Application of IP6 synthesis related kinase inhibitor in tumor radiotherapy sensitization

Abstract

The invention relates to application of an IP6 synthesis related kinase inhibitor in tumor radiotherapy sensitization. According to the application, CRISPR / Cas9 is used in vitro for screening important kinases in cells, it is found that the important kinases IPPK and ITPK1 participating in IP6 synthesis are related to radiotherapy sensitivity, and the research value of IPPK and ITPK1 serving as radiotherapy sensitization targets is further verified through cell colony formation experiments, mouse tumor-bearing induced radiotherapy tolerant cells and patient transcriptome data. In the aspect of mechanism and effect, it is found that targeted inhibition of IPPK and ITPK1 can promote lipid peroxidation induced by Fenton reaction by improving the level of free ferrous ions, so that ferroptosis is easier to occur, and sensitization can be achieved on radiotherapy which also causes lipid peroxidation. The invention provides a potential strategy for comprehensive treatment of malignant tumors, and has important practical significance for formulation of an accurate treatment scheme of malignant tumors clinically.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to the application of IP6 synthesis-related kinase inhibitors in tumor radiosensitization. Background Technology

[0002] Radiotherapy, as one of the core methods of comprehensive cancer treatment, plays an irreplaceable role in neoadjuvant therapy and local control of unresectable lesions. However, after neoadjuvant chemoradiotherapy, a considerable number of cases still show partial or no response, failing to achieve the ideal level of tumor regression. Furthermore, radiotherapy may lead to serious complications in some patients, thus reducing their quality of life. Simply increasing the radiation dose or prolonging the radiotherapy course cannot achieve the initial goal of improving patient prognosis. Therefore, exploring the molecular mechanisms that may lead to radiotherapy tolerance and developing targeted drugs as radiosensitizers for combined use is a worthwhile research and translational approach, aligning with the modern medical trend towards personalized and precise treatment.

[0003] In radiotherapy, the killing effect of ionizing radiation (IR) on tumor cells is a complex biological process triggered by a variety of physicochemical factors. The lethal effect of ionizing radiation is usually a chain reaction resulting from changes in biochemical metabolism, physiological function, and morphology, building upon its indirect and direct damaging effects. The specific biological characteristics exhibited vary depending on the radiation dose, cellular genetic background, and phenotype. From a molecular mechanism perspective, apoptosis, necrosis, and emerging ferroptosis are all possible cell death mechanisms that ultimately lead to the killing effect of radiotherapy.

[0004] Unlike apoptosis and necrosis, ferroptosis does not involve specific death-executing proteins or membrane pore formation. Instead, it is caused by lipid peroxidation of polyunsaturated fatty acid-containing phospholipids (PUFA-PLs) on the cell membrane, leading to decreased membrane fluidity and abnormal permeability, resulting in cell death. The energy from ionizing radiation can directly excite water molecules to generate a large number of oxygen free radicals, which directly participate in the peroxidation of PUFA-PLs. Simultaneously, damage to organelles such as lysosomes leads to the continuous accumulation of ferrous ions, exacerbating the Fenton reaction and resulting in the generation of even more free radicals. Furthermore, intracellular lipid peroxidation easily produces metabolic byproducts such as 4-hydroxy-2-nonenal (4-HNE). These products not only have direct cytotoxicity but can also act as signaling molecules, exacerbating radiotherapy-induced cell damage. In the long term, radiation-induced DNA damage occurs on the one hand by activating ATM and downregulating xCT along the cGAS / STING / ATF3 axis, thus inhibiting cysteine ​​supply to the oxygen free radical scavenging system. On the other hand, it also upregulates ACSL4, a key synthase of PUFA-PLs, further making irradiated cells more susceptible to ferroptosis.

[0005] Inositol hexakisphosphate (IP6) is the most abundant phosphoinositol (IP) compound in mammalian cells. Metabolically, IP6 plays a crucial role in the metabolism of phosphoinositol compounds. IP6 can bind to various proteins via electrostatic interactions, and chemically synthesized IP6 probes can capture dozens of proteins that specifically bind to it. Chemically, IP6 possesses certain antioxidant properties. In vitro studies have shown that adding IP6 can reduce oxygen free radicals generated by lipid peroxidation by interfering with the formation of ATP ferrooxide complexes and inhibiting the activity of xanthine oxidase and superoxide dismutase in intestinal tissue extracts. However, since the ferroptosis hypothesis was proposed, there have been no in-depth reports investigating the link between the IP6 metabolic pathway and cellular ferroptosis. Reports on the effects of the endogenous IP6 metabolic pathway on tumorigenesis, development, and treatment are scarce, and accurate molecular mechanism studies are lacking. Its therapeutic value remains unknown; therefore, more basic research is needed to comprehensively understand the key effects of this metabolic pathway and its products. Summary of the Invention

[0006] The purpose of this invention is to address the problem of unclear radiotherapy tolerance mechanisms in existing technologies. By combining kinase-targeted CRISPR / Cas9 screening technology with high-throughput sequencing, a cell model for screening radiotherapy tolerance targets was established. The screening results revealed for the first time the link between IP6 anabolic kinases IPPK and ITPK1 and radiosensitivity. This expands the understanding of other biological effects caused by radiation and clarifies that IP6 anabolic kinases are targets related to radiotherapy resistance, providing a more comprehensive scientific basis and solution for radiosensitization.

[0007] To solve the above-mentioned technical problems, the present invention is achieved through the following technical solution.

[0008] The first aspect of the present invention provides the use of an IP6 synthesis-associated kinase inhibitor in the preparation of a drug that improves the radiosensitivity of tumors; wherein the IP6 synthesis-associated kinase is selected from one or more of IPPK, ITPK1 and IPMK.

[0009] Preferably, the IP6 synthesis-related kinase inhibitor is selected from one or more of shRNA, sgRNA, siRNA, and small molecule inhibitors designed based on IP6 synthesis-related kinase.

[0010] Preferably, the IP6 synthesis-related kinase inhibitor is selected from one or more of IPPK-based shRNA, ITPK1-based shRNA, IPMK-based shRNA, and IPMK-based sgRNA; most preferably, the IPPK-based shRNA targeting sequence is selected from one or more of SEQ ID NO: 1 (5'-CGGCAAGATCGTCAACTATTA-3') and SEQ ID NO: 2 (5'-GCCGATTCTGTGTGTAGAGAT-3'); the ITPK1-based shRNA targeting sequence is selected from one or more of SEQ ID NO: 3 (5'-CGAGATGGCTATCGTGTTCAA-3') and SEQ ID NO: 4 (5'-CATCTTCTTCAACAGCCACAA-3'); the IPMK-based shRNA targeting sequence is selected from SEQ ID NO: 5 (5'-GCAACACAATAGATGAGGGAT-3') and SEQ ID NO: 2 (5'-GCCGATTCTGTGTGTAGAGAT-3'). NO: 6 (5'-GCAAGTTCATTACTCTTTGTT-3') or one or more of them, and the sgRNA targeting sequence designed based on IPMK is selected from SEQ ID NO: 7 (5'-GCCCGGCCACCTGATGCGAG-3').

[0011] Preferably, the small molecule inhibitor is selected from suramin and / or its derivatives; the derivatives are selected from one or more of suramin pharmaceutical salts, solvates, prodrugs, tautomers, meso compounds, racemates, enantiomers, diastereomers, or mixtures thereof.

[0012] Preferably, the tumor is selected from one or more of the following: colorectal cancer, fibrosarcoma, esophageal cancer, lung cancer, prostate cancer, breast cancer, liver cancer, pancreatic cancer, lymphoma, skin cancer, and head and neck tumors.

[0013] Preferably, the head and neck tumor is selected from one or more of oral cancer, nasopharyngeal cancer, hypopharyngeal cancer, and laryngeal cancer.

[0014] A second aspect of the present invention provides the use of a reagent for detecting the expression level of IP6 synthesis-related kinase in the preparation of products for detecting tumor radiosensitivity and / or predicting efficacy; wherein the IP6 synthesis-related kinase is selected from one or more of IPPK, ITPK1 and IPMK.

[0015] Preferably, the reagent for detecting the expression level of IP6 synthesis-related kinase includes primers for detecting the expression level of IP6 synthesis-related kinase gene and / or reagents for detecting the expression level of IP6 synthesis-related kinase protein.

[0016] Preferably, the primers for detecting the expression level of the IP6 synthesis-related kinase gene are selected from at least one of the following primer pairs:

[0017] Primer pair 1: The upstream sequence for IPPK is shown in SEQ ID NO: 8 (5'-GGTTGAGCGATACCTGGAAGAG-3'), and the downstream sequence is shown in SEQ ID NO: 9 (5'-CGTCATCCTCAGTGGAAAGGTC-3').

[0018] Primer pair 2: The upstream sequence for ITPK1 is shown in SEQ ID NO: 10 (5'-CATCCTTGAAGCCGACCAGAATG-3'), and the downstream sequence is shown in SEQ ID NO: 11 (5'-CTCATAGGACTTGGAGCGGTCA-3').

[0019] Primer pair 3: The upstream sequence for IPMK is shown in SEQ ID NO: 12 (5'-GCAACATCCAGATGGCACAGTTT-3'), and the downstream sequence is shown in SEQ ID NO: 13 (5'-GAAGAACACCATCAAAACAGTCAG-3').

[0020] Preferably, the reagent for detecting the expression level of IP6 synthesis-related kinase protein is selected from IP6 synthesis-related kinase monoclonal antibodies and / or IP6 synthesis-related kinase polyclonal antibodies; for example, Anti-IPPK (HPA020603, Sigma), Anti-ITPK1 (10568-1-AP, Proteintech), Anti-IPMK (30161-1-AP, Proteintech), etc.

[0021] Preferably, the tumor is selected from one or more of the following: colorectal cancer, fibrosarcoma, esophageal cancer, lung cancer, prostate cancer, breast cancer, liver cancer, pancreatic cancer, lymphoma, skin cancer, and head and neck tumors.

[0022] Preferably, the head and neck tumor is selected from one or more of oral cancer, nasopharyngeal cancer, hypopharyngeal cancer, and laryngeal cancer.

[0023] A third aspect of the present invention provides the use of an IP6 synthesis-associated kinase inhibitor in the preparation of a medicament that enhances the sensitivity of ferroptosis inducers to tumor therapy; wherein the IP6 synthesis-associated kinase is selected from one or more of IPPK, ITPK1, and IPMK.

[0024] Preferably, the IP6 synthesis-related kinase inhibitor is selected from one or more of shRNA, sgRNA, siRNA, and small molecule inhibitors designed based on IP6 synthesis-related kinase.

[0025] Preferably, the IP6 synthesis-related kinase inhibitor is selected from one or more of the following: IPPK-based shRNA, ITPK1-based shRNA, IPMK-based shRNA, and IPMK-based sgRNA; most preferably, the IPPK-based shRNA targeting sequence is selected from one or more of SEQ ID NO: 1 and SEQ ID NO: 2; the ITPK1-based shRNA targeting sequence is selected from one or more of SEQ ID NO: 3 and SEQ ID NO: 4; the IPMK-based shRNA targeting sequence is selected from one or more of SEQ ID NO: 5 and SEQ ID NO: 6; and the IPMK-based sgRNA targeting sequence is selected from one or more of SEQ ID NO: 7.

[0026] Preferably, the small molecule inhibitor is selected from suramin and / or its derivatives; the derivatives are selected from one or more of suramin pharmaceutical salts, solvates, prodrugs, tautomers, meso compounds, racemates, enantiomers, diastereomers, or mixtures thereof.

[0027] Preferably, the tumor is selected from one or more of the following: colorectal cancer, fibrosarcoma, esophageal cancer, lung cancer, prostate cancer, breast cancer, liver cancer, pancreatic cancer, lymphoma, skin cancer, and head and neck tumors.

[0028] Preferably, the head and neck tumor is selected from one or more of oral cancer, nasopharyngeal cancer, hypopharyngeal cancer, and laryngeal cancer.

[0029] Preferably, the ferroptosis inducer is selected from one or more of GPX4 inhibitors, System Xc-(SLC7A11) inhibitors, DHODH inhibitors, FSP1 inhibitors, and nano-carbon iron suspension injections; the GPX4 inhibitors include, but are not limited to, one or more of RSL3, ML162, ML210, Fin56, and N6F11; the System Xc-(SLC7A11) inhibitors include, but are not limited to, Erastin and sulfasalazine; the DHODH inhibitors include, but are not limited to, one or more of Emvododstat and buquina; the FSP1 inhibitors include, but are not limited to, iFSP1; and the nano-carbon iron suspension injection is selected from CNSI-Fe.

[0030] The fourth aspect of the present invention provides the use of an IP6 synthesis-related kinase inhibitor and a ferroptosis inducer in the preparation of a medicament for treating tumors and / or promoting tumor radiosensitivity; wherein the IP6 synthesis-related kinase is selected from one or more of IPPK, ITPK1 and IPMK.

[0031] Preferably, the IP6 synthesis-related kinase inhibitor is selected from one or more of shRNA, sgRNA, siRNA, and small molecule inhibitors designed based on IP6 synthesis-related kinase.

[0032] Preferably, the IP6 synthesis-related kinase inhibitor is selected from one or more of the following: IPPK-based shRNA, ITPK1-based shRNA, IPMK-based shRNA, and IPMK-based sgRNA; most preferably, the IPPK-based shRNA targeting sequence is selected from one or more of SEQ ID NO: 1 and SEQ ID NO: 2; the ITPK1-based shRNA targeting sequence is selected from one or more of SEQ ID NO: 3 and SEQ ID NO: 4; the IPMK-based shRNA targeting sequence is selected from one or more of SEQ ID NO: 5 and SEQ ID NO: 6; and the IPMK-based sgRNA targeting sequence is selected from SEQ ID NO: 7.

[0033] Preferably, the small molecule inhibitor is selected from suramin and / or its derivatives; the derivatives are selected from one or more of suramin pharmaceutical salts, solvates, prodrugs, tautomers, meso compounds, racemates, enantiomers, diastereomers, or mixtures thereof.

[0034] Preferably, the tumor is selected from one or more of the following: colorectal cancer, fibrosarcoma, esophageal cancer, lung cancer, prostate cancer, breast cancer, liver cancer, pancreatic cancer, lymphoma, skin cancer, and head and neck tumors.

[0035] Preferably, the head and neck tumor is selected from one or more of oral cancer, nasopharyngeal cancer, hypopharyngeal cancer, and laryngeal cancer.

[0036] Preferably, the ferroptosis inducer is selected from one or more of GPX4 inhibitors, System Xc-(SLC7A11) inhibitors, DHODH inhibitors, FSP1 inhibitors, and nano-carbon iron suspension injections; the GPX4 inhibitors include, but are not limited to, one or more of RSL3, ML162, ML210, Fin56, and N6F11; the System Xc-(SLC7A11) inhibitors include, but are not limited to, Erastin and sulfasalazine; the DHODH inhibitors include, but are not limited to, one or more of Emvododstat and buquina; the FSP1 inhibitors include, but are not limited to, iFSP1; and the nano-carbon iron suspension injection is selected from CNSI-Fe.

[0037] The fifth aspect of the present invention provides a pharmaceutical composition for improving the radiosensitivity of tumors, comprising an IP6 synthesis-associated kinase inhibitor and a ferroptosis inducer; wherein the IP6 synthesis-associated kinase is selected from one or more of IPPK, ITPK1 and IPMK.

[0038] Preferably, the IP6 synthesis-related kinase inhibitor is selected from one or more of shRNA, sgRNA, siRNA, and small molecule inhibitors designed based on IP6 synthesis-related kinase.

[0039] Preferably, the IP6 synthesis-related kinase inhibitor is selected from one or more of the following: IPPK-based shRNA, ITPK1-based shRNA, IPMK-based shRNA, and IPMK-based sgRNA; most preferably, the IPPK-based shRNA targeting sequence is selected from one or more of SEQ ID NO: 1 and SEQ ID NO: 2; the ITPK1-based shRNA targeting sequence is selected from one or more of SEQ ID NO: 3 and SEQ ID NO: 4; the IPMK-based shRNA targeting sequence is selected from one or more of SEQ ID NO: 5 and SEQ ID NO: 6; and the IPMK-based sgRNA targeting sequence is selected from SEQ ID NO: 7.

[0040] Preferably, the small molecule inhibitor is selected from suramin and / or its derivatives; the derivatives are selected from one or more of suramin pharmaceutical salts, solvates, prodrugs, tautomers, meso compounds, racemates, enantiomers, diastereomers, or mixtures thereof.

[0041] Preferably, the tumor is selected from one or more of the following: colorectal cancer, fibrosarcoma, esophageal cancer, lung cancer, prostate cancer, breast cancer, liver cancer, pancreatic cancer, lymphoma, skin cancer, and head and neck tumors.

[0042] Preferably, the head and neck tumor is selected from one or more of oral cancer, nasopharyngeal cancer, hypopharyngeal cancer, and laryngeal cancer.

[0043] Preferably, the ferroptosis inducer is selected from one or more of GPX4 inhibitors, System Xc-(SLC7A11) inhibitors, DHODH inhibitors, FSP1 inhibitors, and nano-carbon iron suspension injections; the GPX4 inhibitors include, but are not limited to, one or more of RSL3, ML162, ML210, Fin56, and N6F11; the System Xc-(SLC7A11) inhibitors include, but are not limited to, Erastin and sulfasalazine; the DHODH inhibitors include, but are not limited to, one or more of Emvododstat and buquina; the FSP1 inhibitors include, but are not limited to, iFSP1; and the nano-carbon iron suspension injection is selected from CNSI-Fe.

[0044] Preferably, the pharmaceutical composition may also contain a pharmaceutically acceptable carrier.

[0045] Preferably, the pharmaceutically acceptable carrier is selected from one or more of fillers, binders, lubricants, disintegrants, flavoring agents, colorants, antioxidants, antibacterial agents, chelating agents, surfactants, and solvents.

[0046] The sixth aspect of the present invention provides the use of the above-described pharmaceutical composition in the preparation of medicaments for treating tumors and / or improving the radiosensitivity of tumors.

[0047] Preferably, the tumor is selected from one or more of the following: colorectal cancer, fibrosarcoma, esophageal cancer, lung cancer, prostate cancer, breast cancer, liver cancer, pancreatic cancer, lymphoma, skin cancer, and head and neck tumors.

[0048] Preferably, the head and neck tumor is selected from one or more of oral cancer, nasopharyngeal cancer, hypopharyngeal cancer, and laryngeal cancer.

[0049] It should be understood that, unless otherwise specified, in the context of this invention, the primers and / or primer pairs refer to PCR primers used to synthesize the cDNA strand of the relevant gene in PCR, thereby detecting the expression level of the mRNA of the relevant gene (e.g., IPPK, ITPK1, and IPMK). In addition to the primers and / or primer pairs listed in this invention, those skilled in the art are fully capable of designing corresponding primers and / or primer pairs based on the sequence of the relevant gene using conventional methods and techniques in the art, including but not limited to molecular biology, and screening the designed primers and / or primer pairs using conventional experimental methods, as long as they can specifically detect the expression level of the relevant gene. The inhibitor refers to a substance that can specifically downregulate the expression level of a specific biomarker (such as IPPK, ITPK1, and IPMK) and / or the transcriptional level of its mature mRNA and / or the expression level of the biomarker protein or its kinase activity. For example, it can use antisense oligonucleotides, siRNA, shRNA, sgRNA, antagomiRs, miRNA sponges, miRNA Erasers, target masking, and / or multiple targets to downregulate the expression level of a specific biomarker, or use small molecule inhibitors to inhibit the kinase activity of the biomarker. Any substance that can reduce the level and / or activity of a specific biomarker is acceptable.

[0050] Cancer is one of the major diseases threatening public health. Radiotherapy, as a routine treatment for advanced mid-to-low rectal cancer and other cancers, has gradually matured. Therefore, based on this, identifying potential targets that may affect radiosensitivity and developing corresponding radiosensitizers can better meet the needs of new drug development in the era of personalized precision medicine. This invention starts from the practical clinical problems raised in the background above, seeking possible radiotherapy tolerance targets and potential radiosensitizers for clinical application. First, in vitro, using a library of sgRNAs targeting kinases, HT29 cells subjected to X-ray irradiation as a survival pressure were screened using CRISPR / Cas9 technology. Subsequently, high-throughput sequencing was performed to compare the degree of sgRNA copy number loss in the irradiated group to obtain candidate genes, and kinases on the IP6 anabolic pathway with significant effects were selected for further study. This study validated the function of IP6 synthesis-related kinases as radiosensitization targets: changes in IP6 protein levels were detected in radiotolerant cell lines; the effects of IPPK, ITPK1, and IPMK knockdown using shRNA on radiotolerance were examined; and the relationship between IPPK and ITPK1 expression levels and colorectal cancer prognosis was analyzed in public transcriptome databases. The study also explored the effects of targeting IP6-related kinases on radiotoxicity: the effects of IPPK or ITPK1 knockdown on radiotoxicity were compared at the cellular level; the effects of IPPK and ITPK1 knockdown on radiosensitization under different inhibitors of cell death pathways were investigated to explore the cell death pathways regulated by IPPK and ITPK1; and the effects of IPPK and ITPK1 on radiotherapy-induced lipid peroxidation and its byproducts were examined to verify the proportion of ferroptosis involved. The mechanism by which inhibiting IP6 synthesis enhances ferroptosis was clarified: the effects of IPPK and ITPK1 on ferroptosis were further verified using the ferroptosis inducer RSL3, and the changes in the content of free ferrous ions and related regulatory proteins caused by the knockdown of IPPK and ITPK1 were examined by the ferrous ion probe FerroOrange and Western blotting experiments, so as to determine the specific mechanism by which targeting IPPK and ITPK1 causes changes in ferroptosis sensitivity.

[0051] In summary, this invention establishes a cell model for screening radiotherapy tolerance targets using kinase-targeted CRISPR / Cas9 technology combined with high-throughput sequencing. The screening results reveal for the first time the link between IP6 anabolic kinases IPPK and ITPK1 and radiosensitivity. At the cellular level, the mechanism link between the function of IPPK and ITPK1 and ferroptosis is validated, providing a richer understanding of the biochemical regulatory mechanisms of ferroptosis. This has significant practical implications for addressing the challenges of inter-individual clinical efficacy differences and gaps in regression / prognostic assessment in cancers, especially colorectal cancer and fibrosarcoma, and for better achieving precision medicine. It provides a new drug therapeutic target for conquering malignant tumors, thus offering a new direction for subsequent drug development and clinical treatment, possessing extremely high social value and market application prospects. Attached Figure Description

[0052] Figure 1 This diagram illustrates the results of screening IP6 anabolic kinases as potential radiosensitizing targets. In Figure A, a volcano plot shows the significance of downregulation (-LogP) of each gene relative to the copy number fold change (LogFC). Green dots represent genes involved in the IP6 anabolic pathway, and dashed lines represent significance thresholds. Figure B shows the effective sgRNA copy number downregulation of IPPK, ITPK1, and IPMK in the irradiated group compared to the untreated group.

[0053] Figure 2 This is a schematic diagram showing the difference in survival rates between induced HT29 radiotherapy-resistant cells and parental cells under irradiation, as detected by colony formation assays; where ***p<0.001; **p<0.01.

[0054] Figure 3 This is a schematic diagram showing the results of Western blot analysis to detect the differences in the levels of IPPK, ITPK1, and IPMK in tolerant cells and parental cells.

[0055] Figure 4 This is a schematic diagram showing the results of Western blotting (WB) experiments to detect protein levels in cells after stable knockout of IPPK, ITPK1, and IPMK.

[0056] Figure 5 A schematic diagram showing the colony formation experiment and colony count results after IPMK knockout in cells.

[0057] Figure 6 A schematic diagram showing the colony count results after knocking out IPPK or ITPK1 in cells.

[0058] Figure 7 This diagram illustrates the colony formation experiment of IPPK knockdown cells under different irradiation doses and the statistical results of cell survival under different dose treatments.

[0059] Figure 8 This is a schematic diagram showing the colony formation experiment of ITPK1 knockdown cells under different irradiation doses and the statistical results of cell survival under different dose treatments.

[0060] Figure 9 The diagram shows the KM survival curve results of the relationship between IPPK and ITPK1 expression levels and the prognosis of colorectal cancer patients; in Figure A, the number of patients is n=1167, and in Figure B, the number of patients is n=1336.

[0061] Figure 10 This is a schematic diagram showing the cell viability of HT29 cells with stable IPPK and ITPK1 knockdown after 6 days of irradiation using the CCK8 assay; where ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05, ns, no significant difference.

[0062] Figure 11 A schematic diagram showing the results of Western blot analysis of 4-HNE levels in IPPK knockdown HT29 cells at 24 h and 48 h after 12 Gy irradiation.

[0063] Figure 12 The diagram shows the flow cytometry fluorescence peaks, lipid peroxidation rate in the irradiated group, and the fold change in average fluorescence intensity of HT29 cells after irradiation with a dose of 6 Gy for 48 h, as well as the results of the C11 BODIPY lipid peroxidation probe. Among them, ****p < 0.0001; **p < 0.01; *p < 0.05.

[0064] Figure 13 This is a schematic diagram showing the correlation between the expression levels of IPPK and ITPK1 in various tumor cells and the tolerance of common drugs in the drug library, analyzed by the CTRP server.

[0065] Figure 14 The diagram shows the dose-response curves of IPPK and ITPK1 knockdown HT29 cells to RSL3 in the CCK8 assay and the relative resistance index results from three experiments; where ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05.

[0066] Figure 15 The diagram shows the dose-response curves of IPPK and ITPK1 knockdown HT1080 cells to RSL3 in the CCK8 assay and the relative resistance index results from three experiments; where ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05.

[0067] Figure 16A schematic diagram of the results of SYTOX Green staining and images of dead cells in HT29 cells after treatment with RSL3 and Fer-1 for 6 hours.

[0068] Figure 17 A schematic diagram showing the results of SYTOX Green staining and images of dead cells in HT1080 cells after treatment with RSL3 and Fer-1 for 6 hours.

[0069] Figure 18 The flow cytometry fluorescence peaks of IPPK and ITPK1 knockdown HT29 cells after 4 h of RSL3 treatment are shown in the diagram. The lipid peroxidation rate of each group and the average fluorescence intensity fold change of the RSL3-treated group after three experiments are also shown. Among them, ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05.

[0070] Figure 19 The flow cytometry fluorescence peaks, lipid peroxidation rates, and average fluorescence intensity fold changes of the RSL3-treated groups after 4 h of treatment with the C11 BODIPY lipid peroxidation probe to detect IPPK and ITPK1 knockdown in HT1080 cells are shown in the diagram. The values ​​are: ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05.

[0071] Figure 20 The flow cytometry fluorescence signal peaks of IPPK and ITPK1 knocked-down HT29 cells detected by the FerroOrange ferrous ion probe are shown in the figure. The average fluorescence intensity fold change of each group after three experiments is also shown in the figure. Among them, ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05.

[0072] Figure 21 The flow cytometry fluorescence signal peaks of HT1080 cells with IPPK and ITPK1 knockdown detected by the FerroOrange ferrous ion probe are shown in the figure. The average fluorescence intensity fold change of each group after three experiments is also shown in the figure. Among them, ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05.

[0073] Figure 22 A schematic diagram showing the results of Western blotting analysis of changes in key proteins associated with ferroptosis in IPPK knockdown HT29 / HT1080 cells.

[0074] Figure 23This is a schematic diagram showing the effect of suramin and IP6 treatment on the concentration of free ferrous ions in HT29 cells using the FerroOrange ferrous ion probe (with DFO as a negative control). The left figure is a representative flow cytometry plot; the right figure is the statistical analysis of three independent experiments. The statistical method was one-way ANOVA, where *p < 0.05 and **p < 0.01.

[0075] Figure 24 This is a schematic diagram showing the effects of suramin and IP6 treatment on the concentration of free ferrous ions in HT1080 cells detected by the FerroOrange ferrous ion probe (with DFO as a negative control). The left figure is a representative flow cytometry plot; the right figure is the statistical analysis of three independent experiments. Statistical methods used were one-way ANOVA; *p<0.05, **p<0.01.

[0076] Figure 25 This is a schematic diagram of the weight growth curve results of an experimental treatment of HT29 subcutaneous xenografts with a combination of suramin and radiotherapy.

[0077] Figure 26 Photograph of the tumor mass at the endpoint of the experimental treatment of HT29 subcutaneous xenograft with suramin combined with radiotherapy.

[0078] Figure 27 This is a schematic diagram showing the comparison of tumor weight at the end of the experimental treatment of HT29 subcutaneous xenografts with a combination of suramin and radiotherapy. Statistical analysis was performed using one-way ANOVA, where **p < 0.01, ns: no significant difference. Detailed Implementation

[0079] To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0080] Unless otherwise specified, the cell lines listed in this invention, including HT29 and HT1080, were cultured according to existing techniques. All cell lines were identified by short tandem repeat analysis at the China Center for Type Culture Collection (Wuhan), and the presence of mycoplasma contamination was verified using a PCR detection kit (Shanghai Biothrive Sci). They were also cryopreserved in liquid nitrogen for subsequent experiments. All reagents used in this invention were commercially available. The experimental methods used in this invention, such as molecular biology, cell biology experiments, and animal experiments, are conventional methods and techniques in the art.

[0081] The expression levels of IPPK, ITPK1, IPMK, etc., in vitro and in vivo can be detected using conventional methods in the field (e.g., PCR, Western blot, etc.). For ease of understanding, Western blot and specific antibodies were used to detect and demonstrate the levels of IPPK, ITPK1, IPMK, etc. in this embodiment of the invention. This invention also verified the relevant primer pairs described in this invention through specific experiments (primer pair 1: upstream sequence for IPPK as shown in SEQ ID NO: 8 (5'-GGTTGAGCGATACCTGGAAGAG-3'), downstream sequence as shown in SEQ ID NO: 9 (5'-CGTCATCCTCAGTGGAAAGGTC-3'); primer pair 2: upstream sequence for ITPK1 as shown in SEQ ID NO: 10 (5'-CATCCTTGAAGCCGACCAGAATG-3'), downstream sequence as shown in SEQ ID NO: 11 (5'-CTCATAGGACTTGGAGCGGTCA-3'); primer pair 3: upstream sequence for IPMK as shown in SEQ ID NO: 9 (5'-CGTCATCCTCAGTGGAAAGGTC-3'); primer pair 3: upstream sequence for IPMK as shown in SEQ ID NO: 9 (5'-CGTCATCCTCAGTGGAAAGGTC-3'); primer pair 4: upstream sequence for IPMK as shown in SEQ ID NO: 9 (5'-CGTCATCCTCAGTGGAAAGGTC-3'); primer pair 5: upstream sequence for IPMK as shown in SEQ ID NO: 9 (5'-CGTCATCCTCAGTGGAAAGGTC-3'); primer pair 6: upstream sequence for IPMK as shown in SEQ ID NO: 9 (5'-CGTCATCCTCAGTGGAAAGGTC-3'); primer pair 7: upstream sequence for IPMK as shown in SEQ ID NO: 9 (5'-CGTCATCCTCAGTGGAGCGGTCA-3'); primer pair 8: upstream sequence for IPPK1 as shown in SEQ ID NO: 9 (5' The specificity of the detection of IPPK, ITPK1, or IPMK expression levels shown in SEQ ID NO: 12 (5'-GCAACATCCAGATGGCACAGTTT-3') and the downstream sequence shown in SEQ ID NO: 13 (5'-GAAGAACACCATCAAAACAGTCAG-3') is not repeated here, as the results are consistent with those of Western blot. For the inhibition of IPPK, ITPK1, IPMK, etc., this invention has verified the specificity of the detection of IPPK, ITPK1, or IPMK expression levels using designed shRNAs (sequences shown in SEQ ID NO: 1-SEQ ID NO: 6) / sgRNAs (sequences shown in SEQ ID NO: 7), which can specifically inhibit the expression levels of IPPK, ITPK1, or IPMK, respectively. Since the detection and inhibition of specific gene / protein expression levels are conventional methods in the art and not the main improvement direction of this invention, some detection results are not specifically presented in this invention. Those skilled in the art can perform detection and verification according to the experimental methods described in this invention or other conventional methods in the prior art as needed.In addition to the primer pairs, antibodies, and shRNA and sgRNA listed in this invention, those skilled in the art can also design relevant primer pairs / antibodies, siRNA, shRNA, and sgRNA based on the IPPK, ITPK1, or IPMK gene sequences and / or protein structures, or obtain commercially available reagents to detect or inhibit the expression levels of IPPK, ITPK1, or IPMK. Therefore, the specific information on primer pairs, antibodies, shRNA, sgRNA, etc., listed in the context of this invention does not constitute a limitation on the actual scope of protection of this invention.

[0082] Representative results from biological experiments were selected from replicates and presented in the contextual figures. Data were displayed as mean ± SD and mean ± SEM as specified in the figures. All in vitro experiments were repeated at least three times, and animal experiments at least twice. Data were analyzed using software such as GraphPad Prism 8.0 or SPSS 22.0. Standard medical statistical methods, such as t-tests, chi-square tests, and analysis of variance, were used to compare differences in means between two or more groups. p < 0.05 was considered statistically significant.

[0083] Example 1

[0084] Tumor cells possess a complex kinase regulatory network. Different kinases alter their signaling regulation and metabolic state by phosphorylating corresponding substrate molecules, thereby executing specific biological effects. Among these kinases may be important targets affecting cell survival under ionizing radiation damage; inhibiting the function of these targets holds promise for providing precise targeted sensitization effects in radiotherapy. To comprehensively identify kinases that may affect cancer cell tolerance during radiotherapy for colorectal cancer, this invention uses Dharmacon... TMThe Edit-RTM human protein kinase lentiviral sgRNA library (containing 2613 sgRNA sequences targeting 697 major kinase targets in human cells) was used as a tool for protein target knockout. To ensure that only one corresponding sgRNA was knocked out per cell, the infection index of the lentivirus and the seeded cells was controlled at 0.3 according to the user guidelines. Furthermore, to apply survival pressure and screen for important radiotherapy-regulated kinases in target cells, cells successfully transfected with the library's sgRNA after puromycin screening were subjected to 5 Gy X-ray irradiation (treatment group) and unirradiated group (control group), respectively. Seven days later, genomic DNA was extracted from all surviving cells in both groups, and nested PCR was performed using corresponding primers to amplify the sgNRA fragment sequences, followed by next-generation high-throughput sequencing. Finally, the MaGECK algorithm was used to normalize the two sets of information in the obtained sequencing data, calculate the Log FC (Logarithmic Fractional Number) of the sgRNA copy number corresponding to each kinase gene in the library between the treatment group and the control group, and rank the genes based on their significance scores. Compared to the untreated group, the more significant the loss (reduction) of sgRNA copy number in the irradiated group, the higher the likelihood of cell death caused by irradiation after knockout. Therefore, the kinase gene corresponding to this sgRNA may be a radiation tolerance factor and could be used as a target for sensitizing radiotherapy.

[0085] Based on the above principles, the statistical ranking results of the copy number decrease after HT29 cell irradiation are as follows: Figure 1 As shown in the figure. The results showed that IPPK, along with two other kinases involved in the conversion of IP3 to IP5 upstream of the IP6 anabolic pathway, ITPK1 and IPMK, ranked highly; among them, IPPK is the only kinase that uses IP5 as a substrate to synthesize IP6. A volcano plot illustrates the significance of the three kinases in the IP6 anabolic pathway in this screening. A specific analysis of the changes in the copy number of each sgRNA targeting these genes also revealed a significant decrease in the copy numbers of sgRNAs targeting IPPK (8), ITPK1 (6), and IPMK (3) in surviving cells after radiation treatment. Therefore, this invention identifies the three kinases in IP6 anabolic metabolism—IPPK, ITPK1, and IPMK—as candidate targets for radiosensitization.

[0086] The above results showed that the copy numbers of IPPK, ITPK1, and IPMK kinases decreased significantly in HT29 cells after irradiation, suggesting that targeted intervention of these kinases in the IP6 anabolic pathway may increase the radiosensitivity of HT29 cells. Therefore, to induce radiotolerant cells, this invention selected HT29 cells stably expressing m-cherry markers for in vivo experiments in nude mice. The specific steps are as follows:

[0087] (1) Take HT29 cells labeled with mCherry in the logarithmic growth phase, digest them, count and resuspend them in PBS, and then count them at a concentration of 5 × 10⁻⁶. 6 One mouse was injected subcutaneously on the right flank of a 4-week-old female BALB / c nude mouse.

[0088] (2) When the tumor volume reaches 100mm 3 Irradiation begins at the above time, with a daily dose of 1 Gy for 5 consecutive days, followed by a two-day break. Then, irradiation continues for another 5 days at the same dose until the total dose reaches 10 Gy, at which point irradiation is stopped.

[0089] (3) Continue to observe for one week. When the tumor growth rate accelerates significantly, remove the tumor, cut it into small pieces, grind it with a 200-mesh sterile sieve, wash it with PBS and resuspend it to obtain a single-cell suspension. Screen the mCherry fluorescence-positive tumor cells by flow cytometry and continue to culture to obtain radiotherapy-resistant cells.

[0090] Subsequently, the aforementioned radiotherapy-resistant cells were used for colony formation experiments. The specific steps are as follows:

[0091] (1) Add 1 mL of the required cell culture medium to the wells of a six-well plate in advance. After digesting and resuspending the cells, count them and then serially dilute them to 10,000 cells / mL. Take 100 μL and add it evenly to each well, and shake the plate along the cross to distribute the cells evenly. Seed 1,000 cells in each well.

[0092] (2) One day after plating, apply a certain dose (0, 2, 4 Gy) of ionizing radiation using an X-ray irradiator. Culture for 10-14 days depending on cell growth until a large number of single colonies of more than 50 cells are formed.

[0093] (3) Discard the culture medium before staining, rinse gently twice with PBS, add 0.5% crystal violet staining solution prepared with methanol and stain for 20 minutes, recover the staining solution and wash the six-well plate with distilled water.

[0094] (4) After staining, the six-well plate was photographed on a white light plate using a digital camera, and the number of colonies in each well was counted using ImageJ software. The cell survival rate corresponding to each well was expressed as the number of colonies in the irradiated group / the average number of colonies in the unirradiated group. The survival rate of different cells under different doses was plotted and compared using GraphPad software.

[0095] Experimental results are as follows Figure 2 As shown in the figure. The results showed that the survival rate of the radiotherapy-tolerant cells under radiation treatment was significantly higher than that of the parent cells, indicating that they have a certain degree of radiotherapy tolerance.

[0096] Furthermore, the above cellular proteins were collected and analyzed by Western blotting (WB). The specific steps are as follows:

[0097] (1) Collect cells for digestion and collection, add lysis buffer, and lyse on ice for 10 min.

[0098] (2) Centrifuge at 12000rpm for 10min at 4℃, take the supernatant and add loading buffer (to 1×), then bathe in a 100℃ metal bath for 5min.

[0099] (3) Take the sample prepared in step (2) and perform protein gel electrophoresis.

[0100] (4) After electrophoresis, take the protein gel and perform the transfer operation (PVDF membrane, 220-300mA constant current transfer for 2h).

[0101] (5) After the transfer is completed, the PVDF membrane is removed and placed in the sealing solution, and sealed at room temperature for 90 min at 5 rpm on a vertical shaker.

[0102] (6) After sealing, clean the PVDF membrane and immerse it in primary antibody (Anti-IPPK (#HPA020603, Sigma, WB=1:1000); Anti-ITPK1 (#10568-1-AP, Proteintech, WB=1:1000); Anti-IPMK (#30161-1-AP, Proteintech; WB=1:1000)) and incubate it on a vertical shaker at 5 rpm and 4℃ for 12-16 h.

[0103] (7) After the primary antibody incubation is complete, clean the PVDF membrane, then immerse it in the secondary antibody and place it on a vertical shaker at 5 rpm for 90 min at room temperature.

[0104] (8) After the secondary antibody incubation is completed, the PVDF membrane is washed and the target protein is detected using an ECL chemiluminescence kit and a chemiluminescence analyzer.

[0105] Experimental results are as follows Figure 3 As shown in the figure. The results showed that, compared with the parental cells that had not been induced to tolerate radiotherapy, the levels of IPPK and ITPK1 proteins in the radiotherapy-tolerant cell line were significantly increased, while the changes in IPMK were less significant.

[0106] The above results further suggest that the radiosensitivity of HT29 cells may be regulated by IP6 synthesis-related kinases, among which the upregulation of IPPK and ITPK1 may be related to radiotherapy tolerance in colorectal cancer.

[0107] Example 2

[0108] To verify whether the aforementioned kinases targeting the IP6 synthesis pathway have a radiosensitizing effect, shRNA or sgRNA were selected to construct stable knockdown cell lines of IPPK, ITPK1, and IPMK in HT29 cells using CRISPR-Cas9 technology, and the knockdown effect was detected by Western blot experiment. The results showed that the shRNA or sgRNA constructed in this invention could stably knock out IPPK (sh1, targeting sequence as shown in SEQ ID NO: 1, 5'-CGGCAAGATCGTCAACTATTA-3'; sh2, targeting sequence as shown in SEQ ID NO: 2, 5'-GCCGATTCTGTGTGTAGAGAT-3'), ITPK1 (sh1, targeting sequence as shown in SEQ ID NO: 3, 5'-CGAGATGGCTATCGTGTTCAA-3'; sh2, targeting sequence as shown in SEQ ID NO: 4, 5'-CATCTTCTTCAACAGCCACAA-3') and IPMK (targeting sequence as shown in SEQ ID NO: 7, 5'-GCCCGGCCACCTGATGCGAG-3'; sg1 / sg2 are two independent single-cell clones) in HT29 cells (see See Figure 4 ).

[0109] Subsequently, the stable knockout cell lines constructed above were used for cell colony formation experiments. The results showed that cells with IPMK knockout were unable to form large colonies (see [link to study]). Figure 5 This means that knocking out IPMK significantly inhibits cell proliferation, indicating that this gene is important for maintaining normal cellular physiological functions. In contrast, knocking out IPPK and ITPK1 has a weaker inhibitory effect on cell proliferation (see [link to original text]). Figure 6 It is more suitable as a target for in-depth research in the development of radiosensitizers.

[0110] To examine the sensitizing effect of different irradiation doses, cells stably knocked down with IPPK and ITPK1 were irradiated with X-rays at doses of 2, 4, and 6 Gy, respectively, in a colony formation assay. Cell viability at each dose was calculated based on the ratio of colony numbers to the unirradiated group. The results showed that, compared to the unirradiated group, both IPPK and ITPK1 knockdown significantly reduced the irradiation tolerance of HT29 cells (see [link to study]). Figure 7-8 Among them, targeted knockdown of IPPK has a significant sensitizing effect on irradiation of all doses.

[0111] To further explore the clinical significance of IPPK and ITPK1, we integrated transcriptome datasets from multiple colorectal cancer patients in public databases using the Kaplan-Meier Plotter server and plotted KM survival curves related to survival time. The specific steps are as follows:

[0112] Kaplan-Meier survival curves were plotted to analyze the correlation between IPPK and ITPK1 expression and survival rate by integrating transcriptome data from multiple databases using the Kaplan-Meier Plotter server. The IPPK analysis included 1167 patients, and the datasets used were GSE12945, GSE14333, GSE143985, GSE17538, GSE29621, GSE31595, GSE33114, GSE37892, GSE38832, GSE39582, GSE41258, and GSE92921. The analysis of ITPK1 included 1336 patients, and the dataset used included GSE12945, GSE14333, GSE143985, GSE17538, GSE29621, GSE31595, GSE33114, GSE37892, GSE38832, GSE39582, GSE41258, and GSE92921. The server URL is as follows: (https: / / kmplot.com / analysis / index.php?p=service&cancer=colon). The results showed that high expression of both IPPK and ITPK1 was positively correlated with poor prognosis in colorectal cancer (see...). Figure 9 ).

[0113] Example 3

[0114] Since the cytotoxic effect of radiotherapy is the result of multiple cell death pathways, to explore the mechanism of action of IPPK and ITPK1-sensitized radiotherapy, cells were treated with the apoptosis inhibitor Z-VAD-FMK (TargetMol, T6013), the necrosis inhibitor Necrostatin-1 (Nec-1) (TargetMol, Necrostatin-1), and the ferroptosis inhibitor Ferrostatin-1 (Fer-1) (TargetMol, Ferrostatin-1) before irradiation. Cell viability was measured and compared in each group 6 days after 6 Gy irradiation using the CCK8 assay to reflect the critical role of different cell death pathways. The results are as follows: Figure 10As shown in the figure. The results showed that, compared with the DMSO control group without any inhibitors, all three cell death inhibitors could improve cell activity after irradiation. Among them, the ferroptosis inhibitor Fer-1 could completely offset the radiosensitizing effect of IPPK and ITPK1 knockdown, while after treatment with the apoptosis inhibitor Z-VAD-FMK and the necrosis inhibitor Nec-1, the activity of the IPPK and ITPK1 knockdown group was still significantly lower than that of the non-knockdown group. This indicates that ferroptosis inhibitors can salvage the damage caused by IPPK and ITPK1 knockdown under radiotherapy, and that targeting IPPK and ITPK1 largely improves the radiosensitivity of HT29 cells by promoting irradiation-induced ferroptosis.

[0115] 4-HNE, as a byproduct of lipid peroxidation, can reflect the severity of ferroptosis to some extent. Analysis of HT29 cell protein levels 24h and 48h after irradiation (12Gy) revealed that 4-HNE gradually accumulated and peaked within 48h in the non-knockdown group, while IPPK knockdown allowed 4-HNE levels to reach levels close to those at 48h within 24h (see [link to study]). Figure 11 This indicates that IPPK knockdown significantly promotes irradiation-induced lipid peroxidation.

[0116] C11 BODIPY 581 / 591 is a lipophilic fluorescent probe for lipid peroxidation. When it enters a cell and undergoes active oxidation on the cell membrane, its emission wavelength changes from the reduced state to the oxidized state. Therefore, flow cytometry is used to detect the oxidized fluorescence signal at 525 nm, with the maximum signal value from the control group cells used as the gating baseline, which reflects the overall membrane lipid peroxidation level of the cell. Thus, the degree of membrane lipid peroxidation in HT29 cells irradiated for 48 hours was detected using the C11 BODIPY reagent. The specific steps are as follows:

[0117] (1) Sow 3×10 seeds in each hole of the 12-hole plate. 5 The cells will adhere to the wall overnight.

[0118] (2) During the test, the culture medium of each well was collected into the corresponding centrifuge tube. The cells in the well were rinsed once with 0.5 mL of HBSS and the corresponding rinsing solution was collected. Then, 0.5 mL of 0.05% trypsin containing EDTA was added to digest until the cells were observed to be completely dispersed under the microscope. The digestion was stopped with the original culture medium and collected back into the centrifuge tube.

[0119] (3) After centrifuging at 220g for 5 min, discard the supernatant, resuspend each tube with 2 mL of HBSS, centrifuge at 220g for 5 min again, and discard the supernatant.

[0120] (4) Dilute C11 BODIPY lipid peroxidation dye (Thermo Scientific) to 2 μM with HBSS, resuspend the cells in 1 mL of staining working solution in each tube, tighten the centrifuge tube cap and incubate in a 37°C incubator in the dark for 30 min.

[0121] (5) Centrifuge at 220g for 5min, discard the staining working solution, resuspend the cells in 500μL HBSS in each tube and transfer them to a new 1.5mL EP tube for flow cytometer.

[0122] Experimental results are as follows Figure 12 As shown in the figure, the results also showed that the lipid peroxidation level in the IPPK and ITPK1 knockdown groups was significantly higher than that in the control group. This indicates that the radiosensitization effect caused by knocking down both IPPK and ITPK1 depends on the occurrence of lipid peroxidation and ferroptosis, and IPPK and ITPK1 may regulate the ferroptosis sensitivity of cells.

[0123] Subsequently, analysis using the CTRP database (The Cancer Therapeutics Response Portal) revealed that drugs with a high positive correlation between tolerability and IPPK and ITPK1 expression levels were mostly ferroptosis inducers, including RSL3, ML162, ML210, and erastin (see [link to relevant documentation]). Figure 13 This further suggests that these two kinases may play an important regulatory role in ferroptosis. To this end, a series of experiments were conducted to study the effects of targeted knockout of IPPK and ITPK1 on ferroptosis sensitivity.

[0124] Specifically, cells (HT29 and HT1080) were treated with different concentrations of RSL3 for 24 h, and cell viability was detected using CCK8 reagent after the reaction. Dose-response curves were plotted, and IC50 was calculated. 50 The relative tolerance index was used to assess the relative tolerance of different cells, thereby quantifying the degree of tolerance of different cells to RSL3 toxicity. The specific steps are as follows:

[0125] (1) Add logarithmic growth phase suspension of cells to a 96-well plate, seed 4000 cells in each well, and set up 3 blank wells without cells as a background control of the culture medium. Incubate overnight in an incubator until the cells are completely attached.

[0126] (2) Prepare RSL3 solutions with a 2-fold concentration gradient (16000, 8000, 4000, 2000, 1000, 500, 250, 125, 62.5, 31.25, 0 nM) in the deep-well plate using antibiotic-free complete culture medium diluted from high to low. Set up 3 replicates for each concentration, and replace the culture medium at the corresponding cell location.

[0127] (3) After RSL3 treatment for 24 hours, cell viability was measured. 1 / 10 of CCK8 was added to serum-free medium to prepare working solution. The original drug solution was removed. 100 μL of working solution was added to each well and incubated in a 37℃ incubator in the dark for 1-4 hours. After the working solution changed color significantly, the 96-well plate was removed and the OD value at 450 nM wavelength was read using an ELISA reader.

[0128] (4) Calculate the cell viability corresponding to each well according to the following formula: (OD value of each dose-treated well - background OD value of working solution) / (average OD value of untreated well - background OD value of working solution). Use Graphpad software to draw the dose-response curve of each group of cells based on cell viability and calculate the IC50 value. The relative resistance index is the ratio of the IC50 value of knockdown cells to the IC50 value of non-knockdown control cells.

[0129] Experimental results are as follows Figure 14-15 As shown in the figure. The results showed that knocking down IPPK and ITPK1 with different shRNAs significantly reduced the cell viability of HT29 and HT1080 cells within a specific RSL3 dose range and increased the cell responsiveness to RSL3. Comparison of relative tolerance index revealed that knocking down IPPK and ITPK1 significantly reduced the relative tolerance index of both cell types to RSL3, with IPPK knockdown showing a particularly significant increase in ferroptosis-inducing cytotoxicity.

[0130] Simultaneously, HT29 and HT1080 cells were treated with 5 μM and 3 μM RSL3, respectively. The number of dead cells treated with the same concentration of RSL3 over a certain period was compared under a microscope using SYTOX Green, a nucleic acid dye sensitive to membrane permeability, to investigate the degree of ferroptosis-induced cell killing. Images obtained from fluorescence microscopy showed that knocking down both IPPK and ITPK1 significantly increased the number of cells killed by RSL3 over a certain period. Cells in the RSL3-treated group exhibited morphological changes including rounding, reduced individual cell volume, and sparser cell arrangement, but no typical nuclear dissolution and fragmentation characteristic of apoptosis, nor typical cytoplasmic swelling characteristic of necrotic cells, were observed. In the control group with the ferroptosis inhibitor Fer-1, the cell killing effect of RSL3 was completely inhibited, and the cells maintained normal morphology and structure, indicating that the large number of cell deaths in the knockdown group were due to ferroptosis caused by lipid peroxidation (see [link to relevant documentation]). Figure 16-17 The above results collectively indicate that inhibiting IPPK and ITPK1 makes cells more susceptible to ferroptosis.

[0131] Since ferroptosis is a consequence of the direct oxidation of phospholipid membrane structures by a large number of oxygen free radicals, comparing the lipid peroxidation levels among different treatment groups can reflect the strength of the effect at a biochemical level. Specifically, ferroptosis was induced in HT29 and HT1080 cells by adding 5 μM and 2 μM RSL3, respectively. The membrane lipid peroxidation level at 4 h was detected using the C11 BODIPY probe. The experimental results are as follows: Figure 18-19 As shown in the figure. The results showed that the intensity of lipid peroxidation fluorescence signals induced by IPPK and ITPK1 knockdown groups in both cell types was higher than that in the non-knockdown group within a certain time period, indicating that targeted inhibition of IPPK and ITPK1 can promote lipid peroxidation. Compared with ITPK1 knockdown, IPPK knockdown showed a more significant increase in lipid peroxidation levels during this period. In this experiment, a negative control group was set up in which the ferroptosis inhibitor Fer-1 (5 μM) and the iron chelator DFO (100 μM) were added to the inducer group. The results showed that both Fer-1 and DFO could effectively inhibit RSL3-induced lipid peroxidation and offset the difference between the IPPK / ITPK1 knockdown group and the non-knockdown group.

[0132] Example 4

[0133] During ferroptosis, lipid peroxidation begins with the release of free Fe2+ within the cell. 2+ Reacting with hydrogen peroxide to generate reactive oxygen species, DFO and other potent iron chelators can effectively prevent ferroptosis by capturing ferrous ions required for lipid peroxidation. Structurally, IP6 molecules possess high electronegativity, and the abundant phosphate groups surrounding its hexa-inositol ring facilitate the electrostatic chelation of ferrous metal ions. Furthermore, IP6 is the most abundant phosphoinositol compound in animal cells, reaching physiological concentrations of up to approximately 100 μM. Based on this, it is speculated that the large amount of IP6 molecules within cells may be due to their chelating properties, capturing excess Fe. 2+ This leads to ferroptosis becoming one of the inhibitory factors. Targeting and knocking down key kinases IPPK and ITPK1 in the IP6 synthesis pathway can directly reduce IP6 levels and decrease Fe²⁺ chelated with IP6. 2+ Indirectly increases intracellular free Fe 2+ This level makes cells more susceptible to lipid peroxidation.

[0134] To verify the chelating effect of IP6 molecules on intracellular free Fe... 2+ To mitigate the influence of the level, this invention employs the iron-specific fluorescent probe FerroOrange to quantitatively compare the free Fe levels between IPPK and ITPK1 knockdown cells and non-knockdown cells using flow cytometry. 2+ The specific steps for addressing level differences are as follows:

[0135] (1) Sow 3×10 seeds in each hole of the 12-hole plate. 5 The cells will adhere to the wall overnight.

[0136] (2) During the test, the culture medium of each well was collected into the corresponding centrifuge tube. The cells in the well were rinsed once with 0.5 mL of HBSS and the corresponding rinsing solution was collected. Then, 0.5 mL of 0.05% trypsin containing EDTA was added to digest until the cells were observed to be completely dispersed under the microscope. The digestion was stopped with the original culture medium and collected back into the centrifuge tube.

[0137] (3) After centrifuging at 220g for 5 min, discard the supernatant, resuspend each tube with 2 mL of HBSS, centrifuge at 220g for 5 min again, and discard the supernatant.

[0138] (4) Dilute FerroOrange ferrous dye (DOJINDO) to 2 μM with HBSS, resuspend cells in 1 mL of staining working solution in each tube, tighten the centrifuge tube cap, and incubate at 37°C in the dark for 30 min. Except for the untreated group, 200 μM IP6 and 100 μM DFO were added to the cells of each group for 8 h as chelating agent control groups to test the chelating effect of IP6.

[0139] (5) Centrifuge at 220g for 5min, discard the staining working solution, resuspend the cells in 500μL HBSS in each tube and transfer them to a new 1.5mL EP tube for flow cytometer.

[0140] Experimental results are as follows Figure 20-21 As shown in the figure. The results showed that, compared with the non-knockdown group, knocking down IPPK and ITPK1 could increase the amount of free Fe in cells to a certain extent. 2+ The content of Fe was similar in both HT29 and HT1080 cells. By adding 200 μM IP6, significantly higher than physiological concentrations, to the cell culture medium of each group, the detectable Fe levels in each group of cells were significantly reduced. 2+ The levels were reduced, and the differences caused by IPPK or ITPK1 knockdown were minimized. This demonstrates that intracellular targeted inhibition of IP6-related kinases can lead to increased intracellular free Fe by reducing IP6 levels. 2+ When the intracellular IP6 content increases, unstable free Fe... 2+ And so it decreased.

[0141] Transferrin receptor (TFRC) and ferritin are key effector molecules regulating cellular iron homeostasis. TFRC is responsible for transporting extracellular iron into the cell, and upregulated TFRC is currently considered a marker of ferroptosis sensitivity. 2+ When there is an excess of ferritin, it can usually be synthesized in a feedback manner to store the excess Fe. 2+This reduces ferroptosis sensitivity. Western blotting analysis of IPPK knockdown cells revealed no significant changes in TFRC and ferritin heavy chain (FTH) levels compared to the non-knockdown group (see [link to study]). Figure 22 This indicates that targeted inhibition of IPPK leads to the release of free Fe. 2+ While the level of IP6 increases, it does not significantly induce compensatory changes in iron homeostasis regulatory proteins. Therefore, in terms of iron metabolism, compared to the protein level regulation of iron homeostasis, IP6 has a more significant effect on Fe... 2+ The regulation of its content is an independent ion buffering mechanism.

[0142] During the research process, this invention discovered that the antiparasitic drug suramin can competitively inhibit the active domain of the IPPK kinase, thereby significantly reducing intracellular IP6 levels. To this end, the intracellular free Fe in HT29 and HT1080 cells after treatment with 40 μM suramin for 16 h was measured. 2+ Content, specific steps are as follows:

[0143] (1) After the tumor cells (HT29, HT1080) to be adherent to the culture vessel reached the logarithmic growth phase, they were cultured in fresh medium and divided into 4 groups: Group 1 (blank group, control) and Group 2 (negative control group, DFO) were added with equal amounts of solvent; Group 3 (suramin group) and Group 4 (suramin + IP group, suramin + IP6) were added with suramin (Sigma-Aldrich, S2671) to a final concentration of 40 μM. The cells were cultured in a 37℃, 5% CO2 cell culture incubator for 12 h.

[0144] (2) Next, equal amounts of solvent were added to groups 1 and 3, iron chelating agent DFO (TargetMol, T18971) was added to group 2 to a final concentration of 100 μM, and IP6 (MCE, HY-N0814) was added to group 4 to a final concentration of 200 μM.

[0145] (3) The cells in each group were incubated in a 37°C, 5% CO2 cell culture incubator for 4 hours. Then, they were digested into a single-cell suspension with 0.05% trypsin containing EDTA and washed twice with HBSS.

[0146] (4) Resuspend the cells in a solution of 2 μM FerroOrange ferrous dye and incubate at 37°C in the dark for 30 min. Detect the fluorescence intensity (MFI) at 585 nm using flow cytometry to reflect the Fe content of different cells. 2+ level.

[0147] The results showed that, compared with the untreated group, suramin treatment induced intracellular free Fe in cells. 2+The content increased significantly, and the addition of exogenous IP6 to the treated cell culture medium could partially reduce the effect of suramin on free Fe. 2+ Improvement of level (see) Figure 23-24 Therefore, the effect of this inhibitor is similar to that of knocking down IPPK or ITPK1, which can reduce free Fe. 2+ The amount increases, and this effect is related to the IP6 content.

[0148] Furthermore, the effect of the IPPK inhibitor suramin on radiotherapy activity was studied in vivo, and the specific steps are as follows:

[0149] (1) Take HT29 cells in good growth condition, digest them, count and resuspend them in PBS, and then add 5×10⁻⁶ cells. 6 One tumor per mouse was injected subcutaneously on the right flank of 4-week-old female BALB / c nude mice. Tumor size was measured every 3 days using calipers, calculated as 0.52 × (long diameter) × (short diameter). 2 Calculate tumor volume (mm) 3 And plot the growth curve.

[0150] (2) Starting from day 4 after inoculation with HT29 cells, when the outline of the subcutaneous tumor was clearly visible, the experimental mice were divided into 4 groups (6 mice in each group): suramin + irradiation group (suramin IR), suramin alone treatment group (suramin non-IR), irradiation alone group (PBS IR), and simulated treatment control group (PBS non-IR). Among them, the irradiation treatment was carried out at 2 Gy / day for 7 consecutive days; the suramin treatment method was based on its administration method and treatment dose in the infectious disease mouse model: intraperitoneal injection, 1 mg / dose, once every two days, for a total of 3 administrations (PBS was used as a solvent control).

[0151] (3) After the course of treatment, stop the administration and irradiation, continue to observe and record the tumor size, draw the growth curve, and sacrifice the mice on the 25th day to collect and weigh the tumor.

[0152] Experimental results are as follows Figure 25-27 As shown. The results indicated that suramin treatment alone had no significant effect on the growth of HT29 xenografts, but suramin treatment enhanced the inhibitory effect of radiation on xenograft growth (see...). Figure 25-26 According to tumor weight (see...) Figure 27 According to calculations, the tumor inhibition rate in the irradiation-only group was approximately 85.2%, while that in the suramin-combined radiotherapy group was approximately 94.6%. Suramin increased the tumor inhibition rate of radiotherapy by approximately 9.4% (the difference was statistically significant), meaning that compared to radiotherapy alone, suramin-combined treatment can further enhance the efficacy of radiotherapy.

[0153] This invention utilizes CRISPR screening of HT29 cells transfected with an sgRNA kinase library. A 5 Gy irradiation treatment group and a no-irradiation control group were used. This process excluded essential genes whose knockout in both groups negatively impacted cell survival, allowing for the screening of genes that significantly contribute to tolerance at a given irradiation dose. Both cell groups were maintained for only 7 days post-treatment without passaging before genome sequencing. This reflects the short-term effects of a single irradiation dose while minimizing the impact of gene knockout-induced proliferation inhibition or compensatory activation of related bypass pathways, thus improving the accuracy of phenotypic screening. Negative screening using scoring in HT29 cells revealed a ranking of significantly lost genes in the irradiated group. The top-ranked genes included IPPK, ITPK1, and IPMK, making this metabolic pathway a novel target for radiosensitization in this study.

[0154] From a metabolic perspective, IPPK is the only kinase that converts IP5 to IP6. ITPK1 and IPMK participate in the multi-step reaction of IP3 to IP5 conversion, and the two cooperate with each other, but the function performed by IPMK is irreplaceable. This invention found that knocking out IPMK severely inhibits the colony-forming ability of HT29 cells, verifying its indispensability for maintaining normal cellular physiological functions. Compared with ITPK1 knockdown, IPPK knockdown resulted in more significant radiosensitization, lipid peroxidation, and free iron ion accumulation effects, possibly because IPMK partially compensates for the function of ITPK1 in the metabolic pathway. By adding inhibitors of apoptosis, necrosis, and ferroptosis and comparing their effects on the degree of radiosensitization of IPPK and ITPK1 knockdown cells under radiotherapy, the results showed that only ferroptosis inhibitors could completely offset the difference in radiotherapy killing effect in the knockdown group, indicating that the radiosensitization effect of targeted inhibition of the above kinases mainly promotes ferroptosis.

[0155] IP6 exhibits strong chelating ability for divalent metal ions, including ferrous ions. In vitro studies have shown that adding IP6 can significantly inhibit lipid peroxidation catalyzed by peroxidase in intestinal extracts by mitigating the Fenton reaction. However, no studies have investigated the impact of endogenous IP6 metabolism on ferroptosis. The physiological concentration of IP6 in normal cells is approximately 10-100 μM, and its chelating effect on various ions in intracellular fluid may represent a significant chemical equilibrium. Considering the complexity of intracellular fluid composition, simply increasing IP6 concentration may easily affect calcium ions, which are more physiologically critical than iron. Therefore, this invention only investigated its effects on lipid peroxidation by inhibiting related kinases to reduce IP6 levels. The results showed that both targeted knockdown of related kinases and inhibitor treatment could lead to a certain degree of free ferrous ion accumulation, while adding exogenous IP6 could reduce this difference, indicating that unstable Fe in cells... 2+ The level was negatively correlated with the change in IP6 content.

[0156] Regardless of whether radiotherapy or ferroptosis inducers are administered, intracellular lipid peroxidation is accompanied by free Fe. 2+ The continuous increase in Fe content drives peroxidation into an irreversible chain reaction. Currently, there are no reports on in-depth studies of the process leading to ferrodegeneration. 2+ The specific reasons for the continuous accumulation. This invention discovers that knocking down IPPK and ITPK1 can lead to the release of free Fe without directly causing lipid peroxidation. 2+ Increased levels. Based on this, regardless of whether radiotherapy or RSL3 induction was applied, cells with impaired IP6 synthesis exhibited more severe lipid peroxidation and more widespread ferroptosis. This means that decreased IP6 levels lead to more Fe... 2+ In a free state available for the Fenton reaction, it can induce a more vigorous peroxidation reaction. In this free Fe... 2+ When the amount of iron increased, ferritin, which is responsible for storing iron, did not show a significant compensatory upregulation, indicating that the amount of free Fe in the intracellular fluid was low. 2+ Chelation with IP6 may be a new balance based on the regulation of iron metabolism homeostasis, while inhibiting IP6 synthesis is a feasible way to promote lipid peroxidation by increasing the level of free ferrous ions.

[0157] In summary, this invention, through extensive research, has clarified that IP6 synthesis-associated kinase is a gene highly correlated with radiotherapy tolerance. By revealing the link between IP6 synthesis-associated kinase and radiotherapy tolerance, this invention has significant practical implications for addressing the challenges of inter-individual differences in clinical efficacy and the gaps in regression / prognostic assessment, thus enabling better precision medicine. It provides a new drug therapeutic target for conquering malignant tumors, offering a new direction for subsequent drug development and clinical treatment, and possesses extremely high social value and market application prospects.

[0158] The above detailed embodiments provide a specific description of the analytical methods involved in this invention. It should be noted that the above description is only intended to help those skilled in the art better understand the methods and ideas of this invention, and is not intended to limit the scope of the invention. Without departing from the principles of this invention, those skilled in the art can make appropriate adjustments or modifications to this invention, and such adjustments and modifications should also fall within the protection scope of this invention.

Claims

1. The application of IP6 synthesis-related kinase inhibitors in the preparation of drugs to improve tumor radiosensitivity, characterized in that, The IP6 synthesis-related kinase is selected from one or more of IPPK, ITPK1, and IPMK.

2. The application according to claim 1, characterized in that, The IP6 synthesis-related kinase inhibitor is selected from one or more small molecule inhibitors of shRNA, sgRNA, and siRNA designed based on IP6 synthesis-related kinase.

3. The use of a reagent for detecting the expression level of IP6 synthesis-related kinases in the preparation of products for detecting tumor radiosensitivity and / or predicting efficacy, characterized in that, The IP6 synthesis-related kinase is selected from one or more of IPPK, ITPK1, and IPMK.

4. The application according to claim 3, characterized in that, The reagents for detecting the expression level of IP6 synthesis-related kinases include primers for detecting the expression level of IP6 synthesis-related kinase genes and / or reagents for detecting the expression level of IP6 synthesis-related kinase proteins.

5. The application of IP6 synthesis-related kinase inhibitors in the preparation of drugs that enhance the sensitivity of ferroptosis inducers to tumor therapy, characterized in that, The IP6 synthesis-related kinase is selected from one or more of IPPK, ITPK1, and IPMK.

6. The application according to claim 5, characterized in that, The IP6 synthesis-related kinase inhibitor is selected from one or more of shRNA, sgRNA, siRNA, and small molecule inhibitors designed based on IP6 synthesis-related kinase.

7. The application of IP6 synthesis-related kinase inhibitors and ferroptosis inducers in the preparation of drugs for treating tumors and / or promoting tumor radiosensitivity, characterized in that, The IP6 synthesis-related kinase is selected from one or more of IPPK, ITPK1, and IPMK.

8. The application according to claim 7, characterized in that, The IP6 synthesis-related kinase inhibitor is selected from one or more of shRNA, sgRNA, siRNA, and small molecule inhibitors designed based on IP6 synthesis-related kinase.

9. A pharmaceutical composition for improving the radiosensitivity of tumors, characterized in that, Including IP6 synthesis-related kinase inhibitors and ferroptosis inducers; the IP6 synthesis-related kinase is selected from one or more of IPPK, ITPK1 and IPMK.

10. The pharmaceutical composition according to claim 9, wherein the IP6 synthesis-related kinase inhibitor is selected from one or more of shRNA, sgRNA, siRNA, and small molecule inhibitors designed based on IP6 synthesis-related kinase.

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