New application of protein kinase CK2 in predicting the efficacy of immunotherapy for KRAS-mutated lung cancer

Abstract

The present invention belongs to the field of medicine, and specifically discloses the use of protein kinase CK2 in predicting the efficacy of immunotherapy for KRAS mutant lung cancer. It relates to the use of a reagent for detecting CK2 expression in the preparation of a product for analyzing the immunotherapy effect of KRAS mutant lung cancer; wherein, the application is at least one of the following: 1) the use of a reagent for detecting CK2 expression in the preparation of a product for detecting the prognostic effect of immunotherapy for KRAS mutant lung cancer, 2) the use of a reagent for detecting CK2 expression in the preparation of a product for predicting the prognostic effect of immunotherapy for KRAS mutant lung cancer. It also relates to the use of CK2 inhibitors in the preparation of immunotherapy drugs for KRAS mutant lung cancer. It also relates to the use of a reagent for detecting KRAS mutations in the preparation of a product for diagnosing whether lung cancer is a hot tumor; wherein, the KRAS mutation is a mutation that induces lung cancer. The present invention can predict the efficacy of immunotherapy for patients with KRAS mutant lung cancer, and can also provide a new target for immunotherapy of KRAS mutant lung cancer.

Description

Technical Field

[0001] The present invention 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 Art

[0002] 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 gene 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 its basic research and clinical treatment. The reason is that the KRAS protein is small in size and has a smooth surface, and its RAS has a very high affinity for GTP, which has led to the failure of most of the research and development of KRAS targeted drugs. Although the latest NCCN guidelines recommend the targeted drug AMG510 for the treatment of the G12C mutation subtype in KRAS (accounting for 13.8% of all KRAS mutation lung cancers), the effective rate is only 37.1%, which is not satisfactory, and most other KRAS mutation subtypes have no targeted drug breakthroughs. Therefore, traditional platinum-containing chemotherapy is still the first choice for the treatment of KRAS mutation lung cancer. Immunotherapy has brought hope to patients with KRAS mutation lung cancer, and its efficacy is significantly better than that of patients with other lung cancers with driver gene mutations. For example, the ImmunoTarget study, the subgroup analysis of the CheckMate057 study, and the Mazieres J study confirmed that the efficacy of single-drug immunotherapy in patients with KRAS-mutated lung cancer can reach 26%, but the problem of immunotherapy resistance remains to be solved. Therefore, exploring the resistance mechanism of KRAS-mutated lung cancer immunotherapy, finding ways to enhance sensitivity, and exploring new models of "immunotherapy +" have important theoretical significance and clinical value.

[0003] At present, the methods for predicting the therapeutic effect of immune checkpoint inhibitors mainly include two aspects. First, the expression level of PD-L1 protein in tumor tissue is detected by immunohistochemistry to understand its level to predict the therapeutic effect of patients. PD-L1 is a protein regulated by effector T cells. Its high expression implies that there are more effector T cells in tumor tissue, which indicates that there may be a stronger immune response, thereby indicating that patients may have a better response to immunotherapy. However, clinical research results show that high PD-L1 expression is only significantly correlated with the effect of immunotherapy in certain specific tumor types, while other tumor types may not have the same correlation. In addition, there are still some patients with insignificant PD-L1 expression who may still benefit from immunotherapy. This shows that PD-L1 expression level cannot completely and accurately predict the effect of immunotherapy. On the other hand, there are currently multiple brands and models of PD-L1 antibodies on the market, and there are differences in their detection methods, staining degrees, and results interpretation methods. This difference may cause a certain degree of interference in doctors and patients' decisions on whether to undergo immunotherapy and how to treat it. Therefore, standardization and unification of PD-L1 detection methods and interpretation standards are very important to ensure the effectiveness of immunotherapy and patients get the best treatment results. This requires the development of consistent guidelines and standards in clinical practice to improve the precision and reliability of immunotherapy.

[0004] The metabolic immune microenvironment is currently a hot topic in the field of tumor research. Metabolic reprogramming of tumor cells affects the function and state of immune checkpoints, further promoting their 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 immunosuppressive functions; at the same time, it can enhance the expression of PD-L1 in tumor cells themselves, induce the exhaustion of cytotoxic T cells and lead to immune escape. Hexokinase-2 (a key enzyme in glycolysis) has been reported to upregulate the expression of PD-L1 in tumor cells by activating the NF-κB pathway, leading to immune escape. In addition, methylenetetrahydrofolate dehydrogenase 2 (a folate cycle metabolic enzyme) promotes the upregulation of PD-L1 expression in tumor cells by efficiently driving the folate cycle, thereby promoting immune escape.

[0005] Protein kinase CK2 is the first protein kinase discovered by humans. It phosphorylates more than 300 substrates and plays an important role in the physiological and pathological metabolic processes of body cells. In January 2022, the FDA approved its specific inhibitor (CX4945) for the clinical treatment of cancer. The relationship between CK2 and cellular lipid metabolism is the current research focus in the CK2 field. Barbara Guerra et al. reported the potential role of CK2 in lipid metabolism and found that in the liver of mice fed a high-fat diet, CK2 can phosphorylate MED17, which 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-terminus of PTEN. Summary of the invention

[0006] In view of the above problems, the present invention provides a new application of protein kinase CK2 in predicting the efficacy of immunotherapy for KRAS mutant lung cancer, mainly to solve the problem that the efficacy of immunotherapy for KRAS mutant lung cancer is difficult to evaluate.

[0007] In order to solve the above problems, the present invention adopts the following technical solutions:

[0008] A first aspect of the present invention The invention relates to the use of a reagent for detecting CK2 expression 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 use of a reagent for detecting CK2 expression in the preparation of a product for detecting the prognostic effect of immunotherapy for KRAS mutant lung cancer, which application is mainly used to evaluate the therapeutic effect after immunotherapy for KRAS mutant lung cancer; 2) the use of a reagent for detecting CK2 expression in the preparation of a product for predicting the prognostic effect of immunotherapy for KRAS mutant lung cancer, which application is mainly used to evaluate the patient before immunotherapy to predict whether the patient will achieve better results during immunotherapy.

[0009] In another combination, CK2 can also be combined with FASN as an important molecular marker in the treatment of KRAS mutant lung cancer. The use of a reagent for detecting the interaction between CK2 and FASN in the preparation and analysis of a product for immunotherapy of KRAS mutant lung cancer; wherein the application is at least one of the following: 1) the use of a reagent for detecting the interaction between CK2 and FASN in the preparation of a product for detecting the prognostic effect of immunotherapy of KRAS mutant lung cancer, 2) the use of a reagent for detecting the interaction between CK2 and FASN in the preparation of a product for predicting the prognostic effect of immunotherapy of KRAS mutant lung cancer. The reagent for detecting the interaction between CK2 and FASN can use existing reagents that can be detected, and the surface plasmon resonance (SPR) technology represented by Biacore can be used to achieve detection. The aforementioned test for detecting the interaction between CK2 and FASN is a broad concept, which is a collection of essential reagents or alternative reagent solutions used in the detection process (such as the S series CM5 chip involved in the product Biacore T200), and its function is to detect protein interactions. And the immunotherapy effect of KRAS mutant lung cancer can be predicted based on the degree of interaction between CK2 and FASN. The stronger the interaction, the worse the prognosis.

[0010] There is also a combination method, the use of CK2, FASN and DHHC3 combined detection reagents in the preparation of products for analyzing the immunotherapy effect of KRAS mutant lung cancer; wherein the application is at least one of the following: 1) the use of a reagent for detecting CK2 expression in the preparation of a product for detecting the prognosis effect of immunotherapy of KRAS mutant lung cancer, 2) the use of a reagent for detecting CK2 expression in the preparation of a product for predicting the prognosis effect of immunotherapy of KRAS mutant lung cancer. The combined detection reagent can be a combination of several or a single one (such as a chip).

[0011] KRAS mutant lung cancer is lung cancer induced by KRAS mutation, which is significantly different from wild type. For example, lung cancer induced by KRAS-LSL-G12D point mutation, and lung cancer caused by other KRAS site mutations should also be equivalent in the present invention.

[0012] For the corresponding application, the following options can be made in specific application:

[0013] First, when making a prognostic judgment, the detected CK2 expression level is negatively correlated with the prognostic effect of immunotherapy for KRAS mutant lung cancer. Therefore, when the CK2 expression level is high, it means that the effect of immunotherapy for patients with KRAS mutant lung cancer will not be obvious, and when the CK2 expression level is low, it means that the effect of immunotherapy for patients with KRAS mutant lung cancer will be more obvious; similarly, the same method is used for prediction. The level of CK2 expression can be judged according to clinical needs, and the CK2 expression level in normal humans can generally be used as a reference;

[0014] Second, as for the reagent for detecting the expression of CK2, the reagent for detecting the expression of CK2 is at least one of an immunohistochemical staining kit, an ELISA kit, and a WB kit, and the function of the kit is to detect the expression level of CK2;

[0015] Third, in terms of immunotherapy, the monoclonal antibodies for PD-1 / PD-L1 immunotherapy for KRAS mutant lung cancer immunotherapy at least for KRAS mutant lung cancer mainly include: carrelizumab, pembrolizumab, durvalumab and tislelizumab.

[0016] As for the product for predicting the prognosis of immunotherapy for KRAS mutant lung cancer, it can be specifically reflected in the use of reagents for detecting CK2 expression in the preparation of products for screening KRAS mutant lung cancer patients suitable for immunotherapy. The prepared product can be used to detect samples obtained from patients, and when the CK2 expression level is obtained, it can be determined whether the patient is suitable for immunotherapy. For example, when the CK2 expression level is significantly higher than that of normal people, it indicates that the patient is not suitable for immunotherapy, or it indicates that CK2 inhibitors need to be used in the treatment process.

[0017] Second aspect of the present invention The invention relates to the use of CK2 inhibitors in the preparation of immunotherapy drugs for KRAS mutant lung cancer.

[0018] For the corresponding application, the following options or explanations can be made in specific applications:

[0019] First, the CK2 inhibitor is used to enhance the immunotherapy response of KRAS mutant lung cancer, thereby enhancing the immunotherapy effect. The effect of the CK2 inhibitor can be further manifested as at least one of the following:

[0020] 1) Down-regulate FASN expression,

[0021] 2) Down-regulate DHHC3 expression,

[0022] 3) Reduce the production of palmitic acid,

[0023] 4) weaken the stability of PD-L1 membrane,

[0024] 5) Generate CD8 in the tumor of KRAS mutant lung cancer mouse model + T cell infiltration increased, and the tumor presented an activated immune microenvironment;

[0025] Second, the CK2 inhibitor is at least any one of CX4945, TBB, kunalizarin, NSC-231634, and DMAT;

[0026] Third, immunotherapy for KRAS-mutated lung cancer is at least PD-1 / PD-L1 immunotherapy for KRAS-mutated lung cancer. Monoclonal antibodies mainly include: carrelizumab, pembrolizumab, durvalumab and tislelizumab;

[0027] Fourthly, KRAS mutant lung cancer is lung cancer induced by KRAS mutation, which is significantly different from wild type. For example, lung cancer induced by KRAS-LSL-G12D point mutation and lung cancer caused by other KRAS site mutations should also be equivalent in the present invention.

[0028] The third aspect of the present invention The invention 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; wherein the KRAS mutation is a mutation that induces lung cancer.

[0029] 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 them difficult to be recognized and attacked by the immune system. Hot tumors have a higher mutation load, and there are a large number of immune cells in the tumor microenvironment, such as T cells and antigen-presenting cells, which are involved 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 the immune response is relatively weak, 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 respond poorly to immunotherapy because they lack the support of immune cells. Therefore, other strategies, such as targeted therapy or chemotherapy, may be needed for cold tumors to enhance the immune response. Hot tumors usually respond more actively to immunotherapy because the immune system has already played a role in tumor resistance. Immunotherapy can enhance these responses and increase the chances of treatment success. Therefore, the initial clarification of the tumor type can enable doctors to choose treatment options more targeted. For cold tumors, other treatments may need to be explored, while for hot tumors, immunotherapy may be a more effective option. Individualized treatment plans are usually combined with the patient's specific situation and tumor characteristics to achieve the best treatment effect. Therefore, based on the differences between cold tumors and hot tumors in terms of properties, immune cells, and treatment effects, different treatment plans are required for the two. The initial clarification of tumor types can facilitate more targeted treatment, thereby providing an auxiliary means of targeted treatment for clinical treatment.

[0030] Among them, the KRAS mutation at least includes the KRAS-LSL-G12D point mutation. Of course, since other types of lung cancer induced by KRAS mutations also have high similarities, their treatment plans are also the same. Therefore, the remaining KRAS mutation-induced lung cancer should also be within the scope of the present invention.

[0031] The beneficial effects of the present invention are:

[0032] The present invention can predict and detect the efficacy of immunotherapy for patients with KRAS mutant lung cancer, and can also provide a new target for immunotherapy of KRAS mutant lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 : Correlation of age with overall survival (OS) in patients with KRAS-mutated lung cancer.

[0034] Figure 2 : Correlation between CK2 expression level and nutritional score in patients with KRAS mutation lung cancer.

[0035] Figure 3 : Correlation of CK2 expression level with overall survival (OS) in patients with KRAS mutation lung cancer.

[0036] Figure 4 : Correlation of CK2 expression level with progression-free survival (PFS) in patients with KRAS mutation lung cancer.

[0037] Figure 5 : The AUC curve showed the ability of CK2 to predict the 1.5-year, 2-year and 3-year overall survival (OS) of patients with KRAS mutation lung cancer, with AUC values ​​of 0.85, 0.89 and 0.83, respectively.

[0038] Figure 6 : The AUC curve showed the ability of CK2 to predict 1-year, 1.5-year and 2-year progression-free survival (PFS) in patients with KRAS mutation lung cancer, with AUC values ​​of 0.86, 0.9 and 1, respectively.

[0039] Figure 7 : The time-dependent ROC curve showed that the AUC value (95% CI) of CK2 for predicting overall survival (OS) in patients with KRAS mutant lung cancer remained stable over time.

[0040] Figure 8 : The time-dependent ROC curve showed that the AUC value (95% CI) of CK2 for predicting progression-free survival (PFS) in patients with KRAS mutant lung cancer remained stable over time.

[0041] Fig. 9 : Correlation of CK2 expression level with response to immunotherapy in patients with KRAS mutant lung cancer.

[0042] Fig.10 :Bioinformatics analysis showed that KRAS mutant lung cancer has a "cold tumor" tumor immune microenvironment compared with wild-type lung cancer. (A) B cells and CD8 + T cells, neutrophils and macrophages were significantly lacking; (B) Immune cell score heat map, where different colors represent the expression trend in different samples, *p<0.05, **p<0.01, ***p<0.001, asterisks represent the degree of significance (*p), the significance of two groups of samples was tested by Wilcox test, and the significance of three or more groups of samples was 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 axis represents the sample, and the vertical axis represents the percentage of immune cell content in a single sample.

[0043] Fig.11 :Compared with wild-type mice, mice with KRAS gene G12D conditional point mutation had significantly higher intratumoral CD8 + There was a marked lack of T cells.

[0044] Fig.12 :Bioinformatics analysis showed that the genes highly expressed in KRAS mutant lung cancer patients were closely related to lipid metabolism compared with wild-type patients. (A) Volcano plot of differentially expressed genes; (B) Heat map of differentially expressed genes; (CD) Functional enrichment: KEGG or GO pathway enrichment results of differentially up-regulated or down-regulated genes.

[0045] Fig.13 : The high and low expression distribution trend of CK2 in 149 KRAS mutant lung cancer samples with different age, gender and AJCC eighth edition lung cancer stage and patient survival.

[0046] Fig.14 : Non-targeted LC-MS metabolomics analysis found that palmitic acid metabolic pathway was significantly enriched after downregulating CK2, which led to reduced palmitic acid production. (A) Differential metabolites were visualized in the form of volcano plots; (B) Differential metabolite pathway enrichment analysis; the redder the circle, the larger the shape, and the higher the significance; (C) Random Forests VIP (Variable Importance in Projection) diagram; indicates the importance of metabolites and their contribution to sample differentiation; (D) PLS-DA loading diagram; reflects the contribution of metabolites to the differentiation between sample groups. The farther from the center point, the greater the contribution to the differentiation of sample groups; (E) Distribution of palmitic acid in si-Vehicle and si-CK2α groups, P < 0.05.

[0047] Fig.15 : Silencing or inhibiting CK2 leads to a significant downregulation of FASN expression and reduced palmitic acid production in KRAS mutant lung cancer; and CK2 interacts with FASN and DHHC3. (A) Volcano plot of differentially expressed genes; (B) Pathway enrichment analysis of differentially expressed genes; (C) RT-qPCR to detect the mRNA level of FASN; (D) Metabolite kit to detect the specific content of palmitic acid; (E) Mass spectrometry analysis of potential interacting proteins of CK2α.

[0048] Fig.16 :KRAS mutant lung cancer has increased intracellular lipid droplet aggregation compared with KRAS wild-type lung cancer; after targeting CK2, KRAS mutant lung cancer has significantly reduced intracellular lipid droplets, while KRAS wild-type lung cancer has no significant changes. (A) Effect of concentration gradient downregulation of CK2 on lipid droplet aggregation in KRAS mutant cell lines and wild-type cell lines, histogram; (BD) Effect of concentration gradient downregulation of CK2 on lipid droplet aggregation in KRAS mutant cell lines and wild-type cell lines, scatter plot; (EG) Effect of concentration gradient downregulation of CK2 on lipid droplet aggregation in KRAS mutant cell lines and wild-type cell lines; fluorescence microscopy detection, 10μm scale, green is intracellular staining of lipid droplets.

[0049] Fig.17 : In KRAS mutant lung cancer, DHHC3 is one of the candidate CK2 interacting proteins, and Ser241, Thr244 and Thr247 of DHHC3 have been identified as potential phosphorylation sites.

[0050] Fig.18 : Immune cell score heat map, distribution of immune scores of each immune cell in 64 patients with high CK2 expression and 85 patients with KRAS mutation lung cancer with low CK2 expression, 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 KRAS mutation lung cancer samples with high CK2 expression have an immunosuppressive microenvironment in the tumor.

[0051] Fig.19 :The percentage abundance of tumor-infiltrating immune cells in 64 patients with KRAS-mutant lung cancer with high CK2 expression and 85 patients with low CK2 expression. The results suggest that KRAS-mutant lung cancer samples with high CK2 expression have an immunosuppressive microenvironment in the tumor.

[0052] Fig. 20:Expression distribution of immune checkpoint genes in 64 patients with KRAS mutation lung cancer with high CK2 expression and 85 patients with low CK2 expression. The horizontal axis represents different groups of samples, the vertical axis represents the distribution of gene expression, and different colors represent the expression trends in different groups and samples. The results suggest that in KRAS mutation lung cancer samples with high CK2 expression, the tumor cells highly express immune checkpoint genes such as PD-L1 (CD274), HAVCR2, PDCD1LG2 and SIGLEC15, leading to tumor immune escape.

[0053] Fig.21 : Correlation analysis between CK2, FASN, DHHC3 and PD-L1 expression and each immune score, as well as correlation analysis between immune scores themselves. The heat map in the schematic diagram represents the correlation analysis of immune scores themselves, red represents positive correlation, blue represents negative correlation, the redder or bluer the color, the greater the correlation between the two, and the larger the circle, the stronger the correlation; the red line in the schematic diagram represents the negative correlation between the model score or gene expression and the immune score, and the green line represents the positive correlation between the two. The results suggest that CK2 is negatively correlated with CD8+T cells with direct killing function in the tumor of patients with KRAS mutant lung cancer, that is, high expression of CK2 induces the formation of an immunosuppressive microenvironment in KRAS mutant lung cancer.

[0054] Fig. 22 :The Spearman correlation analysis between FASN and CK2 expression, CK2 and DHHC3 expression, CK2 and PD-L1 expression, and DHHC3 and FASN expression in KRAS mutant lung cancer samples was performed. The results showed that FASN and CK2 expression were positively correlated (P=0.003), CK2 and DHHC3 expression were positively correlated (P=2.65e-07), CK2 and PD-L1 expression were positively correlated (P=0.001), and there was no statistical difference between DHHC3 and FASN expression (P=0.092); this provides a basis for the prediction of immunotherapy efficacy and survival prognosis value of combined detection of CK2, FASN and DHHC3 in patients with KRAS mutant lung cancer.

[0055] Fig.23: Kaplan-Meier survival curves of CK2, FASN and DHHC3 as "clusters", where the high and low expression groups of CK2 / FASN / DHHC3 were tested by Log-rank, HR (High exp) represents the risk factor of the high expression group relative to the low expression group. If HR>1, it means that the gene is a risk factor (the higher the expression, the worse the prognosis), and if HR<1, it means 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 CK2 / FASN / DHHC3 high expression group is worse than that of the CK2 / FASN / DHHC3 low expression group.

[0056] Fig.24 :Diagram of animal experiment model. DETAILED DESCRIPTION

[0057] The following is further explanation in conjunction with specific research projects.

[0058] Experimental protocol

[0059] (1) Clarify the role of protein kinase CK2 in high expression of PD-L1 and immune escape in KRAS mutant lung cancer

[0060] 1. Cellular level: clarify the effect of CK2 on the expression of key molecules of fatty acid metabolism in KRAS mutant lung cancer cells;

[0061] 2. Cellular level: clarify the effect of CK2 on the expression of immune cells, cytokines and immune checkpoints in KRAS mutant lung cancer;

[0062] 3. Construct a 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 in the progression from normal lung tissue to KRAS mutant lung cancer;

[0063] 4. Use CK2 inhibitors to treat KRAS-LSL-G12D point mutation lung cancer mouse models to determine their effects on key molecules of fatty acid metabolism, immune cells, cytokines, and immune checkpoints in tumor tissue and peripheral blood;

[0064] 5. Use CK2 inhibitors, immune checkpoint inhibitors and fatty acid metabolism enzyme inhibitors to treat the KRAS-LSL-G12D point mutation lung cancer mouse model, and evaluate the efficacy and safety of each treatment group.

[0065] (2) Elucidate the mechanism by which protein kinase CK2 regulates PD-L1 palmitoylation modification through fatty acid metabolic reprogramming and direct phosphorylation of palmitoyltransferase DHHC3

[0066] Pathway 1: CK2 promotes the de novo synthesis of palmitic acid by activating FASN, a key enzyme in fatty acid metabolism. This pathway continuously provides "raw materials" for palmitic acid to activate PD-L1 palmitoylation modification.

[0067] 1. Clarify the interaction between CK2 and endogenous and exogenous FASN proteins, and identify the domains of interaction between CK2 and FASN;

[0068] 2. Verify the effect of silencing or inhibiting FASN on palmitic acid levels in KRAS mutant lung cancer;

[0069] 3. Rescue experiments verified the effects of overexpression of CK2 and silencing or inhibiting FASN on palmitic acid levels in KRAS mutant lung cancer;

[0070] 4. Based on 13 Analysis of the carbon metabolic flux of C-labeled palmitic acid revealed the upstream and downstream mutual regulation mechanism;

[0071] 5. Determine whether blocking palmitoylation can lead to enhanced membrane anchoring of PD-L1 in KRAS mutant lung cancer;

[0072] 6. The Rescue experiment clearly showed the effect of overexpressing CK2 and silencing or inhibiting FASN on the expression of PD-L1 membrane protein in KRAS mutant lung cancer;

[0073] 7. The Rescue experiment clearly demonstrated the effect of silencing or inhibiting CK2 and exogenous intake of palmitic acid on the PD-L1 membrane protein in KRAS mutant lung cancer.

[0074] Pathway 2: CK2 directly phosphorylates the "catalyst" of palmitoylation modification, palmitoyltransferase DHHC3, mediating the palmitoylation modification of PD-L1, leading to enhanced PD-L1 membrane stability.

[0075] 1. Verify the interaction between CK2 and endogenous and exogenous DHHC3 proteins;

[0076] 2. Construction of a gene carrying stable expression of wild-type DHHC3 (DHHC3 WT ), phosphorylation-deficient T244A / T247A double mutant (DHHC3 2A ) and overexpression of phosphorylated T244E / T247E (DHHC3 2E ) KRAS mutant lung cancer cell lines;

[0077] 3. Western Blotting to Detect DHHC3 2E DHHC3 2A and DHHC3 WT Expression of DHHC3 protein in cells;

[0078] 4. Silencing or inhibiting CK2 to DHHC3 2E DHHC3 2A and DHHC3 WT The effect of cellular DHHC3 protein expression levels;

[0079] 5. Further identified DHHC3 as the major palmitoyltransferase for PD-L1 expression in KRAS-mutant lung cancer;

[0080] 6. Clarify the effect of silencing or inhibiting DHHC3 on PD-L1 membrane protein in KRAS mutant lung cancer;

[0081] 7. The Rescue experiment clearly showed the effects of overexpressing CK2 and silencing or inhibiting DHHC3 on the PD-L1 membrane protein in KRAS mutant lung cancer.

[0082] (3) Explore the therapeutic efficacy prediction and survival prognostic value of protein kinases CK2, FASN, and DHHC3 in patients with KRAS-mutated lung cancer

[0083] 1. To clarify the relationship between the expression levels and localization of CK2, FASN and DHHC3 and clinical characteristics such as staging, immunotherapy efficacy, and prognosis of patients with KRAS mutation lung cancer (paraffin tissue samples);

[0084] 2. To clarify the relationship between CK2, fatty acid metabolites, local infiltrating immune cells, cytokines and immune checkpoint levels and the clinical characteristics of patients with KRAS mutation lung cancer (fresh tissue and serum samples);

[0085] 3. Design retrospective and prospective studies to explore the correlation between CK2, FASN and DHHC3 expression and the efficacy of immunotherapy and patient survival prognosis (clinical research).

[0086] Experimental steps (1) Clarify the role of protein kinase CK2 in high expression of PD-L1 and immune escape in KRAS mutant lung cancer

[0087] 1. Cell level: Use lentivirus and CRISPR / Cas9-mediated gene site-directed knockout technology to change the expression level of CK2, and detect its effects on the metabolic level (Seahorse metabolic instrument detection, important metabolite kit), lipid storage level (Oil Red O staining and Bodipy staining), and fatty acid oxidation level (fatty acid oxidation kit) of KRAS mutant lung cancer cell lines;

[0088] 2. Cell level: Use lentivirus and CRISPR / Cas9-mediated gene site-directed knockout technology to change the expression level of CK2 and detect its effect on PD-L1 expression and cytokines (cytokine chip and ELISA) in KRAS mutant lung cancer cell lines;

[0089] 3. Construct an Adeno-Cre-induced KRAS-LSL-G12D point mutation lung cancer mouse model and analyze the dynamic changes in the expression of CK2, key targets of fatty acid metabolism, immune cells and cytokines from baseline to KRAS mutation lung cancer;

[0090] 4. Treat the KRAS-LSL-G12D point mutation lung cancer mouse model with CK2 inhibitors, detect changes in fatty acid metabolites (mass spectrometry and related metabolite detection kits), inflammatory factors and chemokines (ELISA, protein chip and multifactor flow cytometry) in lung tissue and peripheral blood, and evaluate the effect of CK2 on the metabolic immune microenvironment of KRAS mutant lung cancer;

[0091] 5. In the KRAS-LSL-G12D point mutation lung cancer mouse model, CK2 inhibitors, immune checkpoint inhibitors or combined with fatty acid metabolism enzyme inhibitors were used for treatment, and indicators such as tumor formation rate, tumor formation time, and survival curve were observed to evaluate the therapeutic efficacy, feasibility, and toxic side effects; peripheral blood PBMCs, lung cancer tissues, and adjacent tissues were collected simultaneously to verify the value of CK2 and key genes of fatty acid metabolism in predicting therapeutic efficacy.

[0092] (2) Elucidate the mechanism by which protein kinase CK2 regulates PD-L1 palmitoylation modification through fatty acid metabolic reprogramming and direct phosphorylation of palmitoyltransferase DHHC3

[0093] Pathway 1: CK2 promotes the de novo synthesis of palmitic acid by activating FASN, a key enzyme in fatty acid metabolism. This pathway continuously provides "raw materials" for palmitic acid to activate PD-L1 palmitoylation modification.

[0094] 1. Clarify the interaction between CK2 and endogenous and exogenous FASN proteins, and identify the domain of interaction between CK2 and FASN: Using HEK293T cells as a model, use the co-IP method to detect the interaction between exogenously expressed CK2 and FASN; Using KRAS mutant lung cancer cell lines A549 and H23 as models, use anti-CK2 or anti-FASN antibodies to perform co-IP to detect the endogenous interaction between the two; Use site-directed mutagenesis to construct different mutants of CK2 and FASN with SFB or Myc tags, and use the co-IP method to detect mutual binding, so as to find out the domain of interaction between CK2 and FASN;

[0095] 2. Verify the effect of silencing or inhibiting FASN on palmitic acid levels in KRAS mutant lung cancer: After silencing or inhibiting FASN in KRAS mutant lung cancer cells, use a palmitic acid content detection kit to detect palmitic acid content;

[0096] 3. Rescue experiment verified the effect of overexpression of CK2 and silencing or inhibiting FASN on palmitic acid levels in KRAS mutant lung cancer cells: In KRAS mutant lung cancer cells overexpressing CK2, after silencing or inhibiting FASN, the palmitic acid content was detected using a palmitic acid content detection kit;

[0097] 4. Based on 13 Analysis of carbon metabolic flux of C-labeled palmitic acid reveals the upstream and downstream mutual regulation mechanism: using stable 13 C isotope tracing technology was used to analyze the isotope labeling patterns of upstream and downstream palmitic acid metabolites, thereby inferring the flow and distribution in the palmitic acid metabolic pathway and the activity of the palmitic acid metabolic pathway in KRAS mutant lung cancer.

[0098] 5. To determine whether blocking palmitoylation can lead to enhanced membrane anchoring of PD-L1 in KRAS mutant lung cancer cells: In KRAS mutant lung cancer cells, 2-BP (a universal palmitoylation inhibitor) was used to treat the cells, and PD-L1 membrane protein expression was detected by Western Blotting, flow cytometry, and immunofluorescence; the palmitoylation of PD-L1 was verified by the Click-IT method;

[0099] 6. The Rescue experiment clarified the effects of overexpressing CK2 and silencing or inhibiting FASN on the expression of PD-L1 membrane protein in KRAS mutant lung cancer: in KRAS mutant lung cancer cells overexpressing CK2, after silencing or inhibiting FASN, Western Blotting, flow cytometry and immunofluorescence were used to detect the expression of PD-L1 membrane protein;

[0100] 7. The Rescue experiment clearly showed the effect of silencing or inhibiting CK2 and exogenous intake of palmitic acid on the PD-L1 membrane protein of KRAS mutant lung cancer: Western Blotting, flow cytometry and immunofluorescence were used to detect the expression of PD-L1 membrane protein in KRAS mutant lung cancer cells with downregulated CK2 after exogenous intake of palmitic acid;

[0101] Pathway 2: CK2 directly phosphorylates the "catalyst" of palmitoylation modification, palmitoyltransferase DHHC3, mediating the palmitoylation modification of PD-L1, leading to enhanced PD-L1 membrane stability.

[0102] 1. Verify the interaction between endogenous and exogenous proteins of CK2 and DHHC3: Using HEK293T cells as a model, the co-IP method was used to detect the interaction between exogenously expressed CK2 and DHHC3; using KRAS mutant lung cancer cell lines A549 and H23 as models, anti-CK2 or anti-FASN antibodies were used for co-IP to detect the endogenous interaction between the two;

[0103] 2. Construction of a gene carrying stable expression of wild-type DHHC3 (DHHC3 WT ), phosphorylation-deficient T244A / T247A double mutant (DHHC3 2A ) and overexpression of phosphorylated T244E / T247E (DHHC3 2E ) KRAS mutant lung cancer cell line: Crispr / Cas9 technology was used to construct a cell line carrying stable expression of wild-type DHHC3 (DHHC3 WT ), phosphorylation-deficient T244A / T247A double mutant (DHHC3 2A ) and overexpression of phosphorylated T244E / T247E (DHHC3 2E ) KRAS mutant lung cancer cell lines;

[0104] 3. Western Blotting to Detect DHHC3 2E DHHC3 2A and DHHC3 WT Expression of DHHC3 protein in cells: After the above three cell lines were constructed, the basal expression of DHHC3 in the above three cells was detected by Western Blotting;

[0105] 4. Silencing or inhibiting CK2 to DHHC3 2E DHHC3 2A and DHHC3 WT Effects on the expression level of DHHC3 protein in cells: After silencing or inhibiting CK2, Western Blotting was used to detect its effects on the expression of DHHC3 protein in the above three types of cells;

[0106] 5. Further identification of DHHC3 as the main palmitoyltransferase expressed by PD-L1 in KRAS mutant lung cancer: co-IP detection of protein interaction between PD-L1 and DHHC3; immunofluorescence detection of co-localization of endogenously expressed DHHC3 with PD-L1 in KRAS mutant lung cancer cell lines and tissues; immunohistochemical staining detection of the correlation between DHHC3 and PD-L1 expression;

[0107] 6. Clarify the effect of silencing or inhibiting DHHC3 on the PD-L1 membrane protein in KRAS mutant lung cancer: Use lentivirus and CRISPR / Cas9-mediated gene site-directed knockout technology to change the expression level of DHHC3, and detect the expression level of PD-L1 membrane protein in KRAS mutant lung cancer by Western Blotting; In addition, use Western Blotting to detect the expression level of PD-L1 membrane protein in KRAS mutant lung cancer induced by IFN-γ;

[0108] 7. The Rescue experiment clarified the effect of overexpressing CK2 and silencing or inhibiting DHHC3 on the PD-L1 membrane protein in KRAS mutant lung cancer: In KRAS mutant lung cancer cells overexpressing CK2, after silencing or inhibiting DHHC3, Western Blotting, flow cytometry and immunofluorescence were used to detect the expression of PD-L1 membrane protein.

[0109] (3) Explore the therapeutic efficacy prediction and survival prognostic value of protein kinases CK2, FASN, and DHHC3 in patients with KRAS-mutated lung cancer

[0110] 1. Obtain paraffin tissue samples of KRAS-mutated lung cancer, select antibodies of different colors and species for immunofluorescence and immunohistochemistry staining; score according to staining intensity and expression area ratio. Clarify the expression relationship of CK2, FASN and DHHC3, whether they are co-expressed and whether their expressions are positively correlated. Further collect the clinical data of the patients, count the clinical indicators such as the general condition, staging, treatment plan and efficacy, disease progression-free survival and overall survival of the patients, and analyze them using SPSS16.0 software;

[0111] 2. Collect tissue and serum samples from patients with KRAS mutant lung cancer in our center, and 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 patients with KRAS mutant lung cancer, KRAS wild-type lung cancer and adjacent lung tissues;

[0112] 3. Prospective observation and retrospective studies divided KRAS mutant lung cancer patients into different treatment subgroups (chemotherapy, targeted therapy, immunotherapy and radiotherapy) to evaluate the association between CK2, FASN and DHHC3 expression and patient efficacy, overall survival, disease-free survival, etc.

[0113] Experimental Results

[0114] 1) Result 1: If Figure 1 As shown in , patients with KRAS-mutated lung cancer aged <= 53 years had better overall survival (OS) than those aged > 53 years.

[0115] 2) Result 2: If Figure 2 As shown in the figure, the nutritional scores of patients with KRAS mutant lung cancer and high CK2 expression were mainly 1-2 points, while those of patients with KRAS mutant lung cancer and low CK2 expression were 0-4 points.

[0116] 3) Result 3: If Figure 3 As shown in , the overall survival (OS) of KRAS mutant lung cancer patients with high CK2 expression is worse, and vice versa.

[0117] 4) Result 4: If Figure 4 As shown in the results, the disease-free survival (PFS) of KRAS mutant lung cancer patients with high CK2 expression is worse, and vice versa.

[0118] 5) Result 5: If Figure 5 As shown in the figure, the AUC curve shows the ability of CK2 to accurately predict the 1.5-year, 2-year and 3-year overall survival (OS) of patients with KRAS mutation lung cancer, with AUC values ​​of 0.85, 0.89 and 0.83, respectively.

[0119] 6) Result 6: If Figure 6 As shown in the figure, the AUC curve shows the ability of CK2 to accurately predict the 1-year, 1.5-year and 2-year progression-free survival (PFS) of patients with KRAS mutation lung cancer, with AUC values ​​of 0.86, 0.9 and 1, respectively.

[0120] 7) Result 7: If Figure 7 As shown, the time-dependent ROC curve showed that the AUC value (95% CI) of CK2 in predicting overall survival (OS) of patients with KRAS mutant lung cancer remained stable over time.

[0121] 8) Result 8: If Figure 8 As shown, the time-dependent ROC curve showed that the AUC value (95% CI) of CK2 in predicting progression-free survival (PFS) in patients with KRAS-mutated lung cancer remained stable over time.

[0122] 9) Result 9: Fig. 9 As shown in the data, patients with KRAS mutant lung cancer with high CK2 expression had a poor response to immunotherapy, while patients with KRAS mutant lung cancer with low CK2 expression had a better response to immunotherapy, with the majority of patients evaluated as SD and PR.

[0123] 10) Result 10: If Fig.10 As shown in the results, bioinformatics analysis showed that KRAS mutant lung cancer had a "cold tumor" immune microenvironment compared to wild-type lung cancer: the TCGA database was used to analyze the immune score status of 149 KRAS mutant lung cancer tissues, 852 KRAS wild-type lung cancer tissues, and 108 normal samples. The results showed that KRAS mutant lung cancer had a "cold tumor" immune microenvironment compared to wild-type lung cancer, with B cells, CD8 + There was a marked deficiency of T cells, neutrophils, and macrophages.

[0124] 11) Result 11: If Fig.11 As shown in Figure 2, mice with KRAS G12D conditional point mutation had significantly higher intratumoral CD8 + The research group used flow cytometry to detect the infiltration and functional status of immune cells in the tumors of mice with KRAS gene G12D conditional point mutation and KRAS wild-type mice. The results showed that the CD8 + There is a significant lack of T cells, and the tumor immune microenvironment is immunosuppressive.

[0125] 12) Result 12: If Fig.12 As shown in the results, bioinformatics analysis showed that the genes highly expressed in KRAS mutant lung cancer patients were closely related to lipid metabolism compared with wild-type patients; the TCGA database was used to analyze the differential gene expression between the two groups of samples in 149 KRAS mutant lung cancer tissues and 852 KRAS wild-type lung cancer tissues, and the potential functions enriched by these up-regulated or down-regulated genes were observed through KEGG pathway and GO pathway functional enrichment. The results showed that the genes highly expressed in KRAS mutant lung cancer patients were closely related to lipid metabolism compared with wild-type patients; and the CK2 gene was significantly highly expressed in the KRAS mutant group, indicating that CK2 may be a potential target for KRAS mutant lung cancer.

[0126] 13) Result 13: If Fig.13 As shown in , CK2 expression is closely related to the age, gender and stage clinical characteristics of patients with KRAS mutation lung cancer, and high CK2 expression is associated with a poor prognosis.

[0127] 14) Result 14: If Fig.14 As shown in , non-targeted LC-MS metabolomics analysis by liquid chromatography-mass spectrometry found that the palmitic acid metabolic pathway was significantly enriched after downregulating CK2, and the production of palmitic acid was reduced; in addition, the reduction 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. Our research group conducted non-targeted metabolomics analysis of KRAS mutant lung cancer cell lines (A549) by LC-MS and found that the palmitic acid metabolic pathway was significantly enriched after downregulating CK2 (siRNA); in addition, downregulating CK2 led to a reduction in palmitic acid production.

[0128] 15) Result 15: If Fig.15As shown in, silencing or inhibiting CK2 leads to a significant downregulation of FASN expression and reduced palmitic acid production in KRAS mutant lung cancer; and CK2 has protein interactions with FASN and DHHC3: Our research group found through transcriptomic analysis that CK2 inhibitors treated A549 cell lines, upregulated 1590 genes and downregulated 1231 genes. Pathway enrichment analysis of these differentially expressed genes found that the fatty acid metabolism pathway was significantly enriched; RT-qPCR, palmitic acid content kit and WB method found that silencing or inhibiting CK2 led to a significant downregulation of FASN expression and reduced palmitic acid synthesis in KRAS mutant lung cancer; mass spectrometry analysis found that CK2, FASN and DHHC3 were potential interacting proteins; co-IP experiments found that CK2 and FASN had protein interactions, suggesting that CK2, on the one hand, promotes de novo palmitic acid synthesis by activating FASN; on the other hand, CK2 can directly phosphorylate palmitoyltransferase DHHC3, ultimately activating downstream protein palmitoylation modification. Therefore, CK2, FASN and DHHC3 have important candidate molecular markers in the treatment of KRAS mutant lung cancer.

[0129] 16) Result 16: If Fig.16 As shown in the figure, KRAS mutant lung cancer has more intracellular lipid droplets than KRAS wild-type lung cancer; after targeting CK2, KRAS mutant lung cancer has significantly reduced intracellular lipid droplets, while KRAS wild-type lung cancer has no significant changes; the corresponding KRAS mutant and wild-type lung cancer cell lines were pretreated with CK2 inhibitor CX4945 (concentration gradient: 1.25μM, 2.5μM, 5μM, 10μM, 20μM) for 24h, and the cells were stained with Bodipy fluorescent dye, and the lipid droplet MFI value was detected by flow cytometry; the lipid droplet fluorescence value and the number of positive lipid droplets were detected using a fluorescence microscope and flow cytometer. The results showed that KRAS mutant lung cancer cell line (A549 KRAS Mutation) had more intracellular lipid droplet aggregation and stronger fluorescence value than KRAS wild-type lung cancer cells (H1975-KRAS-Wild / H292-KRAS-Wild). After downregulating CK2, the intracellular lipid droplets in KRAS mutant lung cancer cells were significantly reduced, while there was no significant change in KRAS wild-type lung cancer cells.

[0130] 17) Result 17: If Fig.17 As shown in: In KRAS mutant lung cancer, DHHC3 is one of the candidate interacting proteins of CK2, and Ser241, Thr244 and Thr247 of DHHC3 are all identified as potential phosphorylation sites.

[0131] 18) Result 18: Fig.18 Shown in: KRAS mutant lung cancer sample with high CK2 expression and an immunosuppressive microenvironment within the tumor.

[0132] 19) Result 19: Fig.19 As shown in: KRAS mutant lung cancer samples with high CK2 expression have a significant decrease in activated immune cells within the tumor and present an immunosuppressive microenvironment.

[0133] 20) Result 20: Fig. 20 As shown in: KRAS mutant lung cancer samples with high expression of CK2, whose tumor cells highly express immune checkpoint genes such as PD-L1, HAVCR2, PDCD1LG2 and SIGLEC15, leading to tumor immune escape.

[0134] 21) Result 21: If Fig.21 As shown in the figure: CK2 is negatively correlated with CD8+T cells with direct killing function in the tumors of patients with KRAS mutant lung cancer, that is, high expression of CK2 induces the formation of an immunosuppressive microenvironment in KRAS mutant lung cancer.

[0135] 22) Result 22: If Fig. 22 As shown in the figure: FASN is positively correlated with CK2 expression, CK2 is positively correlated with DHHC3 expression, and CK2 is positively correlated with PD-L1 expression; this provides a basis for the prediction of immunotherapy efficacy and survival prognosis value of combined detection of CK2, FASN and DHHC3 in patients with KRAS mutation lung cancer.

[0136] 23) Result 23: If Fig.23 As shown in the figure: The overall survival (OS) of the CK2 / FASN / DHHC3 high expression group was worse than that of the CK2 / FASN / DHHC3 low expression group.

[0137] 24) Results 24: As shown in Table 1, the correlation between CK2 expression and clinical pathological variables and immunotherapy efficacy in 20 patients with KRAS mutant lung cancer.

[0138] Table 1

[0139]

[0140]

[0141] 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 response.

[0142] Results Analysis

[0143] 1. The CK2 detection agent can effectively and accurately detect the expression level of CK2, and screen patients with KRAS mutant lung cancer who are effective in immunotherapy through the CK2 expression level. The expression level of CK2 is negatively correlated with the response of patients with KRAS mutant lung cancer to immunotherapy. That is, the higher the CK2 expression level, the worse the immunotherapy effect, and the lower the CK2 expression level, the better the immunotherapy effect; more importantly, the AUC curve of the predictive efficacy evaluation of CK2 and the time-dependent ROC curve analysis show that the above-mentioned CK2 detection agent has excellent predictive efficacy.

[0144] 2. KRAS mutant lung cancer has high expression of PD-L1 compared with wild-type patients, and the microenvironment presents "cold tumor" characteristics: Bioinformatics analysis shows that the genes highly expressed in patients with KRAS mutant lung cancer are closely related to lipid metabolism compared with wild-type patients.

[0145] 3. KRAS mutant lung cancer has abnormally active fatty acid metabolism compared with wild-type: Multi-omics results found that compared with wild-type, KRAS mutant lung cancer has significantly upregulated mRNA levels of FASN, a key regulatory enzyme for fatty acid synthesis, increased accumulation of palmitic acid metabolites, and increased lipid droplet aggregation.

[0146] 4. CK2 and FASN expression are positively correlated, and there is a protein interaction; silencing and inhibiting CK2 reduces palmitic acid and lipid droplet synthesis in KRAS mutant lung cancer: Multi-omics results found that silencing or inhibiting CK2 led to the enrichment of differentially expressed genes in KRAS mutant lung cancer mainly in the fatty acid metabolism pathway; Bodipy experiments and RT-qPCR found that silencing or inhibiting CK2 led to a significant downregulation of FASN mRNA and protein levels in KRAS mutant lung cancer, and reduced palmitic acid and lipid droplet formation; mass spectrometry identification found that CK2 and FASN had a protein interaction, suggesting that CK2 promotes de novo palmitic acid synthesis by activating FASN. Therefore, CK2 combined with FASN has an important candidate molecular marker status in the treatment of KRAS mutant lung cancer.

[0147] 5. CK2 may regulate the membrane stability of PD-L1 in KRAS mutant lung cancer through palmitoylation modification: After targeting CK2, the expression of total PD-L1 protein and membrane protein was still downregulated in KRAS mutant lung cancer cells carrying the point mutation PD-L1-T285A / T290A; Western Blotting and immunofluorescence showed that knocking down DHHC3 could downregulate the expression of PD-L1 protein in KRAS mutant lung cancer and inhibit PD-L1 palmitoylation; mass spectrometry identification and Western Blotting found that DHHC3 was one of the candidate interacting proteins of CK2, and knocking down CK2 could downregulate the expression of DHHC3 protein in KRAS mutant lung cancer; phosphorylation site prediction found that Ser241, Thr 244 and Thr247 of DHHC3 were all identified as potential phosphorylation sites of CK2.

[0148] 6. FASN was positively correlated with CK2 expression, CK2 was positively correlated with DHHC3 expression, and CK2 was positively correlated with PD-L1 expression; in addition, the overall survival (OS) of the CK2 / FASN / DHHC3 high expression group was worse than that of the CK2 / FASN / DHHC3 low expression group. This provides a strong basis for the prediction of immunotherapy efficacy and survival prognosis value of combined detection of CK2, FASN and DHHC3 in patients with KRAS mutant lung cancer.

[0149] In summary, the present invention found that in KRAS mutant lung cancer, protein kinase CK2, on the one hand, promotes de novo synthesis of palmitic acid by activating the key enzyme FASN in fatty acid metabolism, and this pathway continuously provides "raw materials" for palmitic acid to activate PD-L1 palmitoylation modification; on the other hand, CK2 directly phosphorylates the "catalyst" of palmitoylation modification, palmitoyltransferase DHHC3, mediates PD-L1 palmitoylation modification, and leads to enhanced PD-L1 membrane stability. The present invention can not only predict the efficacy of immunotherapy for patients with KRAS mutant lung cancer, but also provide new targets for immunotherapy of KRAS mutant lung cancer.

[0150] It will be clear to those skilled in the art that various modifications to the above embodiments may be made without departing from the overall spirit and concept of the present invention. All of these modifications fall within the scope of protection of the present invention. The protection scheme of the present invention shall be subject to the claims attached to the present invention.

Claims

1. Application of reagents for detecting CK2 expression in the preparation and analysis of immunotherapy effects of KRAS mutant lung cancer; The application is any of the following: The application of reagents for detecting CK2 expression in the preparation of products for detecting the prognostic effect of immunotherapy for KRAS-mutated lung cancer, The use of reagents for detecting CK2 expression in the preparation of products for predicting the prognosis of immunotherapy for KRAS-mutated lung cancer; The reagent for detecting CK2 expression is a reagent for detecting CK2 protein expression level, and the KRAS mutant lung cancer immunotherapy is the PD-1 / PD-L1 immunotherapy for KRAS mutant lung cancer.

2. The use according to claim 1, wherein: The reagent for detecting the expression level of CK2 protein is one of an immunohistochemical staining kit, an ELISA kit, and a WB kit.

3. The use according to claim 1, wherein: CK2 protein expression level is negatively correlated with the prognostic effect of PD-1 / PD-L1 immunotherapy in KRAS mutant lung cancer.

4. Application of CK2, FASN and DHHC3 combined detection reagents in the preparation and analysis of KRAS mutant lung cancer immunotherapy effect products; Among them, The application is the use of a combined detection reagent of CK2, FASN and DHHC3 in the preparation of a product for detecting the prognostic effect of immunotherapy for KRAS-mutated lung cancer; The CK2, FASN and DHHC3 combined detection reagent is a reagent for jointly detecting the expression levels of CK2, FASN and DHHC3 proteins. The prognostic effect of immunotherapy for KRAS mutant lung cancer is the prognostic effect of PD-1 / PD-L1 immunotherapy for KRAS mutant lung cancer.

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