Application of CK2 in analysis of cold and hot attributes of lung cancer and total survival of KRAS mutation lung cancer patients and screening of lung cancer patients suitable for immunotherapy

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses application of CK2 in analysis of cold and hot attributes of lung cancer, total survival of KRAS mutation lung cancer patients and screening of lung cancer patients suitable for immunotherapy. The invention relates to application of a reagent for detecting KRAS mutation in preparation of a product for diagnosing whether lung cancer is a hot tumor or not. The invention also discloses application of the reagent for detecting the expression level of the CK2 protein in preparation of products for screening KRAS mutation lung cancer patients suitable for immunotherapy. The invention also discloses an application of the CK2 protein expression inhibitor in preparation of KRAS mutation lung cancer PD-1 / PD-L1 immunotherapy adjuvant drugs. The invention also relates to application of the reagent for detecting the interaction between the CK2 and the FASN protein in preparation of a product for analyzing the immunotherapy effect of the KRAS mutation lung cancer. The immunotherapy effect of the KRAS mutation lung cancer patient can be predicted, and meanwhile a new target spot can be provided for immunotherapy of the KRAS mutation lung cancer.

Description

[0001] This application is a divisional application of a Chinese invention patent with the application number 202311456700.7, the title of "New Application of Protein Kinase CK2 in Predicting the Efficacy of Immunotherapy for KRAS-Mutated Lung Cancer", and the filing date of November 3, 2023. The present invention incorporates all the content of CN117405894A. Technical Field

[0002] 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-mutated lung cancer. Background Art

[0003] The diagnosis and treatment of lung cancer patients with driver gene positivity is a hot topic in lung cancer research. The KRAS mutation is one of the most common gene mutations in lung cancer, accounting for approximately 25%-30% in lung adenocarcinoma. As early as 1984, the 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 smooth on the surface, and its affinity for RAS and GTP is extremely high, resulting in the failure of most RAS-targeted drug development. 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-mutated lung cancers), the effective rate is only 37.1%, and the curative effect is not satisfactory. Moreover, there are no breakthroughs in targeted drugs for most other KRAS mutation subtypes. Therefore, traditional platinum-containing chemotherapy remains the first choice for the treatment of KRAS-mutated lung cancer. Immunotherapy has brought hope to KRAS-mutated lung cancer patients, and its efficacy is significantly better than that of lung cancer patients with other driver gene mutations. For example, studies such as ImmunoTarget, subgroup analysis of the CheckMate057 study, and Mazieres J et al. have confirmed that the effective rate of single-agent immunotherapy for KRAS-mutated 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-mutated lung cancer, finding sensitization pathways, and exploring new "immunotherapy +" models have important theoretical significance and clinical value.

[0004] 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 tissues is detected by immunohistochemistry to understand its high or low level for predicting the therapeutic 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 tissues, indicating that there may be a stronger immune response, thus predicting 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 unclear PD-L1 expression who may still benefit from immunotherapy. This indicates that the PD-L1 expression level cannot fully 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 result interpretation methods. This difference may cause a certain degree of interference for doctors and patients in deciding whether to conduct immunotherapy and how to conduct treatment. Therefore, standardizing and unifying the PD-L1 detection method and interpretation standard is very important for ensuring the effectiveness of immunotherapy and patients obtaining the best treatment results. This requires formulating consistent guidelines and standards in clinical practice to improve the precision and reliability of immunotherapy.

[0005] The metabolic immune microenvironment is a hot issue in current tumor research. Tumor cell metabolic reprogramming 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 PD-L1 expression of 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 PD-L1 expression of tumor cells by activating the NF-κB pathway, resulting in 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, promoting immune escape.

[0006] Protein kinase CK2 is the first protein kinase discovered in 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 a focus issue in the current research of the CK2 field. Barbara Guerra et al. reported the potential role of CK2 in lipid metabolism and found that in the livers of high-fat diet mice, 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

[0007] In view of the above problems, the present invention provides the use of CK2 in analyzing the cold and hot properties of lung cancer and the overall survival of lung cancer patients with KRAS mutations and screening lung cancer patients suitable for immunotherapy, mainly to solve the problems such as the difficult evaluation of the efficacy of immunotherapy for lung cancer patients with KRAS mutations.

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

[0009] The first aspect of the present invention The application of a reagent for detecting the expression of CK2 in the preparation of a product for analyzing the efficacy of immunotherapy for KRAS-mutated lung cancer; wherein, the application is at least one of the following: 1) The application of a reagent for detecting the expression level of CK2 in the preparation of a product for detecting the prognostic effect of immunotherapy for KRAS-mutated lung cancer, and this application is mainly used to evaluate the treatment effect after immunotherapy for KRAS-mutated lung cancer; 2) The application of a reagent for detecting the expression level of CK2 in the preparation of a product for predicting the prognostic effect of immunotherapy for KRAS-mutated lung cancer, and this application is mainly used to evaluate before the patient undergoes immunotherapy to predict whether the patient will achieve a good effect during immunotherapy.

[0010] In another combination, CK2 can also be combined with FASN as an important molecular marker in the treatment of KRAS mutant lung cancer. The application of a reagent for detecting the interaction between CK2 and FASN 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 application of a reagent for detecting the interaction between CK2 and FASN in the preparation of a product for detecting the prognosis of immunotherapy for KRAS mutant lung cancer, 2) the application of a reagent for detecting the interaction between CK2 and FASN 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 utilize existing detectable reagents, and the surface plasmon resonance (SPR) technology represented by Biacore can achieve the 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 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.

[0011] There is another combination. The application of a combined detection reagent for CK2, FASN, and DHHC3 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 application of a reagent for detecting the expression of CK2 in the preparation of a product for detecting the prognosis of immunotherapy for KRAS mutant lung cancer, 2) the application of a reagent for detecting the expression of CK2 in the preparation of a product for predicting the prognosis of immunotherapy for KRAS mutant lung cancer. The combined 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 differences from the wild type. For example, lung cancer induced by the KRAS-LSL-G12D point mutation, and lung cancer caused by other site mutations of KRAS should also be equivalent in the present invention.

[0013] For the corresponding application, the following choices can be made in its specific application:

[0014] First, when making a prognosis judgment, the detection of the expression level of CK2 is negatively correlated with the prognosis of immunotherapy for KRAS mutant lung cancer. Thus, 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, while when the expression level of CK2 is low, it indicates that the effect of immunotherapy for KRAS mutant lung cancer patients will be relatively obvious; similarly, the same method is used for judgment when making a prediction. The high or low expression level of CK2 can be judged according to clinical needs, and generally, the expression level of CK2 in normal human bodies can be used as a reference;

[0015] Second, regarding the reagent for detecting the CK2 expression level, the reagent for detecting the CK2 expression is at least one of immunohistochemical staining kit, ELISA kit, and WB kit. The function of the kit is to detect the CK2 expression level;

[0016] Third, regarding the immunotherapy, the immunotherapy for KRAS mutant lung cancer is at least the PD-1 / PD-L1 immunotherapy monoclonal antibodies for KRAS mutant lung cancer, mainly including: camrelizumab, pembrolizumab, durvalumab, tislelizumab, etc.

[0017] Regarding the product for predicting the prognosis of immunotherapy for KRAS mutant lung cancer, it can specifically be embodied as the application of the reagent for detecting the CK2 expression in the preparation of a product for screening KRAS mutant lung cancer patients suitable for immunotherapy. The prepared product can detect the samples obtained from patients. 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 suggests that a CK2 inhibitor needs to be used in combination during the treatment.

[0018] The second aspect of the present invention It involves 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 options or explanations can be made during the specific application:

[0020] First, the CK2 inhibitor is used to enhance the immunotherapy response effect of KRAS mutant lung cancer, thereby improving the immunotherapy effect. Among them, the functions 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 production of palmitic acid, 4) weakening the membrane stability of PD-L1, 5) increasing the infiltration of CD8 + T cells in the tumor of the KRAS mutant lung cancer mouse model, presenting an activated immune microenvironment in the tumor;

[0021] Second, the CK2 inhibitor is at least any one of CX4945, TBB, quercetin, NSC-231634, and DMAT;

[0022] Third, the immunotherapy for KRAS mutant lung cancer is at least the PD-1 / PD-L1 immunotherapy for KRAS mutant lung cancer, and the monoclonal antibodies mainly include: camrelizumab, pembrolizumab, durvalumab, tislelizumab, etc.;

[0023] Fourthly, KRAS-mutated lung cancer is lung cancer induced by KRAS mutations, which is significantly different from the wild type. For example, lung cancer induced by the KRAS-LSL-G12D point mutation, and lung cancer caused by other site mutations of KRAS should also be equivalent in the present invention.

[0024] The third aspect of the present invention 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 inducing 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 low mutation burden and do not cause strong immune responses, so it is difficult for the immune system to recognize and attack them. Hot tumors, on the other hand, have a higher mutation burden, and there are a large number of immune cells in the tumor microenvironment, such as T cells and antigen-presenting cells. These immune cells participate in immune responses, making it easier for the immune system to recognize and attack tumors. In cold tumors, the infiltration of immune cells is less 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, and there is an interaction between them and tumor cells, which 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 is already playing a role in tumor resistance. Immunotherapy can enhance these responses and increase the chance of treatment success. Therefore, a preliminary identification of the tumor type can enable doctors to more targeted select treatment options. For cold tumors, other treatment methods may need to be explored, while for hot tumors, immunotherapy may be a more effective option. An individualized treatment plan usually combines the specific situation of the patient and tumor characteristics to obtain the best treatment effect. Thus, based on the differences in nature, immune cells, and treatment effects between cold tumors and hot tumors, it is inevitable that different treatment plans are required for the two, and a preliminary identification of the tumor type can facilitate more targeted treatment, thereby providing an auxiliary means for targeted treatment in clinical practice.

[0026] Wherein, the KRAS mutation at least includes the KRAS-LSL-G12D point mutation. Of course, since lung cancers induced by other types of KRAS mutations also have a high degree of similarity and their treatment plans are the same, lung cancers induced by the remaining KRAS mutations should also be within the scope of the present invention.

[0027] In the present disclosure, the present invention can predict and detect the efficacy of immunotherapy for patients with KRAS-mutated lung cancer, and at the same time can provide new targets for immunotherapy of KRAS-mutated lung cancer. Brief Description of the Drawings

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

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

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

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

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

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

[0034] Figure 7 : The time-dependent ROC curve shows that the AUC value (95% CI) of CK2 in predicting the overall survival (OS) of patients with KRAS-mutant lung cancer remains stable over time.

[0035] Figure 8 : The time-dependent ROC curve shows that the AUC value (95% CI) of CK2 in predicting the progression-free survival (PFS) of patients with KRAS-mutant lung cancer remains stable over time.

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

[0037] Figure 10 : Bioinformatics analysis shows that the tumor immune microenvironment of KRAS-mutant lung cancer presents a "cold tumor" characteristic compared with wild-type. (A) The B cells and CD8 in the tumor immune microenvironment of KRAS-mutant lung cancer compared with wild-type +Significant deficiency of T cells, neutrophils, and macrophages; (B) Heatmap of immune cell scores, where different colors represent expression trends in different samples, *p<0.05, **p<0.01, ***p<0.001, and the asterisks represent the degree of importance (*p). The significance between two groups of samples was tested by the Wilcox test, and the significance of three or more groups of samples was tested by the Kruskal-Wallis test; (C) Percentage abundance of tumor-infiltrating immune cells in each sample, with different colors representing different immune cell types. The abscissa represents the samples, and the ordinate represents the percentage of immune cell content in a single sample.

[0038] Figure 11 : In mice with conditional point mutation of the KRAS gene G12D compared with wild-type mice, CD8 + T cells were significantly deficient.

[0039] Figure 12 : Bioinformatics analysis showed that in patients with KRAS-mutated lung cancer compared with wild-type patients, the highly expressed genes 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 upregulated or downregulated genes.

[0040] Figure 13 : CK2 expression levels and their distribution trends in relation to patient survival according to different clinical characteristics such as age, gender, and AJCC eighth edition lung cancer stage in 149 samples of KRAS-mutated lung cancer.

[0041] Figure 14 : Liquid chromatography-mass spectrometry non-targeted LC-MS metabolomics analysis found that after downregulating CK2, the palmitic acid metabolic pathway was significantly enriched, and the production of palmitic acid was reduced. (A) Differential metabolites were visualized in the form of a volcano plot; (B) Pathway enrichment analysis of differential metabolites; the redder and larger the circle color, the higher the significance; (C) VIP (Variable Importance in Projection) plot of Random Forests; indicating the importance of metabolites and their contribution to sample discrimination; (D) PLS-DA loading plot; reflecting the contribution of metabolites to the discrimination between sample groups, and the farther away from the center point, the greater the contribution to differentiating sample groups; (E) Distribution of palmitic acid in the si-Vehicle and si-CK2α groups, P<0.05.

[0042] Figure 15: Silencing or inhibiting CK2 leads to a significant downregulation of FASN expression and a reduction in palmitic acid production in KRAS-mutant lung cancer; moreover, there is a protein interaction between CK2 and FASN and DHHC3. (A) Volcano plot of differential genes; (B) Enrichment analysis of differential expression gene pathways; (C) RT-qPCR detection of the mRNA level of FASN; (D) Detection of the specific content of palmitic acid using a metabolite kit; (E) Mass spectrometry analysis of potential interacting proteins of CK2α.

[0043] Figure 16 : Intracellular lipid droplet aggregation increases in KRAS-mutant lung cancer compared with KRAS-wild-type lung cancer; after targeting CK2, intracellular lipid droplets in KRAS-mutant lung cancer are significantly reduced, while there is no obvious change in KRAS-wild-type lung cancer. (A) Effects of downregulating CK2 in a concentration gradient on lipid droplet aggregation in KRAS-mutant cell lines and wild-type cell lines, histogram; (B-D) Effects of downregulating CK2 in a concentration gradient on lipid droplet aggregation in KRAS-mutant cell lines and wild-type cell lines, scatter plots; (E-G) Effects of downregulating CK2 in a concentration gradient on lipid droplet aggregation in KRAS-mutant cell lines and wild-type cell lines; fluorescence microscopy detection, 10μm scale bar, green is intracellular staining of lipid droplets.

[0044] Figure 17 : 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.

[0045] Figure 18 : Heatmap of immune cell scores, showing the immune score distribution of each immune cell in 64 KRAS-mutant lung cancer patients with high CK2 expression and 85 patients with low CK2 expression. The abscissa represents the type of immune cell infiltration, and the ordinate represents the distribution of this immune infiltration score in different groups. The results suggest that the tumor microenvironment in KRAS-mutant lung cancer samples with high CK2 expression is immunosuppressive.

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

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

[0048] Figure 21 : Correlation analysis of the expressions of CK2, FASN, DHHC3, and PD-L1 with each immune score and the correlation analysis among the immune scores themselves. In the schematic diagram, the heat map represents the correlation analysis of the immune scores themselves. Red represents positive correlation, and blue represents negative correlation. The redder or bluer the color, the greater the correlation between the two. Similarly, the larger the circle, the stronger the correlation. The red lines in the schematic diagram represent negative correlation between the model score or gene expression and the immune score, and green indicates 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 CK2 expression induces the formation of an immunosuppressive microenvironment in KRAS-mutant lung cancer.

[0049] Figure 22 : Correlation analysis of two genes in KRAS-mutant lung cancer samples, namely 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 there is no statistical difference between DHHC3 and FASN expression (P = 0.092); this provides a basis for the prediction of the efficacy of immunotherapy and the value of survival prognosis of combined detection of CK2, FASN, and DHHC3 in patients with KRAS-mutant lung cancer.

[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 samples. If HR > 1, it indicates that the gene is a risk factor (the higher the expression, the worse the prognosis); if HR < 1, it indicates 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 : Schematic diagram of the animal experiment model.

[0052] Figure 25 : KRAS mutant lung cancer shows high expression of PD-L1 compared to the wild type, and the microenvironment presents the characteristics of a "cold tumor". (A, B) Immunohistochemistry was used to detect the protein expression of CD3, CD4, CD8, CD68, PD-1, and PD-L1 in the tumors of KRAS mutant lung cancer patients and KRAS wild-type lung cancer patients (scale bar, 50 μm); (C, D) Multicolor immunofluorescence was used to detect the protein expression of CD4, CD8, CD68, Foxp3-Treg, and PD-L1 in the tumors of KRAS mutant lung cancer patients and KRAS wild-type lung cancer patients (scale bar, 100 μm).

[0053] Figure 26 : In KRAS gene G12D conditional point mutant mice compared with wild-type mice, the proliferation and activation ability of CD8 + T cells in their tumors is weakened, while the exhaustion function is enhanced. (A) Schematic diagram of preparing single-cell suspensions from tumors obtained in in vivo experiments and running them on a flow cytometer; (B) Flow cytometry was used to detect the proportion of CD8 + T cells (Mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001). (C) Flow cytometry was used to detect the cytotoxicity markers IFN-γ, Ki-67, and TNF-α of CD8 + T cells; the expression of the exhaustion marker PD-1 of CD8 + T cells (Mean ± SD, **P < 0.01, ***P < 0.001); (D) Representative immunofluorescence staining pictures of CD3 + , CD8 + , GZMB + T cells and PD-L1 in the tumor tissues of each group of mice. Detailed implementation methods

[0054] The following is a further explanation in combination with specific research projects.

[0055] Experimental Protocol

[0056] (1) To clarify the role of protein kinase CK2 in the high expression of PD-L1 and immune escape in KRAS-mutant lung cancer

[0057] 1. At the cellular level: To clarify the effect of CK2 on the expression of key molecules in fatty acid metabolism in KRAS-mutant lung cancer cells;

[0058] 2. At the cellular level: To clarify the effect of CK2 on the expression of immune cells, cytokines and immune checkpoints in KRAS-mutant lung cancer;

[0059] 3. To construct a KRAS-LSL-G12D point mutant lung cancer mouse model and analyze the dynamic changes of CK2, key molecules in fatty acid metabolism, immune cells, cytokines and immune checkpoints during the progression from normal lung tissue to KRAS-mutant lung cancer;

[0060] 4. To treat the KRAS-LSL-G12D point mutant lung cancer mouse model with a CK2 inhibitor and clarify its effects on indicators such as key molecules in fatty acid metabolism, immune cells, cytokines and immune checkpoints in tumor tissues and peripheral blood;

[0061] 5. To treat the KRAS-LSL-G12D point mutant lung cancer mouse model with a CK2 inhibitor, an immune checkpoint inhibitor and a fatty acid metabolism enzyme inhibitor, and evaluate the effectiveness and safety of each treatment group.

[0062] (2) To clarify the mechanism by which protein kinase CK2 regulates the palmitoylation modification of PD-L1 through fatty acid metabolism reprogramming and direct phosphorylation of palmitoyltransferase DHHC3

[0063] Pathway 1: CK2 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 activating the palmitoylation modification of PD-L1 by palmitic acid.

[0064] 1. To clarify the endogenous and exogenous protein interactions between CK2 and FASN and identify the domains of interaction between CK2 and FASN;

[0065] 2. To verify the effect of silencing or inhibiting FASN on the palmitic acid level in KRAS-mutant lung cancer;

[0066] 3. Rescue experiments to verify the effect of overexpressing CK2, silencing or inhibiting FASN on the palmitic acid level in KRAS-mutant lung cancer;

[0067] 4. Based on 13 13C-labeled palmitic acid carbon metabolic flux analysis to reveal the upstream and downstream mutual regulation mechanisms;

[0068] 5. Determine whether blocking palmitoylation can lead to enhanced membrane anchoring of PD-L1 in KRAS-mutant lung cancer.

[0069] 6. Use rescue experiments to clarify the effects of overexpressing CK2, silencing or inhibiting FASN on the expression of PD-L1 membrane protein in KRAS-mutant lung cancer.

[0070] 7. Use rescue experiments to clarify the effects of silencing or inhibiting CK2 and exogenous intake of palmitic acid on the PD-L1 membrane protein in KRAS-mutant lung cancer.

[0071] Pathway 2: CK2 directly phosphorylates DHHC3, the "catalyst" for palmitoylation modification, mediating the palmitoylation modification of PD-L1 and resulting in enhanced membrane stability of PD-L1.

[0072] 1. Verify the endogenous and exogenous protein interactions between CK2 and DHHC3.

[0073] 2. Construct KRAS-mutant lung cancer cell lines stably expressing wild-type DHHC3 (DHHC3 WT ), phosphorylation-deficient double mutant T244A / T247A (DHHC3 2A ), and overexpressing phosphorylated T244E / T247E (DHHC3 2E ).

[0074] 3. Detect the protein expression of DHHC3 2E , DHHC3 2A , and DHHC3 WT in cells by Western Blotting.

[0075] 4. Examine the effects of silencing or inhibiting CK2 on the protein expression levels of DHHC3 2E , DHHC3 2A , and DHHC3 WT in cells.

[0076] 5. Further identify that DHHC3 is the main palmitoyltransferase for the expression of PD-L1 in KRAS-mutant lung cancer.

[0077] 6. Determine the effects of silencing or inhibiting DHHC3 on the PD-L1 membrane protein in KRAS-mutant lung cancer.

[0078] 7. Use rescue experiments to clarify the effects of overexpressing CK2, silencing or inhibiting DHHC3 on the PD-L1 membrane protein in KRAS-mutant lung cancer.

[0079] (3) Explore the efficacy prediction and survival prognosis value of protein kinases CK2, FASN, and DHHC3 in patients with KRAS-mutant lung cancer.

[0080] 1. To clarify the relationships between the expression levels and localization of CK2, FASN, and DHHC3 and the clinical features such as the stage, immunotherapy efficacy, and prognosis of patients with KRAS-mutated lung cancer (paraffin tissue samples);

[0081] 2. To clarify the relationships between CK2, fatty acid metabolites, locally infiltrating immune cells, cytokines, and immune checkpoint levels and the clinical features of patients with KRAS-mutated lung cancer (fresh tissue and serum samples);

[0082] 3. To design retrospective and prospective studies to explore the correlations between the expressions of CK2, FASN, and DHHC3 and the immunotherapy efficacy and patient survival prognosis (clinical study).

[0083] Experimental procedures (1) To clarify the role of protein kinase CK2 in the high expression of PD-L1 and immune escape in KRAS-mutated lung cancer

[0084] 1. At the cellular 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 levels (Seahorse metabolic analyzer, important metabolite kits) of KRAS-mutated lung cancer cell lines, lipid storage levels (Oil Red O staining and Bodipy staining), and fatty acid oxidation levels (fatty acid oxidation kit);

[0085] 2. At the cellular 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 expression of PD-L1 and cytokines (cytokine array and ELISA) in KRAS-mutated lung cancer cell lines;

[0086] 3. Construct an Adeno-Cre-induced KRAS-LSL-G12D point-mutated lung cancer mouse model, and analyze the dynamic changes in the expressions of CK2, key targets of fatty acid metabolism, immune cells, and cytokines during the progression from the baseline level to KRAS-mutated lung cancer;

[0087] 4. Treat the KRAS-LSL-G12D point-mutated lung cancer mouse model with a CK2 inhibitor, detect the changes in indicators such as fatty acid metabolites (mass spectrometry and related metabolite detection kits), inflammatory factors, and chemokines (ELISA, protein array, and multi-factor flow cytometry) in lung tissues and peripheral blood, and evaluate the effects of CK2 on the metabolic immune microenvironment of KRAS-mutated lung cancer;

[0088] 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.

[0089] (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

[0090] 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;

[0091] 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;

[0092] 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;

[0093] 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;

[0094] 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.

[0095] 5. Determine whether blocking palmitoylation can lead to enhanced membrane anchoring of PD-L1 in KRAS-mutant lung cancer: In KRAS-mutant lung cancer cells, treat with 2-BP (a general palmitoylation inhibitor), and detect the expression of PD-L1 membrane protein by Western Blotting, flow cytometry and immunofluorescence; verify the palmitoylation of PD-L1 by the Click-IT method;

[0096] 6. Rescue experiments to clarify the effects of overexpressing CK2, 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, detect the expression of PD-L1 membrane protein by Western Blotting, flow cytometry and immunofluorescence;

[0097] 7. Rescue experiments to clarify the effects of silencing or inhibiting CK2 and exogenous intake of palmitic acid on PD-L1 membrane protein in KRAS-mutant lung cancer: In KRAS-mutant lung cancer cells with downregulated CK2, after exogenous intake of palmitic acid, detect the expression of PD-L1 membrane protein by Western Blotting, flow cytometry and immunofluorescence;

[0098] Pathway 2: CK2 directly phosphorylates DHHC3, the "catalyst" for palmitoylation modification, mediates the palmitoylation modification of PD-L1, resulting in enhanced membrane stability of PD-L1.

[0099] 1. Verify the endogenous and exogenous protein interactions between CK2 and DHHC3: Using HEK293T cells as a model, detect the interaction between exogenously expressed CK2 and DHHC3 by co-IP; using KRAS-mutant lung cancer cell lines A549 and H23 as models, perform co-IP with anti-CK2 or anti-FASN antibodies to detect the endogenous interaction between them;

[0100] 2. Construct KRAS-mutant lung cancer cell lines stably expressing wild-type DHHC3 (DHHC3 WT ), phosphorylation-deficient T244A / T247A double mutant (DHHC3 2A ), and overexpressing phosphorylated T244E / T247E (DHHC3 2E ): Use the Crispr / Cas9 technique to construct KRAS-mutant lung cancer cell lines stably expressing wild-type DHHC3 (DHHC3 WT ), phosphorylation-deficient T244A / T247A double mutant (DHHC3 2A ), and overexpressing phosphorylated T244E / T247E (DHHC3 2E );

[0101] 3. Detection of DHHC3 by Western Blotting 2E 、DHHC3 2A and DHHC3 WT Protein expression of DHHC3 in cells: After constructing the above three cell lines, detect the basal expression level of DHHC3 in the above three cells by Western Blotting;

[0102] 4. Effect of silencing or inhibiting CK2 on DHHC3 2E 、DHHC3 2A and DHHC3 WT Effect of protein expression level of DHHC3 in cells: After silencing or inhibiting CK2, detect its effect on the protein expression of DHHC3 in the above three cells by Western Blotting;

[0103] 5. Further identify that DHHC3 is the main palmitoyltransferase for the expression of PD-L1 in KRAS-mutant lung cancer: Detect the protein interaction between PD-L1 and DHHC3 by co-IP; Detect the co-localization of endogenously expressed DHHC3 and PD-L1 in KRAS-mutant lung cancer cell lines and tissues by immunofluorescence; Detect the correlation between the expressions of DHHC3 and PD-L1 by immunohistochemical staining;

[0104] 6. Determine the effect of silencing or inhibiting DHHC3 on the membrane protein of PD-L1 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 the membrane protein of PD-L1 in KRAS-mutant lung cancer by Western Blotting; In addition, detect the expression level of the membrane protein of PD-L1 in KRAS-mutant lung cancer induced by IFN-γ by Western Blotting;

[0105] 7. Rescue experiment to determine the effect of overexpressing CK2 and silencing or inhibiting DHHC3 on the membrane protein of PD-L1 in KRAS-mutant lung cancer: In KRAS-mutant lung cancer cells overexpressing CK2, after silencing or inhibiting DHHC3, detect the expression of PD-L1 membrane protein by Western Blotting, flow cytometry and immunofluorescence.

[0106] (3) Explore the efficacy prediction and survival prognosis value of protein kinases CK2, FASN and DHHC3 in patients with KRAS-mutant lung cancer

[0107] 1. Obtain paraffin tissue samples of KRAS-mutated lung cancer, select antibodies of different colors and species 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 the patients, count the general conditions, stages, treatment regimens and curative effects, progression-free survival and overall survival of the patients and other clinical indicators, and analyze them through SPSS 16.0 software;

[0108] 2. Collect tissue and serum samples of KRAS-mutated lung cancer patients in this center, and detect the expressions of CK2, fatty acid metabolites (palmitic acid, monounsaturated fatty acids and polyunsaturated fatty acids, etc.), locally 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-mutated lung cancer, KRAS wild-type lung cancer and adjacent lung tissues;

[0109] 3. Prospectively observe and retrospectively study, divide KRAS-mutated lung cancer patients into different treatment subgroups (chemotherapy, targeted therapy, immunotherapy and radiotherapy), and evaluate the associations between the expressions of CK2, FASN and DHHC3 and the curative effects, overall survival, disease-free survival, etc. of the patients.

[0110] (4) Compared with the wild type, KRAS-mutated lung cancer has a high expression of PD-L1 and the microenvironment presents the characteristics of "cold tumor".

[0111] Collect tumor tissue samples (50 cases each) of KRAS-mutated and KRAS wild-type lung cancer patients in Wuhan Union Hospital, and analyze the cold and hot properties of the tumor microenvironment.

[0112] (5) Compared with wild-type mice, the proliferation and activation ability of intratumoral CD8+ T cells in KRAS gene G12D conditional point mutant mice are weakened, while the exhaustion function is enhanced.

[0113] Detect the infiltration and functional status of immune cells in the tumors of KRAS gene G12D conditional point mutant mice and KRAS wild-type mice by flow cytometry.

[0114] Experimental results

[0115] 1) Result 1: As Figure 1 shown, the overall survival (OS) of KRAS-mutated lung cancer patients aged <= 53 is better than that of patients aged > 53.

[0116] 2) Result 2: AsFigure 2 As shown, the nutritional scores of KRAS-mutant lung cancer patients with high CK2 expression were mainly 1-2 points, while those of KRAS-mutant lung cancer patients with low CK2 expression ranged from 0 to 4 points.

[0117] 3) Result 3: As Figure 3 shown, for KRAS-mutant lung cancer patients with high CK2 expression, the worse their overall survival (OS), and vice versa.

[0118] 4) Result 4: As Figure 4 shown, for KRAS-mutant lung cancer patients with high CK2 expression, the worse their progression-free survival (PFS), and vice versa.

[0119] 5) Result 5: As Figure 5 shown, the AUC curve demonstrated the ability of CK2 to accurately predict the overall survival (OS) of KRAS-mutant lung cancer patients at 1.5 years, 2 years, and 3 years, with AUC values of 0.85, 0.89, and 0.83, respectively.

[0120] 6) Result 6: As Figure 6 shown, the AUC curve demonstrated the ability of CK2 to accurately predict the progression-free survival (PFS) of KRAS-mutant lung cancer patients at 1 year, 1.5 years, and 2 years, with AUC values of 0.86, 0.9, and 1, respectively.

[0121] 7) Result 7: As Figure 7 shown, the time-dependent ROC curve showed that the AUC value (95% CI) of CK2 for predicting the overall survival (OS) of KRAS-mutant lung cancer patients remained stable over time.

[0122] 8) Result 8: As Figure 8 shown, the time-dependent ROC curve showed that the AUC value (95% CI) of CK2 for predicting the progression-free survival (PFS) of KRAS-mutant lung cancer patients remained stable over time.

[0123] 9) Result 9: As Figure 9 shown, KRAS-mutant lung cancer patients with high CK2 expression had a poor response to immunotherapy; while KRAS-mutant lung cancer patients with low CK2 expression had a better response to immunotherapy, and the patients with SD and PR as the efficacy evaluation accounted for the main proportion.

[0124] 10) Result 10: As Figure 10As shown, bioinformatics analysis revealed that compared with wild-type, lung cancers with KRAS mutations presented a "cold tumor" phenotype in the tumor immune microenvironment: The immune scores of 149 KRAS-mutated lung cancer tissues, 852 KRAS wild-type lung cancer tissues, and 108 normal samples were analyzed using the TCGA database. The results showed that compared with wild-type, lung cancers with KRAS mutations presented a "cold tumor" phenotype in the tumor immune microenvironment, with significant deficiencies in B cells, CD8 + T cells, neutrophils, and macrophages.

[0125] 11) Result 11: As Figure 11 shown, compared with wild-type mice, mice with a conditional point mutation of the KRAS gene G12D had significantly fewer intratumoral CD8 + T cells; our research group detected the infiltration and functional status of immune cells in the tumors of mice with a conditional point mutation of the KRAS gene G12D and KRAS wild-type mice by flow cytometry. The results showed that compared with wild-type mice, mice with KRAS-mutated lung cancers had significantly fewer intratumoral CD8 + T cells, and the tumor immune microenvironment was in an immunosuppressive state.

[0126] 12) Result 12: As Figure 12 shown, bioinformatics analysis revealed that compared with wild-type patients, the genes highly expressed in patients with KRAS-mutated lung cancers were closely related to lipid metabolism; the differential gene expression between the two groups of samples in 149 KRAS-mutated lung cancer tissues and 852 KRAS wild-type lung cancer tissues was analyzed using the TCGA database, and the potential functions enriched by these upregulated or downregulated genes were observed through KEGG pathway and GO pathway functional enrichment. The results showed that compared with wild-type patients, the genes highly expressed in patients with KRAS-mutated lung cancers were closely related to lipid metabolism; and the CK2 gene was significantly highly expressed in the KRAS mutation group, indicating that CK2 might be a potential target for KRAS-mutated lung cancers.

[0127] 13) Result 13: As Figure 13 shown, the expression of CK2 was closely related to the age, gender, and stage clinical characteristics of patients with KRAS-mutated lung cancers, and high expression of CK2 was associated with poor prognosis.

[0128] 14) Result 14: As Figure 14As shown in [reference], liquid chromatography-mass spectrometry (LC-MS) non-targeted metabolomics analysis revealed that after downregulating CK2, the palmitic acid metabolic pathway was significantly enriched, leading to a decrease in palmitic acid production. In addition, the decrease in palmitic acid content inhibited protein palmitoylation modification, resulting in weakened membrane anchoring and ultimately enhancing the efficacy of PD-1 / PD-L1 immunotherapy. Our research group conducted non-targeted metabolomics analysis on the KRAS mutant lung cancer cell line (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 decrease in palmitic acid production.

[0129] 15) Result 15: As Figure 15 shown in [reference], silencing or inhibiting CK2 led to a significant downregulation of FASN expression and a decrease in palmitic acid production in KRAS mutant lung cancer. Moreover, there was a protein interaction between CK2 and FASN as well as DHHC3: Our research group found through transcriptomic analysis that after treating the A549 cell line with a CK2 inhibitor, 1590 genes were upregulated and 1231 genes were downregulated. Pathway enrichment analysis of these differentially expressed genes revealed a significant enrichment of the fatty acid metabolic pathway. Through RT-qPCR, palmitic acid content kits, and WB methods, it was found that silencing or inhibiting CK2 led to a significant downregulation of FASN expression and a decrease in 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 a protein interaction between CK2 and FASN, suggesting that on the one hand, CK2 promoted de novo palmitic acid synthesis by activating FASN; on the other hand, CK2 could directly phosphorylate the palmitoyltransferase DHHC3, ultimately activating downstream protein palmitoylation modification. Therefore, CK2, FASN, and DHHC3 have important candidate molecular marker status in the treatment of KRAS mutant lung cancer.

[0130] 16) Result 16: As Figure 16As shown, the intracellular lipid droplet aggregation in KRAS-mutant lung cancer is increased compared with that in KRAS-wild-type lung cancer; after targeting CK2, the intracellular lipid droplets in KRAS-mutant lung cancer are significantly reduced, while there is no obvious change in KRAS-wild-type lung cancer; the corresponding KRAS-mutant and wild-type lung cancer cell lines were pretreated with CK2 inhibitor CX4945 (concentration gradient: 1.25 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) for 24 h, and the cells were stained with Bodipy fluorescent dye, and the MFI value of lipid droplets was detected by flow cytometry; the fluorescence value of lipid droplets and the number of positive lipid droplets were detected by fluorescence microscopy and flow cytometry. The results showed that the intracellular lipid droplet aggregation in the KRAS-mutant lung cancer cell line (A549 KRAS Mutation) was increased compared with that in the KRAS-wild-type lung cancer cells (H1975-KRAS-Wild / H292-KRAS-Wild), and the fluorescence value was stronger. After downregulating CK2, the intracellular lipid droplets in KRAS-mutant lung cancer cells were significantly reduced, while there was no obvious change in KRAS-wild-type lung cancer cells.

[0131] 17) Result 17: As Figure 17 shown: 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.

[0132] 18) Result 18: As Figure 18 shown: The KRAS-mutant lung cancer samples with high CK2 expression showed an immunosuppressive microenvironment in the tumor.

[0133] 19) Result 19: As Figure 19 shown: In the KRAS-mutant lung cancer samples with high CK2 expression, the activated immune cells in the tumor were significantly reduced and showed an immunosuppressive microenvironment.

[0134] 20) Result 20: As Figure 20 shown: The KRAS-mutant lung cancer samples with high CK2 expression had high expression of immune checkpoint genes such as PD-L1, HAVCR2, PDCD1LG2, and SIGLEC15 in tumor cells, resulting in the formation of tumor immune escape.

[0135] 21) Result 21: As Figure 21 shown: CK2 was negatively correlated with CD8 + T cells with direct killing function in the tumor of KRAS-mutant lung cancer patients, that is, high CK2 expression induced the formation of an immunosuppressive microenvironment in KRAS-mutant lung cancer.

[0136] 22) Result 22: As Figure 22As shown: there was a positive correlation between FASN and CK2 expression, a positive correlation between CK2 and DHHC3 expression, and a positive correlation between CK2 and 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-mutated lung cancer.

[0137] 23) Result 23: As Figure 23 shown: the overall survival (OS) of the high CK2 / FASN / DHHC3 expression group was worse than that of the low CK2 / FASN / DHHC3 expression group.

[0138] 24) Result 24: As shown in Table 1, the correlation between CK2 expression and clinicopathological variables and immunotherapy efficacy in 20 patients with KRAS-mutated lung cancer.

[0139] Table 1

[0140]

[0141]

[0142] 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.

[0143] 25) Result 25: The expression of immune markers of tumor-infiltrating cells (CD3, CD4, CD8, CD68, and FoxP3) and immune checkpoint markers (PD-1 and PD-L1) was evaluated by immunohistochemistry ( Figure 25 A - B in Figure 25 ) and multiplex immunofluorescence ( C - D in ); it was found that PD-L1 was highly expressed in KRAS-mutated lung cancer compared with wild type, and the microenvironment presented the characteristics of "cold tumor". + 26) Result 26: It was found that the proliferation (Ki-67) and activation (IFN-γ and TNF-α) functions of intratumoral CD8 Figure 26 T cells in KRAS-mutated lung cancer mice were significantly weakened compared with wild-type mice, while the exhaustion index (PD-1) was significantly enhanced ( + A - C in + ). Immunofluorescence detection found that the infiltration of CD3 + , CD8 Figure 26 and GZMB T cells in the tumor of KRAS-mutated lung cancer mice was reduced compared with wild-type mice, while the expression of PD-L1 was upregulated ( D in ); this result indicates that PD-L1 is upregulated in KRAS-mutated lung cancer and the tumor microenvironment is in an immunosuppressive state.

[0145]

[0145] Result Analysis

[0146] 1. The CK2 detection agent can effectively and accurately detect the expression level of CK2, and screen patients with effective immunotherapy for KRAS-mutant lung cancer 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; the lower the CK2 expression level, the better the immunotherapy effect. More importantly, the analysis of the AUC curve and time-dependent ROC curve for the prediction efficacy evaluation of CK2 shows that the above CK2 detection agent has excellent prediction efficacy.

[0147] 2. Compared with wild-type, KRAS-mutant lung cancer shows high expression of PD-L1 and the microenvironment presents the characteristics of "cold tumor": Bioinformatics analysis shows that compared with wild-type patients, the genes with high expression in patients with KRAS-mutant lung cancer are closely related to lipid metabolism.

[0148] 3. Compared with wild-type, fatty acid metabolism in KRAS-mutant lung cancer is abnormally active: Multi-omics results show that compared with wild-type, the mRNA level of the key regulatory enzyme FASN for fatty acid synthesis is significantly up-regulated, the accumulation of palmitic acid metabolites increases, and lipid droplet aggregation increases in KRAS-mutant lung cancer.

[0149] 4. CK2 is positively correlated with FASN expression and there is protein interaction; silencing and inhibiting CK2 reduces palmitic acid and lipid droplet synthesis in KRAS-mutant lung cancer: Multi-omics results show that silencing or inhibiting CK2 leads to the enrichment of differential genes in KRAS-mutant lung cancer mainly in the fatty acid metabolism pathway; Bodipy experiments and RT-qPCR show that silencing or inhibiting CK2 leads to a significant decrease in the mRNA and protein levels of FASN and a reduction in palmitic acid and lipid droplet formation in KRAS-mutant lung cancer; mass spectrometry identification shows that there is protein interaction between CK2 and FASN, which suggests that CK2 promotes de novo synthesis of palmitic acid by activating FASN. Therefore, CK2 combined with FASN has an important status as a candidate molecular marker in the treatment of KRAS-mutant lung cancer.

[0150] 5. CK2 may regulate the membrane stability of PD-L1 in KRAS mutant lung cancer through the palmitoylation modification pathway: After targeting CK2, the expression of total and membrane PD-L1 proteins in KRAS mutant lung cancer cells carrying point mutations PD-L1-T285A / T290A was still downregulated; Western Blotting and immunofluorescence revealed 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.

[0151] 6. There was a positive correlation between FASN and CK2 expression, a positive correlation between CK2 and DHHC3 expression, and a positive correlation between CK2 and PD-L1 expression; in addition, the overall survival (OS) of the high CK2 / FASN / DHHC3 expression group was worse than that of the low CK2 / FASN / DHHC3 expression group. This provides strong evidence for the value of combined detection of CK2, FASN, and DHHC3 in predicting the efficacy of immunotherapy and survival prognosis in patients with KRAS mutant lung cancer.

[0152] 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 of 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 DHHC3, the "catalyst" for palmitoylation modification - palmitoyltransferase, mediating the occurrence of PD-L1 palmitoylation modification, resulting in enhanced membrane stability of PD-L1. The present invention can not only predict the efficacy of immunotherapy in patients with KRAS mutant lung cancer, but also provide new targets for immunotherapy of KRAS mutant lung cancer.

[0153] Those skilled in the art can clearly understand that various modifications to the above embodiments can be made without departing from the general spirit and concept of the present invention. All of them fall within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims attached to the present invention.

Claims

1. 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, and the microenvironment of lung cancer induced by the KRAS mutation exhibits cold tumor characteristics.

2. The use according to claim 2; Wherein, The KRAS mutation includes the KRAS-LSL-G12D point mutation.

3. 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.

4. The use according to claim 3; Wherein, The immunotherapy is PD-1 / PD-L1 immunotherapy.

5. The use according to claim 4; Wherein, The expression level of CK2 protein is negatively correlated with the response of KRAS mutant lung cancer patients to PD-1 / PD-L1 immunotherapy.

6. Use of a CK2 protein expression inhibitor in the preparation of an adjuvant drug for PD-1 / PD-L1 immunotherapy of KRAS mutant lung cancer; Wherein, The CK2 protein expression inhibitor enhances the response effect of KRAS mutant lung cancer to PD-1 / PD-L1 immunotherapy.

7. The use according to claim 6; Wherein, The CK2 inhibitor is at least one of CX4945, TBB, quercetin, NSC231634, DMAT; the action of the CK2 inhibitor is at least one of the following: 1) Down-regulate the expression of FASN, 2) Down-regulate the expression of DHHC3, 3) Reduce the production of palmitic acid, 4) Weaken the membrane stability of PD-L1, 5) Increase the intratumoral infiltration of CD8 + T cells in a mouse model of KRAS mutant lung cancer, and present an activated immune microenvironment in the tumor.

8. Use of a reagent for detecting the interaction between CK2 and FASN proteins in the preparation of a product for analyzing the immunotherapy effect of KRAS mutant lung cancer; Wherein, The use is at least one of the following: 1) Use of a reagent for detecting the interaction between CK2 and FASN proteins in the preparation of a product for detecting the prognostic effect of PD-1 / PD-L1 immunotherapy for KRAS mutant lung cancer, 2) Use of a reagent for detecting the interaction between CK2 and FASN proteins in the preparation of a product for predicting the prognostic effect of PD-1 / PD-L1 immunotherapy for KRAS mutant lung cancer; The stronger the interaction between CK2 and FASN proteins, the worse the prognostic effect of PD-1 / PD-L1 immunotherapy for KRAS mutant lung cancer.

9. Use of CK2 in the preparation of a product for predicting the survival prognosis of KRAS mutant lung cancer patients; Wherein, The survival prognosis is the overall survival period or the progression-free survival period; moreover, the higher the expression level of CK2 protein, the worse the overall survival of KRAS mutant lung cancer patients, and the higher the expression level of CK2 protein, the worse the disease-free survival of KRAS mutant lung cancer patients.

10. Use of a combined detection reagent for CK2, FASN and DHHC3 in the preparation of a product for predicting the survival prognosis of KRAS mutant lung cancer patients; Wherein, The combined detection reagent for CK2, FASN and DHHC3 is a reagent for jointly detecting the expression levels of CK2, FASN and DHHC3 proteins; The survival prognosis is the overall survival period or the progression-free survival period; The overall survival of the high CK2 / FASN / DHHC3 expression group was worse than that of the low CK2 / FASN / DHHC3 expression group.

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