NMT1 inhibitor and application thereof in preparation of solid tumor treatment medicine

Abstract

According to the application of the inhibitor in preparation of the medicine for treating solid tumor cancers, NMT1 activity is blocked, myristicylation of CHP1 is reduced, combination of CHP1 and PD-L1 is interfered, and finally localization of PD-L1 on tumor cell membranes is reduced. The up-regulation of the NMT1 under the hypoxia condition and the regulation effect of the NMT1 on PD-L1 membrane localization are verified through various research and analysis means such as bioinformatics analysis and clinical case review as well as cell and animal levels, the key effect of the NMT1 in treatment of hypoxia-related cancers is disclosed for the first time, and the NMT1 is used as a core target of a combined therapy and is used for treating the hypoxia-related cancers. And a new means is provided for cancer immunotherapy and development and preparation of cancer drugs.

Description

Technical Field

[0001] The present invention belongs to the field of tumor immunotherapy, and specifically relates to a composition for reducing the localization of PD-L1 on tumor cell membranes to achieve a scheme for treating solid cancers. Background Art

[0002] The response rate of immunotherapy for solid tumor cancer is limited, and the hypoxic or low oxygen state of solid cancer is the main biochemical factor for the low response rate of immunotherapy (hypoxia or low oxygen, hereinafter collectively referred to as hypoxia). The hypoxic state in the tumor microenvironment (TME) is an important driving factor for the progression and treatment resistance of many malignant tumors (such as head and neck squamous cell carcinoma, HNSCC). Hypoxia promotes tumor immune escape through various mechanisms, one of the key mechanisms is to upregulate the expression of the immune checkpoint molecule PD-L1 (programmed death ligand 1). PD-L1 binds to PD-1 (programmed death receptor 1) on the surface of T cells, inhibits the anti-tumor activity of T cells, and allows tumor cells to escape the attack of the immune system. In recent years, immune checkpoint inhibitors (such as anti-PD-1 or anti-PD-L1 antibodies) have shown significant efficacy in the treatment of various cancers, such as non-small cell lung cancer, melanoma, etc. However, in hypoxia-related cancers, the efficacy of immune checkpoint inhibitors is often limited. This is partly attributed to the hypoxia-induced high expression of PD-L1 and its excessive localization on the cell membrane, which enhances the immunosuppressive effect and makes it difficult for single immunotherapy to fully exert its effect.

[0003] Currently, the treatment of hypoxia-related cancers still faces many challenges, especially the problem of immunotherapy resistance needs to be solved urgently. Although traditional chemotherapy or radiotherapy can control tumor growth to a certain extent, its side effects are large and it is difficult to reverse hypoxia-induced immune escape. Therefore, it is of great clinical significance to develop a combination therapy that can overcome the limitations of the hypoxic microenvironment and enhance the effect of immunotherapy. Summary of the invention

[0004] One object of the present invention is to provide a composition comprising a plurality of inhibitors, which can be used as a combined therapeutic approach to treat hypoxia-related cancers.

[0005] Another object of the present invention is to provide a compound for use in preparing a drug for treating hypoxia-related cancers.

[0006] It has been verified that hypoxia not only increases the expression of PD-L1 through transcriptional regulation, but also significantly affects its subcellular localization through post-translational modification. N-myristoylation is an important lipid modification process catalyzed by the N-myristoyltransferase (NMT) family. Among them, NMT1 is particularly active in tumor cells, especially under hypoxic conditions. NMT1 regulates the membrane localization and biological functions of specific proteins by catalyzing the N-myristoylation of these proteins. The latest research has found that in a hypoxic environment, NMT1 promotes the interaction between CHP1 (calcineurin B homologous protein 1) and PD-L1 through myristoylation modification of CHP1. This interaction enhances the localization of PD-L1 on the tumor cell membrane, significantly improving its binding ability to PD-1 on T cells, thereby exacerbating the immunosuppressive effect. Although PD-1 / PD-L1 antibodies can block the signaling pathway between the two, under hypoxic conditions, the high membrane localization of PD-L1 may lead to the inability of the antibody to completely neutralize all exposed PD-L1 molecules, thus limiting the therapeutic effect.

[0007] The present invention takes NMT1 as a novel therapeutic target. By blocking the activity of NMT1, reducing the myristoylation of CHP1, and interfering with the binding of CHP1 and PD-L1, the localization of PD-L1 on the tumor cell membrane is ultimately reduced. Through various research and analysis methods such as bioinformatics analysis and clinical case review, as well as verification at the cellular and animal levels, the upregulation of NMT1 under hypoxic conditions and its regulatory effect on the membrane localization of PD-L1 have been verified. For the first time, the key role of NMT1 in the treatment of hypoxia-related cancers has been revealed, and it is used as the core target of combination therapy, providing new means for the development and preparation of cancer immunotherapy and cancer drugs. For example, the (blood drug) concentration of the NMT1 inhibitor provided in vivo reaches or is maintained at 0.1 - 1 μmol / L.

[0008] The composition of the present invention includes an NMT1 inhibitor and an immune checkpoint inhibitor, and the dosage ratio of the two is, for example, 1:5 to 5:1.

[0009] By jointly using an N-myristoyltransferase 1 (NMT1) inhibitor and an immune checkpoint inhibitor, it is aimed to enhance the anti-tumor immune response and improve the therapeutic effect. The core of this method lies in using the NMT1 inhibitor to block the myristoylation modification of CHP1, reduce the localization of PD-L1 on the tumor cell membrane, and at the same time combine with the immune checkpoint inhibitor to block the PD-1 / PD-L1 signaling pathway, realizing the dual mechanisms of "spatial blockade" and "signal inhibition", effectively overcoming tumor immune escape, improving the efficacy of the immune checkpoint inhibitor, and providing a new treatment strategy for patients with hypoxia-related cancers.

[0010] The NMT1 inhibitor of the present invention, PCLX-001 (chemical name: DDD85646), is a small molecule compound that can specifically inhibit the enzymatic activity of NMT1 (target activity: 5 nM for NMT1 and 8 nM for NMT2), blocking the protein N-myristoylation process mediated by it. Experiments have shown that PCLX-001 can effectively inhibit NMT1 activity at low doses (0.1 - 1 μmol / L), reduce the myristoylation of CHP1, and then interfere with the localization of PD-L1 on the cell membrane without causing obvious cytotoxicity. This selective effect makes PCLX-001 a potential immunotherapy sensitizer. Based on this, the combined use of an NMT1 inhibitor to reduce PD-L1 membrane localization and an immune checkpoint inhibitor to block the PD-1 / PD-L1 signaling pathway may significantly enhance the anti-tumor immune response through the dual mechanisms of "spatial blockade" and "signal inhibition".

[0011] The molecular weight of PCLX-001 is: 537.51, and the molecular formula is C 24 H 30 Cl2N6O2S, and the structural formula is as follows:

[0012]

[0013] Immune checkpoint inhibitors block the interaction between PD-1 and PD-L1, relieve T cell inhibition, and restore their anti-tumor immune function, such as but not limited to: the PD-1 antibody Pembrolizumab or the PD-L1 antibody Atezolizumab, and these substances are used alone or in combination in the present invention. By targeting and blocking the interaction between PD-1 and PD-L1, the inhibitory state of T cells is directly relieved, and their ability to kill tumor cells is restored. The blockade of this signaling pathway and the spatial regulation effect of the NMT1 inhibitor complement each other to jointly enhance the anti-tumor immune response. Through the dual mechanisms of "spatial blockade" (reducing PD-L1 membrane localization) and "signal inhibition" (blocking PD-1 / PD-L1 interaction), the present invention can maximize the anti-tumor activity of cytotoxic T cells and significantly improve the therapeutic effect.

[0014] Based on a large amount of experimental data, including studies at the cellular level and animal models, the present invention demonstrates that NMT1 inhibitors can significantly reduce the membrane localization of PD-L1 and synergistically enhance the anti-tumor immune response with immune checkpoint inhibitors. These findings provide a solid scientific basis for the clinical application of the present invention. For example, histological observation shows that the apoptosis rate of tumor cells in the combination treatment group increases and the necrotic area expands, further demonstrating the effectiveness of the treatment. Another example is that in a mouse subcutaneous xenograft tumor model, the combined use of an NMT1 inhibitor (such as PCLX-001) and a PD-1 antibody significantly inhibits tumor growth, and the tumor volume reduction rate is increased by 50%-70% compared with the single drug treatment group. Immunohistochemical analysis shows that the infiltration of CD8+ T cells in the tumor tissue of the combination treatment group increases significantly, the expression level of Granzyme B is up-regulated, indicating enhanced cytotoxic T cell activity, and at the same time, the expression of PD-1 is reduced, and the immunosuppressive state is relieved.

[0015] As a medicament, the composition of the present invention is used for combined therapy for hypoxia-related cancers and is applicable to a variety of solid tumor cancers, including but not limited to: head and neck squamous cell carcinoma, melanoma, lung cancer, esophageal squamous cell carcinoma, cervical squamous cell carcinoma, breast cancer, etc.

[0016] Clinically, these cancers often show a significant hypoxic microenvironment, high expression of PD-L1, and resistance to known immunotherapies. The combined therapy of the present invention can effectively overcome these problems through a dual mechanism, providing new treatment hope for patients.

[0017] Compared with the prior art, the technical solution of the present invention has the following characteristics:

[0018] 1) The present invention first proposes and verifies a combined treatment strategy of "spatial blockade + signal inhibition". The NMT1 inhibitor achieves "spatial blockade" by reducing the membrane localization of PD-L1, while the immune checkpoint inhibitor achieves "signal inhibition" by blocking the PD-1 / PD-L1 pathway. This two-pronged approach significantly improves the treatment effect and solves the difficult problem of hypoxia-induced immune escape, especially in hypoxia-related cancers with high expression of PD-L1. The synergistic effect of the dual mechanism can significantly improve the treatment response rate of patients with hypoxia-related cancers, especially in patients with high expression of PD-L1 or resistant to immunotherapy.

[0019] 2) The NMT1 inhibitor specifically targets NMT1 activity, avoiding non-specific inhibition of other enzymes, having high specificity, and reducing the risk of potential side effects.

[0020] 3) Low-dose high efficiency: The NMT1 inhibitor used in the present invention can efficiently inhibit the myristoylation of CHP1 at a low dose (0.1 - 1 μmol / L), avoiding the potential toxic and side effects that may be brought by high-dose drugs, and improving the safety of treatment and the tolerance of patients.

[0021] 4) For patients who have received immunotherapy with immune checkpoint inhibitors but have poor efficacy, the combination therapy of the present invention can reactivate the anti-tumor immune response through a dual mechanism, breaking through the drug resistance barrier. The enhanced anti-tumor immune response is expected to extend the progression-free survival (PFS) and overall survival (OS) of patients, and strive for a longer life time for patients.

[0022] 5) The combination therapy can take effect at a low dose, and no significant weight loss, abnormal liver and kidney functions or other serious adverse reactions are observed, showing good safety, reducing the occurrence of toxic and side effects, and helping to improve the quality of life of patients.

[0023] 6) The inhibitor of the present invention can be composed of existing drugs (such as PCLX-001 and PD-1 antibodies), with low development costs and a clear clinical transformation path, and has high application potential.

[0024] In particular, the present invention has significant therapeutic advantages for the following patient groups: Description of the Drawings

[0025] Figure 1 It is a result graph of a clinical retrospective study; among them, A is an example of high expression of NMT1 in immunohistochemical staining of tumor tissue sections, B is a quantitative analysis graph of immunohistochemistry in 72 OSCC specimens, C is a Kaplan-Meier survival curve of immunohistochemical results of tumor pathological sections of 100 patients, and D is a survival analysis result graph of the TCGA cohort evaluating the hypoxia status by stratifying 519 patients according to NMT1 expression and combining the "HALLMARK_HYPOXIA" gene set in MSigDB;

[0026] Figure 2 It is a result graph of the spatial co-localization network of NMT1 / HIF1α / PD-L1 and the inhibition analysis of CD8 + T cell function; among them, A is a result graph of bioinformatics analysis based on TCGA data, B is a result graph of bioinformatics analysis based on TCGA data, and the high and low hypoxia scores are related to poor prognosis of patients, and C is a graph of the correlation between hypoxia scores and various immunosuppressive molecules in bioinformatics analysis based on TCGA data;

[0027] Figure 3 It is a result graph of the spatial co-localization network of HIF1α / NMT1 / PD-L1 and CD8 +Graph of T cell function inhibition results; among them, A is the analysis result graph of 72 OSCC samples, B is the correlation analysis graph of the high expression of HIF1α and prognosis of 72 samples, and C is the immunohistochemical staining result graph of the pathological tissue sections of 72 OSCC patients.

[0028] Figure 4 is the NMT1 / HIF1α / PD-L1 spatial co-localization network and CD8 + Graph of fluorescence staining results of T cell function inhibition;

[0029] Figure 5 is the graph of the dynamic change of PD-L1 membrane localization mediated by hypoxia in a time-dependent manner and the cross-cell line consistency analysis; among them, A is the immunofluorescence microscopy observation graph of the PD-L1 expression distribution in the SCC7 cell line under normoxia and hypoxia conditions, B is the quantitative analysis result graph of the immunofluorescence image of the yellow line in the Merge image of Normoxia 12h, and C is the quantitative analysis result graph of the immunofluorescence image of the yellow line in the Merge image of Hypoxia12h.

[0030] Figure 6 is the verification result graph of the aggregation of PD-L1 in tumor cells to the cell membrane under hypoxia after knocking down NMT1; among them, A is the immunofluorescence staining observation graph of the localization of PD-L1 in tumor cells after knocking down NMT1 under hypoxia conditions, B is the graph of the expression trend of PD-L1 and F-actin quantified according to the yellow line in the Merge image of the control group in Figure A (the cell membrane position is marked together), and C is the graph of the expression trend of PD-L1 and F-actin quantified according to the yellow line in the Merge image of the NMT1 knockdown group in Figure A (the cell membrane position is marked together);

[0031] Figure 7 is the verification result graph of the inhibitory effect of PCLX-001 at various concentrations on the N-myristoylation modification level of SCC7 cells; among them, A is the electrophoresis graph of the N-myristoylation of tumor cells by PCLX-001 at various concentrations, and B is the statistical graph of the results of the CCK8 experiment to evaluate the proliferation of tumor cells by PCLX-001 at various concentrations;

[0032] Figure 8 is the experimental graph of the effect of the combination of PCLX-001 and anti-PD-1 monoclonal antibody on tumor growth; among them, A is the schematic diagram of the experimental route, and B is the ex vivo bright-field photos of the final tumor formation in each group of animals.

[0033] Figure 9 is the statistical graph of the results of the combination of PCLX-001 and anti-PD-1 monoclonal antibody on tumor growth; among them, A is the statistical graph of the final tumor weight of each group of animals, B is the statistical graph of the final tumor volume of each group of animals, C is the statistical graph of the tumor growth curve of each group of animals, and D is the statistical graph of the body weight growth curve of each group of animals;

[0034] Figure 10 It is the co-staining result diagram of Granzyme B, PD-1 and CD8a; among them, A is the three-label immunofluorescence staining field view of Granzyme B, PD-1 and CD8a after fixing and sectioning the tumor tissues of each group of animals, B is the statistical chart of the expression intensity of Granzyme B in the immunofluorescence staining of the tumor tissue sections of each group of animals, and C is the statistical chart of the expression intensity of PD-1 in the immunofluorescence staining of the tumor tissue sections of each group of animals. Specific implementation manner

[0035] The experimental scheme adopted in the embodiments of the present invention is as follows:

[0036] 1) NMT1 inhibitor treatment

[0037] Compound: The NMT1 inhibitor used in this embodiment is preferably PCLX-001 (chemical name: DDD85646), purchased from Shanghai TargetMol Biotech Co., Ltd. It is a small molecule compound with the ability to specifically inhibit NMT1 activity.

[0038] Dose range: In vitro experiment: 0.1 - 1 μmol / L; In vivo experiment: 0.1 - 10 mg / kg, and the specific dose is adjusted according to the patient's body weight, tumor type and treatment stage.

[0039] Alternative options: In addition to PCLX-001, other NMT1 inhibitors include chemical analogs of DDD85646 (such as IMP-1088 from TargetMol, MCE, etc.), siRNA (from GenePharma), shRNA (from GenePharma) or other molecules that can specifically inhibit NMT1 expression or activity.

[0040] Mechanism of action: PCLX-001 inhibits NMT1 activity, blocks the myristoylation of CHP1, reduces the binding of CHP1 to PD-L1, and thus reduces the localization of PD-L1 on the cell membrane.

[0041] Immune checkpoint inhibitor: PD-1 antibody, such as Pembrolizumab, Nivolumab.

[0042] Drug administration plan: Follow clinical standards, for example: Pembrolizumab 200 mg is intravenously injected once every 3 weeks, or Nivolumab 240 mg is intravenously injected once every 2 weeks.

[0043] Mechanism of action: Blocks the interaction between PD-1 and PD-L1, and restores the anti-tumor activity of T cells.

[0044] 2) Combined treatment plan

[0045] Administration method of NMT1 inhibitor: It can be administered orally, intravenously or by intratumoral injection.

[0046] Immune checkpoint inhibitor: Administered by intravenous injection.

[0047] Administration sequence and time: NMT1 inhibitor is preferably administered 24 - 48 hours before the administration of immune checkpoint inhibitor to ensure sufficient reduction of PD-L1 membrane localization. It can also be adjusted to simultaneous administration or administration after NMT1 inhibitor according to clinical needs.

[0048] Treatment cycle: Each cycle is 2 - 4 weeks, and repeated treatment is carried out until the clinical endpoint is reached (such as tumor shrinkage or disease stability). The specific cycle is adjusted according to the patient's response and tolerance.

[0049] 3) Immunofluorescence of tissue sections

[0050] Deparaffinize paraffin sections to water: Place the sections successively into environment-friendly dewaxing solution for 10 min - environment-friendly dewaxing solution for 10 min - environment-friendly dewaxing solution for 10 min - absolute ethanol Ⅰ for 5 min - absolute ethanol Ⅱ for 5 min - absolute ethanol Ⅲ for 5 min - distilled water wash.

[0051] Antigen retrieval: Place the tissue sections in a repair box filled with EDTA antigen retrieval buffer (pH 8.0) and perform antigen retrieval in a microwave oven. Heat to boiling at medium heat for 8 min, turn off the fire for 8 min, then turn to medium-low heat for 7 min. During this process, prevent excessive evaporation of the buffer and do not let the slices dry. After natural cooling, place the slides in PBS (pH 7.4) and shake and wash on a decolorizing shaker 3 times, 5 min each time. The retrieval solution and retrieval conditions are determined according to the tissue.

[0052] Draw a circle: After slightly drying the slices, use a histochemical pen to draw a circle around the tissue (to prevent the antibody from flowing away).

[0053] Serum blocking: Drop BSA in the circle and incubate for 30 min. (If the primary antibody is from goat, add donkey serum)

[0054] Add primary antibody: Gently shake off the blocking solution, drop the primary antibody prepared by mixing PBS in a certain proportion on the slices. Mix two primary antibodies in a certain dilution ratio and drop them on the tissue. Place the slices flat in a wet box and incubate overnight at 4℃. (Add a small amount of water in the wet box to prevent antibody evaporation)

[0055] Add secondary antibody: Place the slides in PBS (pH 7.4) and shake and wash on a decolorizing shaker 3 times, 5 min each time. After slightly drying the slices, drop the secondary antibody corresponding to the species of the primary antibody prepared by mixing in a certain proportion in the circle to cover the tissue and incubate at room temperature for 50 min. (Mix two secondary antibodies and incubate according to a certain proportion)

[0056] DAPI counterstaining of cell nuclei: After gently shaking off the excess liquid from the sections, add DAPI staining solution within the marked area and incubate at room temperature for 10 min in the dark. Spontaneous fluorescence quenching: After gently shaking off the excess liquid from the sections, add the spontaneous fluorescence quenching agent within the marked area for 5 min, and then rinse with running water for 10 min. Mounting: Place the slides in PBS (pH 7.4) and shake on a shaker for 3 times, 5 min each time. After gently shaking off the excess liquid from the sections, mount them with an anti-fluorescence quenching mounting medium.

[0057] Microscopic examination and photography: Place the sections under a scanner to acquire images. (The ultraviolet excitation wavelength of DAPI is 330 - 380 nm, the emission wavelength is 420 nm, emitting blue light; the excitation wavelength of FITC is 465 - 495 nm, the emission wavelength is 515 - 555 nm, emitting green light; the excitation wavelength of CY3 is 510 - 560 nm, the emission wavelength is 590 nm, emitting red light; the excitation wavelength of CY5 is 608 - 648 nm, and the emission wavelength is 672 - 712 nm. The cell nuclei stained with DAPI appear blue under ultraviolet excitation, and positive expression shows the corresponding red or green light emitted by the fluorescently labeled substances.)

[0058] 4) Immunofluorescence staining of cells

[0059] Sample pretreatment: Remove the culture medium and gently rinse the cells twice with room temperature TBS for 5 seconds each time; Cell fixation: Cover the cells with an adequate amount of 4% neutral formaldehyde fixative (prepared with TBS buffer) and fix at 4°C for 15 minutes; Wash the fixative: Remove the fixative and rinse 3 times with pre-cooled TBS buffer at 4°C for 5 minutes each time. Note: Avoid large temperature differences or sudden temperature changes during the experiment; Ensure an adequate amount of fixative, and it can be used in excess; Formaldehyde is toxic, and the addition of the fixative and the washing after fixation should be carried out in a fume hood.

[0060] Staining procedure: Completely cover the sample with 5% blank goat serum. For sections, place them in a humid chamber. For cell culture plates, seal the plates directly and incubate in a 37°C constant temperature and humidity incubator for 30 minutes; Primary antibody incubation: Remove the blocking solution, directly add the primary antibody working solution prepared with TBS buffer dropwise onto the sample, completely cover the sample. For sections, place them in a humid chamber. For cell culture plates, seal the plates directly and incubate at 4°C overnight; 1:100 overnight, 150 μl; Rewarming: Place the sample at room temperature and rewarm for 15 minutes. Remove the antibody working solution and wash once with buffer TBST for 5 minutes; Wash 3 times with buffer TBS, 5 minutes each time; Secondary antibody incubation: Dropwise add the fluorescent secondary antibody working solution corresponding to the species of the primary antibody onto the sample, completely cover the sample, protect from light, and incubate at 37°C for 1 hour; Remove the secondary antibody working solution and wash once with buffer TBST for 5 minutes; Wash 3 times with buffer TBS, 5 minutes each time; 1:2000, protect from light; Cytoskeleton incubation: Dilute phalloidin 1:200 and incubate at 37°C for 1 hour, protect from light; Nuclear staining: Dropwise add the DAPI working solution onto the sample, prepared with 0.01M pH 7.2 ± 0.2 TBS buffer, volume ratio of DAPI solution to TBS buffer is 1:500, protect from light, and incubate at room temperature for 10 minutes; Remove the DAPI working solution and wash once with buffer TBST for 5 minutes; Wash 3 times with buffer TBS, 5 minutes each time; 1:5000; For cell culture plates, directly add the anti-fluorescence quenching mounting medium and then observe and collect images under a fluorescence microscope; For cell smears, dropwise add the anti-fluorescence quenching mounting medium, then cover with a coverslip and seal, and then observe and collect images under a fluorescence microscope; For cell slides, take them out, cover them on a glass slide with the anti-fluorescence quenching mounting medium dropwise added, and then observe and collect images under a fluorescence microscope. Note: During the experiment, all reagent drops should be accurate, fast, and sufficient, and there should be no dry slides; Starting from the secondary antibody incubation, all subsequent steps need to pay attention to light protection operations; Observe and collect images in a timely manner after staining to avoid drying and quenching of fluorescent substances.

[0061] 5) Analysis of immunofluorescence staining results

[0062] Steps for measuring photometric values in ImageJ: Open the ImageJ software and import the immunofluorescence co-localization images. Convert to 8-bit grayscale: Convert the color image to 8-bit grayscale according to the labeled fluorescence channels. For the green fluorescence channel (Alexa Fluor 488), select "Image-Color-Split Channels-Green" and then perform grayscale conversion; the same applies to the red fluorescence channel (Cy3). Perform background subtraction on the grayscale image of each channel. "Process-Subtract Background", and adjust parameters such as "Rolling ball radius" according to the actual situation of the staining. Determine the region of interest (ROI): You can use tools such as straight lines, rectangles, ellipses, or free-drawn ROIs to select the regions with obvious fluorescent positive signals. In "Analyze-Set Measurements", check the grayscale value-related parameters such as "Mean gray value". Then measure each ROI and record the grayscale values corresponding to the red and green fluorescence.

[0063] Prism 10.0 statistical analysis and graphing: Import the data measured in ImageJ into the Prism 10.0 software, either by copying and pasting or directly opening the corresponding file format. Select an appropriate analysis method according to the statistical design. If comparing the fluorescence intensity differences between different samples, such as between multiple experimental groups, one-way analysis of variance (One-Way ANOVA) can be used, and further select a post hoc test method (such as: Tukey's HSD or Bonferroni's multiple comparisons) to analyze the specific differences between groups. If the data does not conform to the normal distribution or the sample size is small, non-parametric tests, such as the Kruskal-Wallis test, may be more appropriate. Create a graph based on the statistical analysis results. You can use a bar graph to visually display the mean values of the red and green fluorescence photometric values and their differences in different experimental groups. Set elements such as appropriate graph titles, axis labels, and error bars to clearly convey the data information. You can also adjust the color combination and style of the graph to enhance its readability.

[0064] 6) Biotinylated protein enrichment

[0065] Calculate the amount of NeutrAvidin beads used according to the total amount of precipitated protein (30 μL / 1 mg protein), and take 1.2 times the volume of NeutrAvidin beads. Add 5 times the volume of HEPES wash buffer A to the taken beads, mix vertically by rotation at room temperature for 2 min, centrifuge at 3000 g for 4 min at room temperature, aspirate the supernatant, and repeat the washing process 2 more times. After the washing is completed, resuspend the beads in HEPES wash buffer A to make the total volume reach the volume taken in the first step. Centrifuge the protein sample at 17000 g for 5 min at room temperature, take about 90% of the supernatant and transfer it to the beads, trying not to aspirate the precipitate. Incubate by vertical rotation at room temperature for 3 h. After the incubation, centrifuge at 3000 g for 4 min at room temperature and aspirate the supernatant. Add an excess of HEPES wash buffer A (1 mL buffer / 30 μL beads) to the beads, mix vertically by rotation at room temperature for 2 min, centrifuge at 3000 g for 4 min at room temperature, aspirate the supernatant, and repeat the washing process 2 more times. Add an excess of HEPES wash buffer B (1 mL buffer / 30 μL beads) to the beads, mix vertically by rotation at room temperature for 2 min, centrifuge at 3000 g for 4 min at room temperature, aspirate the supernatant, and repeat the washing process 2 more times. Add an excess of HEPES solution (1 mL buffer / 30 μL beads) to the beads, mix vertically by rotation at room temperature for 2 min, centrifuge at 3000 g for 4 min at room temperature, aspirate the supernatant, and repeat the washing process 2 more times.

[0066] 7) Digestion and Protein Electrophoresis

[0067] Add a solution of DTT with a final concentration of 10 mM to the beads, mix vertically by rotation at 37 °C for 1 h, centrifuge at 3000 g for 4 min, and aspirate the supernatant. Add a solution of IAA with a final concentration of 25 mM to the beads, mix vertically by rotation in the dark at room temperature for 15 min, centrifuge at 3000 g for 4 min, and aspirate the supernatant. Add trypsin solution to the beads at a ratio of trypsin:protein = 1:50, mix vertically by rotation overnight at 37 °C for 16 h. Centrifuge at 3000 g for 4 min and collect the supernatant. Add 3 μL of formic acid (FA) to the supernatant. Dry at 50 - 60 °C in a centrifugal concentrator for 2 - 3 h. Add 20 - 30 μL of 0.1% FA solution to the dried sample and oscillate for 5 - 10 min to redissolve. Desalt with Pierce C18 Spin Tips, and dry the desalted sample at 50 - 60 °C in a centrifugal concentrator for 5 - 10 min. Add protein dissolution buffer to the dried sample, add 5×Loading Buffer in a volume ratio of 1:4, and load for electrophoresis.

[0068] 8) Cell proliferation detection

[0069] The cell counting kit-8 (CCK-8) was used to detect the cell proliferation ability, and the cell growth differences under different treatment conditions (such as NMT1 knockdown) were evaluated by measuring the cell metabolic activity. The CCK-8 reagent contains a tetrazolium salt (WST-8), which is reduced by the dehydrogenase of living cells to generate a water-soluble orange-yellow product (formazan). Its absorbance (OD value) is proportional to the number of living cells at 450 nm.

[0070] 9) Preparation of experimental materials

[0071] Animal preparation and drug preparation: C3H / He mice, generally healthy male mice aged 4-6 weeks, were selected. The mice were housed in a specific pathogen-free (SPF) animal room, given appropriate feed and water, and allowed to adapt to the environment for one week. Before the experiment, the mice were numbered, weighed, and their health status was checked.

[0072] Cell line preparation: The SCC7 murine cell line was selected and cultured in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. When the cells grew to the logarithmic growth phase (cell density reached about 80-90%), the fresh medium was replaced one day before collecting the cells. The PCLX-001 and anti-PD-1 monoclonal antibody drug solutions were accurately prepared to ensure that the drug concentrations were accurate and met the experimental requirements.

[0073] Cell inoculation for tumor formation: SCC7 cells were digested with trypsin until the cells became round and the cell gaps became larger. The digestion was terminated with serum-containing medium, and the cell suspension was collected into a centrifuge tube. The cells were centrifuged at 1000-1200 rpm for 5-8 minutes, and the supernatant was removed. The cells were resuspended with serum-free medium. The cell suspension concentration was adjusted to an appropriate inoculation concentration using a cell counting chamber or an automatic cell counter. The skin on the left axilla of C3H / He mice was disinfected with an alcohol cotton ball, and then an appropriate amount of cell suspension was aspirated with a 1 mL syringe (100-200 μL per mouse, and the cell amount was about 1-5×106 cells) and slowly injected into the subcutaneous tissue.

[0074] Observation of tumor formation and drug administration: The inoculated mice were returned to the cage for continued feeding. The mental state, diet, and whether there were redness, induration, etc. at the inoculation site of the mice were observed every day. The long diameter (a) and short diameter (b) of the tumor were measured every 1-2 days using a caliper. According to the formula (V = a*b 2(2) Calculate the tumor volume. When the tumor volume reaches 50 cubic millimeters, tumor formation is considered successful, and subsequent drug interventions are initiated. After tumor formation, the mice are divided into an experimental group (intratumoral injection of PCLX-001 once every 48 hours, and intraperitoneal injection of anti-PD-1 monoclonal antibody once every 72 hours) and a control group (administered an equal volume of normal saline or solvent). During the drug intervention process, continue to observe the status of the mice and measure the tumor volume according to the above method, and record the experimental data.

[0075] Data statistical analysis and subsequent processing: Statistically analyze data such as the tumor volume, changes in mouse body weight, and survival time of mice in the experimental group and the control group at different time points. Use appropriate statistical methods (such as t-tests, analysis of variance, etc.) to analyze the experimental results and determine the effects of PCLX-001 and anti-PD-1 monoclonal antibody on the subcutaneous tumor formation of SCC7 cells in C3H / He mice. After the experiment, euthanize the mice and remove the tumor tissues. Part of the tumor tissues are fixed with formalin for pathological examinations, such as HE staining, immunohistochemistry, etc.; part of the tumor tissues are cryopreserved in liquid nitrogen for subsequent molecular biology experiments, such as gene expression analysis, protein extraction, etc.

[0076] Example 1

[0077] All data are from the Department of Oral and Maxillofacial Head and Neck Oncology, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine. According to the established time range (from April 15, 2018, to June 29, 2018), patients who were diagnosed and received surgical treatment during this period were screened from the department's medical record system. Patients who had received chemotherapy or immunotherapy were strictly excluded to ensure the independence of the study. The clinical retrospective study was approved by the Medical Ethics Committee of Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, and the ethical review approval number is SH9H-2024-T388-1. At the same time, the clinical stages of the patients were labeled according to the 8th edition of the staging manual of the American Joint Committee on Cancer (AJCC).

[0078] Retrieve the clinical information of the corresponding patients from the hospital's sample library, including the time of admission and discharge, the time of diagnosis, etc., and record the basic information such as the time and type of specimen collection (such as tissue biopsy specimens, etc.). Enter the detailed pathological examination information of the specimens, including the histological type and degree of differentiation of the tumor. Collect the general demographic data of the patients, such as age, gender, weight, height, smoking history, drinking history, etc. Record the detailed information related to the patients' surgeries, including the surgery date, surgical method (such as the resection range, whether reconstruction is involved, etc.), intraoperative conditions (such as the amount of bleeding, whether there are complications, etc.). Collect comprehensive postoperative follow-up information of the patients. During the follow-up period of up to 60 months, regularly record the survival status of the patients (survival, recurrence, metastasis, etc.), quality of life indicators, and other subsequent treatments received (if any). Establish a dedicated data management system, enter all data in a unified format, and conduct two checks during the input process to ensure the accuracy of the data. And classify and store the data for convenient subsequent analysis. Determine the main analysis indicators according to the research purpose. For example, when analyzing the survival rate differences among patients with different clinical stages, set the survival rate as the main observation indicator and the clinical stage as the grouping variable. Select appropriate statistical analysis methods. For example, the Kaplan-Meier method can be used for survival rate analysis. Use professional statistical software for data analysis. First, conduct descriptive statistics to display the distribution of the basic characteristics and clinical characteristics of the patients. Conduct in-depth analysis according to the predetermined analysis method to explore the correlations among various factors and their impacts on the patients' prognosis.

[0079] The results of the clinical retrospective study are as Figure 1 shown. Figure 1 A shows an example of high expression of NMT1 in immunohistochemical staining of tumor tissue sections (IRS≥1) (scale bar = 2mm and 400μm). In the immunohistochemical quantitative analysis of 72 OSCC specimens, the expression of NMT1 in tumor tissue was significantly higher than that in adjacent normal tissue (p = 0.0439), see Figure 1 B.

[0080] Based on the immunohistochemical results of the tumor pathological sections of 100 patients, survival analysis (clinical retrospective study cohort) was performed using the ciber sort deconvolution method in Rstudio with Prism grahpad 10.0 and the transcriptome data of the TCGA database, and the Kaplan-Meier survival curve was plotted. As Figure 1 C shows, there was a significant difference in the survival rate between the high-expression group and the low-expression group of NMT1 (p = 0.002174, hazard ratio [low / high] = 0.3767, 95% confidence interval: 0.1722 - 0.6792).

[0081] Survival analysis of the TCGA cohort: 519 patients were stratified by NMT1 expression, and the hypoxia status was evaluated by combining with the "HALLMARK_HYPOXIA" gene set in MSigDB. The results are as Figure 1 shown in Figure D. The prognosis of the high NMT1 expression group was worse (p<0.0001, HR(L / H) = 0.4986, 95% CI = 0.3598 to 0.6908).

[0082] The above experimental results indicate that NMT1 can be used as a prognostic marker for OSCC.

[0083] Example 2

[0084] Data acquisition and preprocessing: Use the `TCGAbiolinks` package to download the required mRNA-seq data and corresponding clinical information from the TCGA data portal; remove low-quality reads, align to the reference genome, and calculate gene expression levels; organize the spatial transcriptome data of the research group, and use the `Seurat` package for data cleaning and format conversion to ensure the compatibility of the two data sources.

[0085] Data exploratory analysis: Use the basic statistical functions of R (such as `mean()`, `median()`, `sd()`) for descriptive statistical analysis to understand the overall distribution characteristics of the data; use the `ggplot2` package to create box plots and histograms to show the distribution of gene expression levels and identify potential outliers and skewness.

[0086] Differential expression analysis: Use the `DESeq2` package to perform differential expression analysis on the TCGA mRNA data and spatial transcriptome data, set appropriate thresholds, and screen out genes with significant differential expression; use the `ggplot2` package to create volcano plots and heatmaps to visually show the changes in gene expression.

[0087] Functional annotation and enrichment analysis: Use the `BiocManager` package to install and use `org.Hs.eg.db` for gene annotation, and then use the `GOstats` and `KEGGREST` packages for GO enrichment analysis and KEGG pathway analysis; use the `ggplot2` package to create bar charts and bubble charts to show the enrichment results and evaluate the functional significance of differentially expressed genes

[124] .

[0088] Visualization of spatial transcriptome data: Use the `Seurat` package to preprocess and analyze the spatial transcriptome data to create spatial expression maps; use the visualization function of `Seurat` to show the spatial distribution of genes on tissue sections, and analyze the spatial heterogeneity of gene expression and its association with tissue structure.

[0089] Co-expression analysis and network construction: Use the `WGCNA` package to perform co-expression analysis on differentially expressed genes, construct a gene co-expression network; identify key modules and hub genes in the network, and explore their roles in the gene regulatory network.

[0090] Integrative analysis and comprehensive evaluation: Deeply integrate TCGA mRNA data with spatial transcriptome data, and use the multimodal function of `Seurat` for analysis; comprehensively evaluate the potential value of differentially expressed genes in biological functions and clinical applications.

[0091] NMT1 / HIF1α / PD-L1 spatial co-localization network and CD8 + Analysis results of CD8 T cell function inhibition Figure 2 as shown Figure 2 A. It can be seen that the hypoxia score is positively correlated with effector Treg cells, M0 macrophages and exhausted T cells, and negatively correlated with CD8 + T cells. Bioinformatics analysis based on TCGA data further shows that high and low hypoxia scores are associated with poor patient prognosis (p = 0.0072, HR(L / H) = 0.6909, 95% CI = 0.5275 to 0.9048). The hypoxia score is positively correlated with immunosuppressive molecules such as VEGFA and CD274, see Figure 2 C.

[0092] These analyses indicate that the NMT1 / HIF1α / PD-L1 spatial co-localization network is correlated with CD8 + T cell function inhibition.

[0093] In 72 clinical OSCC samples, the protein expression level of HIF1α was negatively correlated with the immunofluorescence intensity of the CD8 + T cell marker CD8a (R = -0.4384, p = 0.0001; see Figure 3 A and Figure 3 C). Survival analysis further confirmed that the 5-year overall survival rate of patients with high HIF1α expression was significantly lower than that of the low-expression group (HR = 0.3473, 95% CI = 0.1511 - 0.7776, p = 0.0101; see Figure 3 B), indicating that HIF1α is not only a core regulatory factor for hypoxia adaptation, but also an important driving molecule for immune escape.

[0094] In further experiments on the NMT1 / HIF1α / PD-L1 spatial co-localization network and CD8 + T cell function inhibition, immunofluorescence staining was performed on tumor tissue sections of OSCC patients, and the results of regional expression intensity were as Figure 4As shown, the localization of CD8a, NMT1, PD-L1, and PD-1 in cells or tissue regions is visually presented through fluorescence channels of different colors. Below each fluorescence channel, the regional expression intensity is analyzed. The phenotypic map shows the positional relationship of the four markers. The high expression of NMT1 at the tissue level inhibits the infiltration of CD8+ T cells, while the high expression of PD-1 and PD-L1 on the side with high NMT1 expression indicates the formation of an immunosuppressive microenvironment. (scale bar = 200μm).

[0095] Analysis of regional expression intensity shows that the fluorescence signal intensities of NMT1 are significantly positively correlated with those of PD-L1 and PD-1, but show a spatial separation characteristic from the CD8a signal. This distribution pattern suggests: Spatial synergy of the immunosuppressive molecular network: NMT1 and PD-L1 / PD-1 form an immunosuppressive microenvironment in the tumor core area, restricting CD8 + T cell infiltration through a physical barrier effect; Dynamic regulation of functional partitioning: The hypoxic core area continuously upregulates PD-L1 through the HIF1α-NMT1 axis, while peripheral CD8 + T cells are disabled due to lack of co-stimulatory signals and exposure to the inhibitory molecular network.

[0096] Example 3

[0097] The experimental results of the dynamic change of PD-L1 membrane localization mediated by hypoxia in a time-dependent manner and the cross-cell line consistency verification are as Figure 5 shown. Immunofluorescence microscopy was used to observe the PD-L1 expression distribution in the SCC7 cell line under normoxic and hypoxic conditions. See Figure 5 A. The PD-L1 signal intensity on the cell membrane surface of the hypoxic treatment group of SCC7 was significantly higher than that of the normoxic group, indicating that NMT1 is a key protein regulating the plasma membrane distribution of PD-L1.

[0098] For Figure 5 the quantitative analysis results of the immunofluorescence image of the yellow line in the Merge image of Normoxia 12h in A are shown in Figure 5 B. The quantitative analysis results of the immunofluorescence image of the yellow line in the Merge image of Hypoxia 12h in 5B are shown in Figure 5 C. Figure 5 B and Figure 5 C indicate that under hypoxic conditions, PD-L1 has a tendency to aggregate towards the cell membrane, and the tendency of PD-L1 to aggregate towards the cell membrane under hypoxic conditions is statistically significant.

[0099] Example 4

[0100] After NMT1 knockdown, the verification results of the tendency of PD-L1 to aggregate towards the cell membrane in tumor cells under hypoxic conditions are as Figure 6As shown in the figure. Immunofluorescence staining was used to observe the localization of PD-L1 in tumor cells after knocking down NMT1 under hypoxic conditions. See Figure 6 A. For the Figure 6 quantitative analysis of the expression of PD-L1 and F-actin by quantifying the immunofluorescence image of the yellow line in the Merge image of shNC of A, the results are shown in Figure 6 B. For the quantitative analysis of the expression of PD-L1 and F-actin by quantifying the immunofluorescence image of the yellow line in the Merge image of shNMT1 of 6A, the results are shown in Figure 6 C.

[0101] Under hypoxic conditions, after knocking down NMT1, the localization of PD-L1 in tumor cells changed significantly. In the control group, PD-L1 was mainly distributed on the cell membrane, while in the group with knocked-down NMT1, PD-L1 was more distributed in the cytoplasm. This phenomenon was further confirmed by quantitative analysis ( Figure 6 B and Figure 6 C). In the control group, the expression trend of PD-L1 was similar to that of F-actin (cytoskeletal protein), mainly concentrated near the cell membrane; while in the NMT1 knockdown group, the expression trend of PD-L1 was different from that of F-actin, and it was more distributed in the cytoplasm.

[0102] The above results indicate that knocking down NMT1 can reverse the tendency of PD-L1 to aggregate on the cell membrane in tumor cells under hypoxic conditions.

[0103] Example 5

[0104] Evaluate the inhibitory effect of various concentrations of PCLX-001 on the N-myristoylation modification level in SCC7 cells. By Western blot analysis, it was observed that with the increase in the concentration of PCLX-001, the level of N-myristoylation modification decreased significantly. Specifically, the treatments with 0.1 μmol, 0.5 μmol, and 1 μmol of PCLX-001 all showed obvious inhibitory effects, and the higher the concentration, the more significant the inhibitory effect ( Figure 7 A). β-actin was used as an internal reference protein, and its expression level remained consistent among groups to ensure the reliability of the experimental results. However, a higher concentration exceeding 10 μmol was required to inhibit cell proliferation, indicating an obvious difference between its enzyme inhibitory effect and cytotoxic effect ( Figure 7 B). This selective effect at sub-toxic levels provides a basis for its therapeutic application. These results indicate that PCLX-001 can effectively inhibit N-myristoylation modification in SCC7 cells, providing strong experimental evidence for subsequent studies on its combined application with PD-1 monoclonal antibody drugs as an N-myristoylation inhibitor.

[0105] Example 6

[0106] The effect of the combination of PCLX-001 and anti-PD-1 monoclonal antibody on tumor growth is as Figure 8 shown below.

[0107] The experimental method is as Figure 8 shown in A. Briefly, tumors were subcutaneously implanted in the back of C3H / He mice. Tumor formation was confirmed on the seventh day. After tumor formation, PCLX-001 was injected into the tumor every 2 days, and anti-PD-1 monoclonal antibody was injected intraperitoneally every 3 days to evaluate the efficacy of the combination of the two drugs.

[0108] Figure 8 As shown in B, there were significant differences in tumor size among the control group (IgG2a + DMSO), single inhibitor group (IgG2a + PCLX-001), single anti-PD-1 monoclonal antibody group (PD-1mAb + DMSO), and combination treatment group (PD-1mAb + PCLX-001). The tumor volume in the combination treatment group was the smallest, indicating that the combined use of PCLX-001 and anti-PD-1 monoclonal antibody had a significant inhibitory effect on tumor growth. The tumor growth rate in the combination treatment group was significantly slower than that in other groups, especially in the later stage of the experiment, and the growth of tumor volume almost stagnated. This result strongly supported the superiority of the combination of the two drugs in inhibiting tumor growth. Although the tumor growth of mice in the combination treatment group was significantly inhibited, there was no significant difference in body weight gain compared with other groups, indicating that this treatment regimen had good safety. The results of statistical analysis showed that the tumor weight and volume in the combination treatment group were significantly lower than those in other groups, further verifying the strong anti-tumor effect of the combined use of PCLX-001 and anti-PD-1 monoclonal antibody.

[0109] Figure 8 The statistical results of the weight of each tumor shown in B are shown in Figure 9 A, the statistical results of the final volume of each group of tumors are shown in Figure 9 C, and the statistical results of the body weight of each group of animals are shown in Figure 9 D. These experimental results showed that in the mouse model, the combined use of PCLX-001 and anti-PD-1 antibody significantly inhibited tumor growth, and the tumor volume reduction rate was increased by about 50% - 70% compared with the single drug group. In the mouse model, no significant weight loss, abnormal liver and kidney functions, or other serious adverse reactions were observed in the combination treatment. The apoptosis rate of tumor cells in the combination treatment group increased, and the necrotic area expanded.

[0110] The results of the co-staining experiment of Granzyme B, PD-1, and CD8a are as Figure 10 shown below. Figure 10 A is the immunofluorescence staining map of the fixed sections of the tumor tissues of four groups of mice. The corresponding statistical results of the expression intensity of Granzyme B are shown in Figure 10 B, and the statistical results of the expression intensity of PD-1 are shown in Figure 10 C.

[0111] Immunofluorescence staining was performed on tumor tissue sections of four groups of mice, and Granzyme B, PD-1, and CD8a were labeled respectively to further explore the underlying mechanism by which the combination of NMT1 inhibitor and anti-PD-1 monoclonal antibody showed a stronger effect on inhibiting tumor growth. Granzyme B is usually associated with the activity of cytotoxic T cells, and its expression level can reflect the intensity of the immune response in tumor tissues. PD-1 is an immune checkpoint protein, and its expression level is related to immunosuppression. There were significant differences in the expression intensity of Granzyme B among different groups. The expression of Granzyme B in the combination treatment group (PD-1mAb+PCLX-001) was significantly higher than that in other groups, indicating that there might be a stronger immune response in the tumor tissues of the combination treatment group. The lower expression of PD-1 suggested a higher response rate to anti-PD-1 monoclonal antibody in the combination treatment group. By comparing the expression levels of Granzyme B and PD-1, the immune status of the tumor microenvironment (TME) in each group of mice could be inferred. Figure 10 A, Figure 10 B, and Figure 10 C). As shown by these experimental results, there was a significant increase in the infiltration of CD8 + T cells in the tumor tissues of the combination treatment group, and the expression of Granzyme B (granzyme B) was upregulated, indicating enhanced cytotoxic T cell activity. The decrease in the expression level of PD-1 suggested the alleviation of immunosuppression.

[0112] Combined with the previous research results, it can be known that the NMT1 inhibitor PCLX-001 enhances the activity and function of T cells by inhibiting the interaction between PD-1 and PD-L1 and reducing T cell exhaustion. This enables T cells to more effectively recognize and kill tumor cells, further suggesting that NMT1 enhances the efficacy of anti-PD-1 monoclonal antibody by regulating the tumor immune microenvironment.

Claims

1. Use of an inhibitor in the preparation of a medicament for treating solid tumor cancers, wherein the inhibitor is an NMT1 inhibitor, and the inhibitor reduces the localization of PD-L1 on the cell membrane and cooperates with an immune checkpoint inhibitor to block the PD-1 / PD-L1 signaling pathway.

2. The application according to claim 1, wherein The inhibitor is as shown in the following formula.

3. The application according to claim 1, characterized in that The concentration of the NMT1 inhibitor provided in the body by the medicament reaches or is maintained at 0.1 - 1 μmol / L.

4. A composition, characterized in that The NMT1 inhibitor and the immune checkpoint inhibitor.

5. The composition according to claim 4, characterized in that The NMT1 inhibitor is as shown in the following formula.

6. The composition according to claim 5, wherein Provide that the concentration of the NMT1 inhibitor in the body reaches or is maintained at 0.1 - 1 μmol / L.

7. The composition according to claim 4, wherein The immune checkpoint inhibitor is used to block the interaction between PD-1 and PD-L1, relieve T cell inhibition, and restore its anti-tumor immune function.

8. The composition according to claim 4, wherein The immune checkpoint inhibitor is selected from one or more of a PD-1 antibody and a PD-L1 antibody.

9. Use of the composition according to claim 4 in the preparation of a medicament for treating hypoxia-related cancers.

10. The application according to claim 9, wherein The hypoxia-related cancers are head and neck squamous cell carcinoma, melanoma, lung cancer, esophageal squamous cell carcinoma, cervical squamous cell carcinoma, and breast cancer.

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