation, which has obvious difference from wild type. For example, KRAS-LSL-G12D point mutation induced lung cancer, lung cancer caused by other KRAS mutations should be equivalent in this application.

[0013] For the corresponding application, the following choices can be made when it is applied:

[0014] First, when making prognosis, the detection of CK2 expression level is negatively correlated with the prognosis of immunotherapy for KRAS mutant lung cancer. Therefore, when the expression level of CK2 is high, it indicates that the effect of immunotherapy for KRAS mutant lung cancer patients will not be obvious, and when the expression level of CK2 is low, it indicates that the effect of immunotherapy for KRAS mutant lung cancer patients will be more obvious. Similarly, the same method is used for prediction. The high and low of CK2 expression level can be judged according to the clinical demand, and the CK2 expression level in normal human body can be used as reference;

[0015] Secondly, as for the reagent for detecting the expression level of CK2, the reagent for detecting the expression of CK2 is at least one of at least one of an immunohistochemical staining kit, an ELISA kit, and a WB kit. The role of the kit is to detect the expression level of CK2.

[0016] Thirdly, as for the immunotherapy, the immunotherapy of KRAS mutant lung cancer is at least the PD-1 / PD-L1 immunotherapy of KRAS mutant lung cancer, and the monoclonal antibody mainly includes: cari Li Zhu single antibody, pa Bo Li Zhu single antibody, Du Fa Li You single antibody, and replacement Li Zhu single antibody, etc.

[0017] As for the product for predicting the prognosis effect of the immunotherapy of KRAS mutant lung cancer, it can be embodied as the application of the reagent for detecting the expression of CK2 in the preparation of the product for screening KRAS mutant lung cancer patients suitable for immunotherapy. The prepared product can detect the sample obtained from the patient's body. When the expression level of CK2 is obtained, it can be judged whether the patient is suitable for immunotherapy. For example, when the expression level of CK2 is significantly higher than that of normal people, it means that the patient is not suitable for immunotherapy, or it is suggested that the CK2 inhibitor should be used in the treatment process.

[0018] A second aspect of the present application The application relates to the application of a CK2 inhibitor in the preparation of an immunotherapy drug for KRAS mutant lung cancer.

[0019] For the corresponding application, the following selection or explanation can be made in the specific application:

[0020] Firstly, the CK2 inhibitor is used to improve the response effect of the immunotherapy of KRAS mutant lung cancer, thereby improving the immunotherapy effect. The effect of the CK2 inhibitor can be further embodied as at least one of the following: 1) down-regulating the expression of FASN, 2) down-regulating the expression of DHHC3, 3) reducing the generation of palmitic acid, 4) weakening the membrane stability of PD-L1, 5) increasing the intratumoral CD8 + T cell infiltration of KRAS mutant lung cancer mouse model, and forming an activated immune microenvironment in the tumor;

[0021] Secondly, the CK2 inhibitor is at least any one of CX4945, TBB, Kunzinc, NSC-231634, and DMAT.

[0022] Thirdly, the immunotherapy of KRAS mutant lung cancer is at least the PD-1 / PD-L1 immunotherapy of KRAS mutant lung cancer, and the monoclonal antibody mainly includes: cari Li Zhu single antibody, pa Bo Li Zhu single antibody, Du Fa Li You single antibody, and replacement Li Zhu single antibody, etc.

[0023] Fourthly, KRAS mutant lung cancer refers to lung cancer induced by KRAS mutation, which has obvious difference from wild type. For example, KRAS-LSL-G12D point mutation induced lung cancer, lung cancer caused by KRAS mutation at other sites should also be equivalent in the present application.

[0024] A third aspect of the present application The application of the reagent for detecting KRAS mutation in the preparation of a product for diagnosing whether the lung cancer is a hot tumor; wherein the KRAS mutation is a mutation that induces lung cancer.

[0025] Cold tumors generally refer to those tumors that lack active immune cells and immune responses in the tumor microenvironment. They usually have a lower mutation load and do not cause a strong immune response, making it difficult for the immune system to recognize and attack them. Hot tumors, on the other hand, have a higher mutation load and a large number of immune cells, such as T cells and antigen-presenting cells, in the tumor microenvironment. These immune cells participate in the immune response, making it easier for the immune system to recognize and attack the tumor. In cold tumors, there is less infiltration of immune cells and a relatively weak immune response, which reduces the effectiveness of immunotherapy. In hot tumors, there are more active immune cells around the tumor, which interact with tumor cells and may respond more actively to immunotherapy. Cold tumors usually have a poor response to immunotherapy because they lack the support of immune cells. Therefore, other strategies such as targeted therapy or chemotherapy may be needed to enhance the immune response for cold tumors. Hot tumors usually have a more positive response to immunotherapy because the immune system has already played a role in tumor resistance. Immunotherapy can enhance these responses and improve the chances of successful treatment. Therefore, preliminary identification of tumor type can help doctors more targetedly select treatment options. For cold tumors, other treatment methods may need to be explored, while for hot tumors, immunotherapy may be a more effective choice. Individualized treatment plans usually take into account the patient's specific situation and tumor characteristics to achieve the best treatment effect. Thus, based on the differences between cold and hot tumors in terms of nature, immune cells, and treatment effect, different treatment options are needed for the two types of tumors, and preliminary identification of tumor type can facilitate more targeted treatment, thus providing an auxiliary means for targeted treatment in clinical treatment.

[0026] Among them, the KRAS mutation at least includes KRAS-LSL-G12D point mutation, of course, due to the high similarity between lung cancers induced by other types of KRAS mutations, their treatment options are also the same, so the rest of the KRAS mutation induced lung cancer should also be within the scope of the present application.

[0027] In the present disclosure, the present application can predict and detect the efficacy of immunotherapy for KRAS mutant lung cancer patients, and also provide new targets for KRAS mutant lung cancer immunotherapy. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Correlation of age with overall survival (OS) in KRAS mutant lung cancer patients.

[0029] Figure 2 Correlation of CK2 expression level with nutritional score grade in KRAS mutant lung cancer patients.

[0030] Figure 3 Correlation of CK2 expression level with overall survival (OS) in KRAS mutant lung cancer patients.

[0031] Figure 4 Correlation of CK2 expression level with progression free survival (PFS) in KRAS mutant lung cancer patients.

[0032] Figure 5 AUC curve showing the ability of CK2 to predict overall survival (OS) at 1.5 years, 2 years and 3 years in KRAS mutant lung cancer patients, with AUC values of 0.85, 0.89 and 0.83, respectively.

[0033] Figure 6 AUC curve showing the ability of CK2 to predict progression free survival (PFS) at 1 year, 1.5 years and 2 years in KRAS mutant lung cancer patients, with AUC values of 0.86, 0.9 and 1, respectively.

[0034] Figure 7 Time-dependent ROC curve showing that the AUC values (95% CI) of CK2 to predict overall survival (OS) in KRAS mutant lung cancer patients remain stable over time.

[0035] Figure 8 Time-dependent ROC curve showing that the AUC values (95% CI) of CK2 to predict progression free survival (PFS) in KRAS mutant lung cancer patients remain stable over time.

[0036] Figure 9 Correlation of CK2 expression level with response to immunotherapy in KRAS mutant lung cancer patients.

[0037] Figure 10 Bioinformatics analysis showing that KRAS mutant lung cancer presents a "cold tumor" signature in the tumor immune microenvironment compared to wild type. (A) B cells, CD8 T cells and CD4 T cells in the tumor immune microenvironment of KRAS mutant lung cancer compared to wild type. +T cell, neutrophil and macrophage were significantly depleted; (B) Heatmap of immune cell score, different colors represent the expression trend in different samples, *p < 0.05, **p < 0.01, ***p < 0.001, the asterisk represents the importance (*p), the significance of two groups of samples is tested by wilcox test, and the significance of three or more groups of samples is tested by Kruskal-Wallis test; (C) The percentage abundance of tumor-infiltrating immune cells in each sample, different colors represent different immune cell types, the horizontal coordinate represents the sample, and the vertical coordinate represents the percentage of immune cells in the individual sample.

[0038] Figure 11 : The intratumoral CD8 T cell in KRAS G12D conditional point mutation mice was significantly lower than that in wild-type mice. + T cell was significantly depleted.

[0039] Figure 12 : Bioinformatics analysis showed that the highly expressed genes in KRAS mutant lung cancer patients were closely related to lipid metabolism. (A) Volcano plot of differential genes; (B) Heatmap of differential gene expression; (C-D) Functional enrichment: KEGG or GO pathway enrichment results of differentially up-regulated or down-regulated genes.

[0040] Figure 13 : The distribution trend of CK2 expression in 149 KRAS mutant lung cancer samples with different clinical characteristics such as age, gender and AJCC eighth edition lung cancer staging and patient survival.

[0041] Figure 14 : Liquid chromatography-mass spectrometry non-target LC-MS metabolomics analysis found that the palmitic acid metabolic pathway was significantly enriched after down-regulating CK2, and the production of palmitic acid was reduced. (A) Differential metabolites can be visualized in the form of volcano plot; (B) Differential metabolite pathway enrichment analysis; the more red the circle color is, the larger the shape is, and the higher the significance is; (C) VIP (Variable Importance in Projection) plot of Random Forests; represents the importance of metabolites and their contribution to sample classification; (D) PLS-DA loading plot; reflects the contribution of metabolites to the classification of sample groups, the farther from the center point, the greater the contribution to the classification of sample groups; (E) Distribution of palmitic acid in si-Vehicle and si-CK2a groups, P < 0.05.

[0042] Figure 15: Silencing or inhibiting CK2 leads to significant down-regulation of FASN expression in KRAS mutant lung cancer, and reduces palmitic acid production; and CK2 has protein interactions with FASN and DHHC3. (A) volcano plot of differential genes; (B) pathway enrichment analysis of differentially expressed genes; (C) RT-qPCR detection of the mRNA level of FASN; (D) metabolite kit detection of the specific content of palmitic acid; (E) mass spectrometry analysis of the potential interaction proteins of CK2a.

[0043] Figure 16 : Intracellular lipid droplets are more aggregated in KRAS mutant lung cancer than in KRAS wild-type lung cancer; after targeting CK2, intracellular lipid droplets in KRAS mutant lung cancer are significantly reduced, while there is no significant change in KRAS wild-type lung cancer. (A) Effect of down-regulating CK2 at different concentrations on lipid droplet aggregation in KRAS mutant cell lines and wild-type cell lines, histogram; (B-D) Effect of down-regulating CK2 at different concentrations on lipid droplet aggregation in KRAS mutant cell lines and wild-type cell lines, scatter plot; (E-G) Effect of down-regulating CK2 at different concentrations on lipid droplet aggregation in KRAS mutant cell lines and wild-type cell lines; fluorescence microscope detection, 10 pm scale, green for intracellular lipid droplet staining.

[0044] Figure 17 : In KRAS mutant lung cancer, DHHC3 is one of the candidate interaction proteins of CK2, and Ser241, Thr244 and Thr247 of DHHC3 are determined as potential phosphorylation sites.

[0045] Figure 18 : Immune cell score heatmap, distribution of immune scores of each immune cell in 64 cases of CK2 high expression and 85 cases of CK2 low expression KRAS mutant lung cancer patients, where the horizontal axis represents the type of immune cell infiltration, and the vertical axis represents the distribution of the immune infiltration score in different groups. The results suggest that the CK2 high expression KRAS mutant lung cancer sample has an immunosuppressive microenvironment in the tumor.

[0046] Figure 19 : Percentage abundance of tumor-infiltrating immune cells in 64 cases of CK2 high expression and 85 cases of CK2 low expression KRAS mutant lung cancer patients. The results suggest that the CK2 high expression KRAS mutant lung cancer sample has an immunosuppressive microenvironment in the tumor.

[0047] Figure 20: The expression distribution of immune checkpoint genes in 64 cases of CK2 high expression and 85 cases of CK2 low expression KRAS mutant lung cancer patients. Among them, the abscissa represents different groups of samples, and the ordinate represents the expression distribution of the gene. Different colors represent different groups and expression trends in different samples. The results suggest that CK2 high expression KRAS mutant lung cancer samples have high expression of PD-L1 (CD274), HAVCR2, PDCD1LG2, and SIGLEC15 immune checkpoint genes, leading to tumor immune escape.

[0048] Figure 21 : Correlation analysis between CK2, FASN, DHHC3 and PD-L1 expression and immune scores, and correlation analysis between immune scores. The heat map in the schematic diagram represents the correlation analysis between immune scores. Red represents positive correlation, blue represents negative correlation, and the redder or bluer the color, the greater the correlation between the two. Similarly, the larger the circle, the stronger the correlation. The red line in the schematic diagram represents a negative correlation between model scores or gene expression and immune scores, and green represents a positive correlation. The results suggest that CK2 is negatively correlated with intratumoral CD8 + T cells in KRAS mutant lung cancer patients, that is, high expression of CK2 induces the formation of an immunosuppressive microenvironment in KRAS mutant lung cancer.

[0049] Figure 22 : Two-gene correlation analysis in KRAS mutant lung cancer samples, respectively, Spearman correlation analysis between FASN and CK2 expression, CK2 and DHHC3 expression, CK2 and PD-L1 expression, and DHHC3 and FASN expression. The results suggest that FASN and CK2 expression are positively correlated (P=0.003), CK2 and DHHC3 expression are positively correlated (P=2.65e-07), CK2 and PD-L1 expression are positively correlated (P=0.001), and DHHC3 and FASN expression have no statistical difference (P=0.092); This provides a basis for the joint detection of CK2, FASN and DHHC3 in the prediction of immunotherapy efficacy and survival prognosis in KRAS mutant lung cancer patients.

[0050] Figure 23: Kaplan-Meier survival curves of CK2, FASN and DHHC3 as a "cluster", where the high and low expression groups of CK2 / FASN / DHHC3 were tested by Log-rank, HR (High exp) represents the risk coefficient of the high expression group relative to the low expression group sample, if HR > 1 represents that the gene is a risk factor (the higher the expression, the worse the prognosis), if HR < 1 represents that the gene is a protective factor (the higher the expression, the better the prognosis). The results suggest that the overall survival (OS) of the high expression group of CK2 / FASN / DHHC3 is worse than that of the low expression group of CK2 / FASN / DHHC3.

[0051] Figure 24 : Animal experiment mode diagram. DETAILED DESCRIPTION

[0052] Further explanation will be made in combination with specific research projects.

[0053] Experimental scheme

[0054] (1) To clarify the role of protein kinase CK2 in the high expression of PD-L1 and immune escape of KRAS mutant lung cancer

[0055] 1. Cell level: To clarify the effect of CK2 on the expression of key molecules of fatty acid metabolism in KRAS mutant lung cancer cells;

[0056] 2. Cell level: To clarify the effect of CK2 on the expression of immune cells, cytokines and immune checkpoints in KRAS mutant lung cancer;

[0057] 3. To construct KRAS-LSL-G12D point mutation lung cancer mouse model, and analyze the dynamic changes of CK2, key molecules of fatty acid metabolism, immune cells, cytokines and immune checkpoints from normal lung tissue to KRAS mutant lung cancer;

[0058] 4. To use CK2 inhibitor to treat KRAS-LSL-G12D point mutation lung cancer mouse model, and to clarify its effect on tumor tissue and peripheral blood fatty acid metabolism key molecules, immune cells, cytokines and immune checkpoints and other indicators;

[0059] 5. To use CK2 inhibitor, immune checkpoint inhibitor and fatty acid metabolism enzyme inhibitor to treat KRAS-LSL-G12D point mutation lung cancer mouse model, to evaluate the effectiveness and safety of each treatment group.

[0060] (2) To clarify the mechanism of protein kinase CK2 regulating PD-L1 palmitoylation modification through fatty acid metabolism reprogramming and direct phosphorylation of palmitoyltransferase DHHC3

[0061] Approach 1: CK2 promotes de novo palmitate synthesis by activating the key enzyme FASN of fatty acid metabolism, which provides "raw materials" for palmitoylation modification of PD-L1 activated by palmitate.

[0062] 1. To determine the interaction between CK2 and FASN endogenous and exogenous proteins, and to identify the domains of CK2 and FASN interaction;

[0063] 2. To verify the effect of silencing or inhibiting FASN on the palmitate level of KRAS mutant lung cancer;

[0064] 3. Rescue experiment to verify the effect of overexpression of CK2, silencing or inhibiting FASN on the palmitate level of KRAS mutant lung cancer;

[0065] 4. Based on 13 C-labeled palmitate carbon metabolic flux analysis, to reveal the up and down stream mutual regulation mechanism;

[0066] 5. To determine whether blocking palmitoylation can lead to enhanced PD-L1 membrane anchoring in KRAS mutant lung cancer;

[0067] 6. Rescue experiment to determine the effect of overexpression of CK2, silencing or inhibiting FASN on the expression of PD-L1 membrane protein in KRAS mutant lung cancer;

[0068] 7. Rescue experiment to determine the effect of silencing or inhibiting CK2, exogenous intake of palmitate on PD-L1 membrane protein in KRAS mutant lung cancer.

[0069] Approach 2: CK2 directly phosphorylates the "catalyst" of palmitoylation modification, palmitoyl transferase DHHC3, mediates the occurrence of PD-L1 palmitoylation modification, leading to enhanced PD-L1 membrane stability.

[0070] 1. To verify the interaction between CK2 and DHHC3 endogenous and exogenous proteins;

[0071] 2. To construct KRAS mutant lung cancer cell lines carrying stable expression of wild-type DHHC3 (DHHC3 WT ), phosphorylation-deficient T244A / T247A double mutant (DHHC3 2A ) and overexpression of phosphorylation T244E / T247E (DHHC3 2E );

[0072] 3. Western Blotting to detect DHHC3 2E , DHHC3 2A and DHHC3 WT protein expression in DHHC3

[0073] 4. Silencing or inhibiting CK2 affects DHHC3 2E , DHHC3 2A and DHHC3 WT cell DHHC3 protein expression levels;

[0074] 5. Further identify DHHC3 as the main palmitoyl transferase of KRAS mutant lung cancer PD-L1 expression;

[0075] 6. Determine the effect of silencing or inhibiting DHHC3 on KRAS mutant lung cancer PD-L1 membrane protein;

[0076] 7. Rescue experiment to determine the effect of overexpression of CK2, silencing or inhibiting DHHC3 on KRAS mutant lung cancer PD-L1 membrane protein.

[0077] (3) Explore the efficacy prediction and survival prognosis value of protein kinase CK2, FASN and DHHC3 in KRAS mutant lung cancer patients.

[0078] 1. Determine the relationship between CK2, FASN and DHHC3 expression levels and localization and clinical characteristics of KRAS mutant lung cancer patients, such as stage, immunotherapy efficacy and prognosis (paraffin tissue samples);

[0079] 2. Determine the relationship between CK2, fatty acid metabolites, local infiltration of immune cells, cytokines and immune checkpoint levels and clinical characteristics of KRAS mutant lung cancer patients (fresh tissue and serum samples);

[0080] 3. Design retrospective and prospective studies to explore the correlation between CK2, FASN and DHHC3 expression and immunotherapy efficacy and patient survival prognosis (clinical research).

[0081] Experimental procedures

[0082] (1) Determine the role of protein kinase CK2 in KRAS mutant lung cancer PD-L1 high expression and immune escape

[0083] 1. Cell level: Use lentivirus and CRISPR / Cas9-mediated gene site knockout technology to change the expression level of CK2, and detect its effect on KRAS mutant lung cancer cell line metabolism (Seahorse metabolic instrument detection, important metabolite kit), lipid storage level (oil red O staining and Bodipy staining), fatty acid oxidation level (fatty acid oxidation kit);

[0084] 2. Cell level: Use lentivirus and CRISPR / Cas9-mediated gene site knockout technology to change the expression level of CK2, and detect its effect on KRAS mutant lung cancer cell line PD-L1 expression and cytokines (cytokine chip and ELISA);

[0085] 3. Construct Adeno-Cre induced KRAS-LSL-G12D point mutation lung cancer mouse model, analyze the expression dynamic changes of CK2, key target of fatty acid metabolism, immune cells and cytokines in the baseline level to KRAS mutant lung cancer progression;

[0086] 4. Use CK2 inhibitor to treat KRAS-LSL-G12D point mutation lung cancer mouse model, detect the changes of fatty acid metabolites (mass spectrometry and related metabolite detection kit), inflammatory factors and chemotactic factors (ELISA, protein chip and multi-factor flow) and other indicators in lung tissue and peripheral blood, evaluate the influence of CK2 on KRAS mutant lung cancer metabolic immune microenvironment;

[0087] 5. In KRAS-LSL-G12D point mutation lung cancer mouse model, use CK2 inhibitor, immune checkpoint inhibitor or combined with fatty acid metabolism enzyme inhibitor for treatment, observe the indexes such as tumorigenic rate, tumorigenic time and survival curve, evaluate the treatment efficacy, feasibility and side effects; collect peripheral blood PBMC, lung cancer tissue and paracancerous tissue synchronously, verify the value of CK2 and key gene of fatty acid metabolism in predicting treatment efficacy.

[0088] (2) Clarify the mechanism of protein kinase CK2 regulating PD-L1 palmitoylation modification through fatty acid metabolism reprogramming and direct phosphorylation of palmitoyltransferase DHHC3

[0089] Pathway 1: CK2 promotes de novo synthesis of palmitic acid by activating key enzyme FASN of fatty acid metabolism, which provides "raw materials" for palmitoylation modification of activated PD-L1 from palmitic acid;

[0090] 1. Identify the interaction of CK2 and FASN endogenous and exogenous proteins, and identify the domains of CK2 and FASN interaction: use co-IP method to detect the interaction of exogenous CK2 and FASN in HEK293T cells as a model; use co-IP method to detect the endogenous interaction between A549 and H23 KRAS mutant lung cancer cell lines with anti-CK2 or anti-FASN antibody; use site-directed mutagenesis to construct CK2 and FASN mutants with SFB or Myc tag, and use co-IP method to detect the interaction, so as to find out the domains of CK2 and FASN interaction;

[0091] 2. Verify the influence of silencing or inhibiting FASN on palmitic acid level in KRAS mutant lung cancer: after silencing or inhibiting FASN in KRAS mutant lung cancer cells, use palmitic acid content detection kit to detect palmitic acid content;

[0092] 3. Rescue experiment verifies the effect of overexpression of CK2, silencing or inhibiting FASN on the level of palmitic acid in KRAS mutant lung cancer: after silencing or inhibiting FASN in KRAS mutant lung cancer cells overexpressing CK2, the content of palmitic acid is detected using a palmitic acid content detection kit;

[0093] 4. Based on 13 C-labeled palmitic acid carbon metabolic flow analysis reveals the mutual regulation mechanism of upstream and downstream: stable 13 C isotope tracing technology is used to analyze the isotope labeling mode of upstream and downstream palmitic acid metabolites, so as to calculate the flow direction and distribution in the palmitic acid metabolic pathway and the activity of the palmitic acid metabolic pathway in KRAS mutant lung cancer;

[0094] 5. It is clear whether blocking palmitoylation can lead to enhanced PD-L1 membrane anchoring in KRAS mutant lung cancer: in KRAS mutant lung cancer cells, 2-BP (a general palmitoylation inhibitor) is used for treatment, and Western Blotting, flow cytometry and immunofluorescence are used to detect PD-L1 membrane protein expression; Click-IT method is used to verify the palmitoylation of PD-L1;

[0095] 6. Rescue experiment to determine the effect of overexpression of CK2, silencing or inhibiting FASN on the expression of PD-L1 membrane protein in KRAS mutant lung cancer: after silencing or inhibiting FASN in KRAS mutant lung cancer cells overexpressing CK2, Western Blotting, flow cytometry and immunofluorescence are used to detect the expression of PD-L1 membrane protein;

[0096] 7. Rescue experiment to determine the effect of silencing or inhibiting CK2 and exogenous intake of palmitic acid on PD-L1 membrane protein in KRAS mutant lung cancer: after exogenous intake of palmitic acid in KRAS mutant lung cancer cells with down-regulated CK2, Western Blotting, flow cytometry and immunofluorescence are used to detect the expression of PD-L1 membrane protein;

[0097] Pathway 2: CK2 directly phosphorylates the "catalyst" of palmitoylation modification - palmitoyl transferase DHHC3, mediates the occurrence of PD-L1 palmitoylation modification, and leads to the enhancement of PD-L1 membrane stability.

[0098] 1. Verify the endogenous and exogenous protein interaction between CK2 and DHHC3: using HEK293T cells as a model, co-IP method is used to detect the interaction between exogenous CK2 and DHHC3; using KRAS mutant lung cancer cell lines A549 and H23 as models, co-IP is used to detect the endogenous interaction between CK2 and DHHC3 using anti-CK2 or anti-FASN antibodies;

[0099] 2. Construct KRAS mutant lung cancer cell lines carrying stable expression of wild-type DHHC3 (DHHC3 WT ), phosphorylation-deficient T244A / T247A double mutant (DHHC3 2A ), and overexpression of phosphorylation T244E / T247E (DHHC3 2E ): Construct KRAS mutant lung cancer cell lines carrying stable expression of wild-type DHHC3 (DHHC3 WT ), phosphorylation-deficient T244A / T247A double mutant (DHHC3 2A ), and overexpression of phosphorylation T244E / T247E (DHHC3 2E ) using Crispr / Cas9 technology;

[0100] 3. Western Blotting to detect DHHC3 2E , DHHC3 2A and DHHC3 WT protein expression in cells: After the construction of the above three cell lines, Western Blotting was used to detect the basal expression of DHHC3 in the three cells;

[0101] 4. Effect of silencing or inhibiting CK2 on DHHC3 2E , DHHC3 2A and DHHC3 WT protein expression level in cells: After silencing or inhibiting CK2, Western Blotting was used to detect its effect on DHHC3 protein expression in the above three cells;

[0102] 5. Further identification of DHHC3 as the main palmitoyl transferase for KRAS mutant lung cancer PD-L1 expression: co-IP to detect protein interaction between PD-L1 and DHHC3; immunofluorescence to detect the co-localization of endogenous DHHC3 with PD-L1 in KRAS mutant lung cancer cell lines and tissues; immunohistochemical staining to detect the correlation between DHHC3 and PD-L1 expression;

[0103] 6. To determine the effect of silencing or inhibiting DHHC3 on KRAS mutant lung cancer PD-L1 membrane protein: Lentivirus and CRISPR / Cas9-mediated gene knockout technology to change the expression level of DHHC3, Western Blotting to detect the expression level of KRAS mutant lung cancer PD-L1 membrane protein; In addition, Western Blotting was used to detect the expression level of KRAS mutant lung cancer PD-L1 membrane protein induced by IFN-γ;

[0104] 7. Rescue experiment to determine the effect of overexpression of CK2, silencing or inhibition of DHHC3 on PD-L1 membrane protein in KRAS mutant lung cancer: after silencing or inhibiting DHHC3 in KRAS mutant lung cancer cells overexpressing CK2, the expression of PD-L1 membrane protein was detected using Western Blotting, flow cytometry and immunofluorescence.

[0105] (3) Explore the efficacy prediction and survival prognosis value of protein kinases CK2, FASN and DHHC3 in KRAS mutant lung cancer patients

[0106] 1. Obtain KRAS mutant lung cancer paraffin tissue samples, select different color and species antibodies for immunofluorescence and immunohistochemical staining; score according to staining intensity and expression area ratio. Determine the expression relationship of CK2, FASN and DHHC3, whether they are co-expressed and whether the expression is positively correlated. Further collect the clinical data of patients, including general condition, stage, treatment regimen, efficacy, disease progression-free survival and overall survival, etc. and analyze them by SPSS16.0 software;

[0107] 2. Collect tissue and serum samples of KRAS mutant lung cancer patients in our center, detect the expression of CK2, fatty acid metabolites (palmitic acid, monounsaturated fatty acids and polyunsaturated fatty acids, etc.), local infiltrating immune cells (CD45, CD3, CD4, CD8, CD107a, CD45RO and CD137, etc.) and cytokines (IFN-γ, CXCL1, TNF-α, CCL2, IL-12, CCL5, IL-1β, CXCL10, GM-CSF, IL-10, IFN-β, IFN-α and IL6, etc.) in KRAS mutant lung cancer, KRAS wild type lung cancer and paracancerous lung tissue;

[0108] 3. Prospective observation and retrospective study divide KRAS mutant lung cancer patients into different treatment subgroups (chemotherapy, targeted therapy, immunotherapy and radiotherapy), and evaluate the correlation between CK2, FASN and DHHC3 expression and patient efficacy, overall survival, disease-free survival, etc.

[0109] Experimental results

[0110] 1) Result 1: As shown in Figure 1 , KRAS mutant lung cancer patients with age <= 53 have better overall survival (OS) than patients with age > 53.

[0111] 2) Result 2: As shown in Figure 2 , the nutritional score of KRAS mutant lung cancer patients with high expression of CK2 is mainly 1-2 points, while the nutritional score of KRAS mutant lung cancer patients with low expression of CK2 is 0-4 points.

[0112] 3) Result 3: As shown in Figure 3 Figure 3, the worse the overall survival (OS) of the KRAS mutant lung cancer patients with high expression of CK2, the better.

[0113] 4) Result 4: As shown in Figure 4 Figure 4, the worse the progression-free survival (PFS) of the KRAS mutant lung cancer patients with high expression of CK2, the better.

[0114] 5) Result 5: As shown in Figure 5 Figure 5, the AUC curve shows the ability of CK2 to accurately predict the overall survival (OS) of KRAS mutant lung cancer patients for 1.5 years, 2 years and 3 years, and the AUC values are 0.85, 0.89 and 0.83, respectively.

[0115] 6) Result 6: As shown in Figure 6 Figure 6, the AUC curve shows the ability of CK2 to accurately predict the progression-free survival (PFS) of KRAS mutant lung cancer patients for 1 year, 1.5 years and 2 years, and the AUC values are 0.86, 0.9 and 1, respectively.

[0116] 7) Result 7: As shown in Figure 7 Figure 7, the time-dependent ROC curve shows that the AUC value (95% CI) of CK2 to predict the overall survival (OS) of KRAS mutant lung cancer patients remains stable over time.

[0117] 8) Result 8: As shown in Figure 8 Figure 8, the time-dependent ROC curve shows that the AUC value (95% CI) of CK2 to predict the progression-free survival (PFS) of KRAS mutant lung cancer patients remains stable over time.

[0118] 9) Result 9: As shown in Figure 9 Figure 9, the KRAS mutant lung cancer patients with high expression of CK2 have a poor response to immunotherapy; while the KRAS mutant lung cancer patients with low expression of CK2 have a good response to immunotherapy, and the patients with SD and PR are the main proportion.

[0119] 10) Result 10: As shown in Figure 10 Figure 10, bioinformatics analysis shows that KRAS mutant lung cancer presents a "cold tumor" feature in the tumor immune microenvironment compared to wild type: the immune score status between 149 cases of KRAS mutant lung cancer tissue, 852 cases of KRAS wild type lung cancer tissue and 108 cases of normal samples was analyzed using TCGA database. The results show that KRAS mutant lung cancer presents a "cold tumor" feature in the tumor immune microenvironment compared to wild type, and B cells, CD8 + T cells, neutrophils and macrophages are significantly lacking.

[0120] 11) Result 11: As shown in Figure 11 Figure 11, KRAS G12D conditional point mutation mice have significantly less intratumoral CD8 + T cells than wild-type mice; the intratumoral immune cell infiltration and functional status of KRAS G12D conditional point mutation mice and KRAS wild-type mice were detected by flow cytometry, and the results showed that KRAS mutant lung cancer mice had significantly less intratumoral CD8 + T cells than wild-type mice, and the tumor immune microenvironment was in an immunosuppressive state.

[0121] 12) Result 12: As shown in Figure 12 Figure 12, bioinformatics analysis showed that the highly expressed genes in KRAS mutant lung cancer patients were closely related to lipid metabolism; the differential gene expression between 149 KRAS mutant lung cancer tissues and 852 KRAS wild-type lung cancer tissues was analyzed using the TCGA database, and the potential functions of the up-regulated or down-regulated genes enriched by KEGG pathway and GO pathway functional enrichment were observed. The results showed that the highly expressed genes in KRAS mutant lung cancer patients were closely related to lipid metabolism, and CK2 gene was significantly highly expressed in the KRAS mutant group, indicating that CK2 may be a potential target for KRAS mutant lung cancer.

[0122] 13) Result 13: As shown in Figure 13 Figure 13, CK2 expression was closely related to the age, sex, and stage of KRAS mutant lung cancer patients, and high expression of CK2 had a poor prognosis.

[0123] 14) Result 14: As shown in Figure 14 Figure 14, liquid chromatography-mass spectrometry (LC-MS) non-targeted metabolomics analysis found that the palmitic acid metabolic pathway was significantly enriched after down-regulation of CK2, and the production of palmitic acid was reduced; in addition, the decrease in palmitic acid content can inhibit the occurrence of protein palmitoylation modification, thereby weakening membrane anchoring and ultimately enhancing the efficacy of PD-1 / PD-L1 immunotherapy. Non-targeted metabolomics analysis of KRAS mutant lung cancer cell line (A549) by LC-MS found that the palmitic acid metabolic pathway was significantly enriched after down-regulation of CK2 (siRNA); in addition, down-regulation of CK2 led to a decrease in palmitic acid production.

[0124] 15) Result 15: As shown in Figure 15As shown, silencing or inhibiting CK2 significantly downregulated FASN expression and reduced palmitic acid production in KRAS-mutant lung cancer. Furthermore, CK2 interacts with FASN and DHHC3. Transcriptomic analysis revealed that CK2 inhibitor treatment of the A549 cell line upregulated 1590 genes and downregulated 1231 genes. Pathway enrichment analysis of these differentially expressed genes showed significant enrichment of the fatty acid metabolism pathway. RT-qPCR, palmitic acid content assays, and Western blotting showed that silencing or inhibiting CK2 significantly downregulated FASN expression and reduced palmitic acid synthesis in KRAS-mutant lung cancer. Mass spectrometry analysis revealed that CK2 interacts with FASN and DHHC3. Co-IP experiments showed protein-protein interactions between CK2 and FASN, suggesting that CK2 promotes de novo palmitic acid synthesis by activating FASN. Additionally, CK2 can directly phosphorylate palmityltransferase DHHC3, ultimately activating palmitoylation of downstream proteins. Therefore, CK2, FASN, and DHHC3 hold important candidate molecular marker status in the treatment of KRAS-mutant lung cancer.

[0125] 16) Result 16: As Figure 16 As shown, KRAS-mutant lung cancer cells exhibited increased intracellular lipid droplet aggregation compared to KRAS wild-type lung cancer cells. Targeting CK2 significantly reduced intracellular lipid droplets in KRAS-mutant lung cancer cells, while no significant change was observed in KRAS wild-type lung cancer cells. KRAS-mutant and wild-type lung cancer cell lines were pretreated with the CK2 inhibitor CX4945 (concentration gradients: 1.25 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) for 24 h, stained with Bodipy fluorescent dye, and their lipid droplet MFI values ​​were detected by flow cytometry. Fluorescence microscopy and flow cytometry were used to detect lipid droplet fluorescence values ​​and the number of positive lipid droplets. The results showed that the KRAS-mutant lung cancer cell line (A549 KRAS Mutation) exhibited increased intracellular lipid droplet aggregation and stronger fluorescence values ​​compared to KRAS wild-type lung cancer cells (H1975-KRAS-Wild / H292-KRAS-Wild). Downregulation of CK2 significantly reduced intracellular lipid droplets in KRAS-mutant lung cancer cells, while no significant change was observed in KRAS wild-type lung cancer cells.

[0126] 17) Result 17: As Figure 17 As shown: In KRAS-mutant lung cancer, DHHC3 is one of the candidate interacting proteins of CK2, and Ser241, Thr244 and Thr247 of DHHC3 have been identified as potential phosphorylation sites.

[0127] 18) Result 18: As Figure 18 The image shows a KRAS-mutated lung cancer sample with high CK2 expression, exhibiting an immunosuppressive microenvironment within the tumor.

[0128] 19) Result 19: As shown in Table 19: KRAS mutant lung cancer samples with high expression of CK2, the intratumoral activated state of immune cells was significantly reduced and presented an immunosuppressive microenvironment. Figure 19

[0129] 20) Result 20: As shown in Table 20: KRAS mutant lung cancer samples with high expression of CK2, the tumor cells highly expressed immune checkpoint genes such as PD-L1, HAVCR2, PDCD1LG2 and SIGLEC15, leading to the formation of tumor immune escape. Figure 20

[0130] 21) Result 21: As shown in Table 21: CK2 was negatively correlated with intratumoral CD8 + T cells in KRAS mutant lung cancer patients, that is, high expression of CK2 induced the formation of an immunosuppressive microenvironment in KRAS mutant lung cancer. Figure 21

[0131] 22) Result 22: As shown in Table 22: FASN was positively correlated with CK2 expression, CK2 was positively correlated with DHHC3 expression, and CK2 was positively correlated with PD-L1 expression; this provides a basis for the value of CK2, FASN and DHHC3 combined detection in predicting the efficacy of immunotherapy and survival prognosis in KRAS mutant lung cancer patients. Figure 22

[0132] 23) Result 23: As shown in Table 23: The overall survival (OS) of the CK2 / FASN / DHHC3 high expression group was worse than that of the CK2 / FASN / DHHC3 low expression group. Figure 23

[0133] 24) Result 24: As shown in Table 1, the correlation between CK2 expression and clinical pathological variables and the efficacy of immunotherapy in 20 KRAS mutant lung cancer patients.

[0134] Table 1

[0135]

[0136]

[0137] Abbreviations: BMI - body mass index; PS - performance status; TP53 - tumor protein 53; PD-L1 - programmed death ligand 1; ICB - immune checkpoint blockade; PD - progressive disease; SD - stable disease; PR - partial remission.

[0138] Result analysis

[0139] ​​​​​1. The CK2 detection agent can effectively and accurately detect the expression level of CK2, and screen KRAS mutant lung cancer patients who are effective for immunotherapy through the expression level of CK2. The expression level of CK2 is negatively correlated with the response of KRAS mutant lung cancer patients to immunotherapy. That is, the higher the expression level of CK2, the worse the effect of immunotherapy, and the lower the expression level of CK2, the better the effect of immunotherapy; more importantly, the prediction performance evaluation AUC curve and time-dependent ROC curve analysis of CK2 show that the above-mentioned CK2 detection agent has excellent prediction performance.

[0140] 2. KRAS mutant lung cancer has high expression of PD-L1 compared with wild type, and the microenvironment presents "cold tumor" characteristics: bioinformatics analysis shows that the highly expressed genes of KRAS mutant lung cancer patients are closely related to lipid metabolism compared with wild type patients.

[0141] 3. KRAS mutant lung cancer has abnormal active fatty acid metabolism compared with wild type: multi-omics results show that the mRNA level of FASN, a key regulator of fatty acid synthesis, is significantly up-regulated in KRAS mutant lung cancer compared with wild type, and the accumulation of palmitic acid metabolites and lipid droplets increases.

[0142] 4. CK2 is positively correlated with FASN expression, and there is protein interaction; silencing and inhibiting CK2 reduces the synthesis of palmitic acid and lipid droplets in KRAS mutant lung cancer: multi-omics results show that silencing or inhibiting CK2 leads to differential genes in KRAS mutant lung cancer mainly enriched in fatty acid metabolism pathway; Bodipy experiment and RT-qPCR show that silencing or inhibiting CK2 leads to significant down-regulation of FASN mRNA and protein levels in KRAS mutant lung cancer, and reduction of palmitic acid and lipid droplet formation; mass spectrometry identification shows that CK2 and FASN have protein interaction, which suggests that CK2 promotes de novo synthesis of palmitic acid by activating FASN. Therefore, CK2 combined with FASN has an important candidate molecular marker status in the treatment of KRAS mutant lung cancer.

[0143] 5. CK2 may regulate the membrane stability of PD-L1 in KRAS mutant lung cancer through palmitoylation modification pathway: after targeting CK2, KRAS mutant lung cancer cells carrying PD-L1-T285A / T290A point mutation still have down-regulation of PD-L1 total protein and membrane protein expression; Western Blotting and immunofluorescence show that knocking down DHHC3 can down-regulate the expression of PD-L1 protein in KRAS mutant lung cancer and inhibit the palmitoylation of PD-L1; mass spectrometry identification and Western Blotting show that DHHC3 is one of the candidate interacting proteins of CK2, and knocking down CK2 can down-regulate the expression of DHHC3 protein in KRAS mutant lung cancer; phosphorylation site prediction shows that Ser241, Thr244 and Thr247 of DHHC3 are determined as potential phosphorylation sites of CK2.

[0144] 6.FASN is positively correlated with CK2 expression, CK2 is positively correlated with DHHC3 expression, and CK2 is positively correlated with PD-L1 expression; in addition, the overall survival (OS) of the CK2 / FASN / DHHC3 high expression group is poorer than that of the CK2 / FASN / DHHC3 low expression group. This provides a strong basis for the value of CK2, FASN and DHHC3 joint detection in predicting the efficacy of immunotherapy and survival prognosis in KRAS mutant lung cancer patients.

[0145] In summary, the present application finds that in KRAS mutant lung cancer, protein kinase CK2 promotes de novo synthesis of palmitic acid by activating fatty acid metabolism key enzyme FASN, and this pathway continuously provides "raw materials" for palmitoylation modification of activated PD-L1; on the other hand, CK2 directly phosphorylates palmitoyl transferase DHHC3, the "catalyst" of palmitoylation modification, to mediate the occurrence of PD-L1 palmitoylation modification, resulting in enhanced PD-L1 membrane stability. The present application can not only predict the efficacy of immunotherapy in KRAS mutant lung cancer patients, but also provide a new target for immunotherapy of KRAS mutant lung cancer.

[0146] Those skilled in the art can clearly make various modifications to the above embodiments without departing from the overall spirit and concept of the present application. All fall within the scope of the present application. The protection scheme of the present application is subject to the claims attached to the present application.

Claims

1. Use of a reagent for detecting the expression level of CK2 protein in the preparation of a product for screening KRAS mutant lung cancer patients suitable for immunotherapy; wherein, The immunotherapy is PD-1 / PD-L1 immunotherapy.

2. Use according to claim 1; wherein, The CK2 protein expression level is negatively correlated with the response of the KRAS mutant lung cancer patient to the PD-1 / PD-L1 immunotherapy.

3. The use of the reagent for detecting the expression level of CK2 protein in the preparation of a product for predicting the survival prognosis of a KRAS mutant lung cancer patient; wherein, The survival prognosis is overall survival or progression-free survival. The higher the CK2 protein expression level is, the worse the overall survival of the KRAS mutant lung cancer patient is, and the higher the CK2 protein expression level is, the worse the progression-free survival of the KRAS mutant lung cancer patient is.

4. The combined detection reagent of CK2, FASN and DHHC3 in the preparation of a product for predicting the survival prognosis of a KRAS mutant lung cancer patient; wherein, The CK2 / FASN / DHHC3 combined detection reagent is a reagent for combined detection of CK2, FASN and DHHC3 protein expression levels. The survival prognosis is overall survival, and the overall survival of the CK2 / FASN / DHHC3 high expression group is worse than that of the CK2 / FASN / DHHC3 low expression group.

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