Application of IRAK1 inhibitor combined with PARP inhibitor in the preparation of anti-tumor drugs

Abstract

The present invention provides an application of an IRAK1 inhibitor in combination with a PARP inhibitor in the preparation of an anti-tumor agent, and relates to the field of biomedicine technology. The present invention finds that in tumors with high expression of IRAK1, IRAK1 can promote the occurrence of NBS1 phosphorylation, thereby improving the efficiency of DNA homologous recombination repair, resulting in insensitivity to PARP inhibitor treatment; if the IRAK1 enzyme activity is inhibited, NBS1 phosphorylation will be reduced, and the homologous recombination repair efficiency will decrease. At this time, the use of a PRAP inhibitor in combination has an obvious synthetic lethal effect. The present invention has found a new treatment plan for tumors with high expression of IRAK1.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of an IRAK1 inhibitor in combination with a PARP inhibitor in the preparation of an anti-tumor medicament. Background Art

[0002] Gastric cancer (GC) is a malignant tumor that originates from gastric mucosal epithelial cells and mainly occurs in the lesser curvature of the gastric antrum and the pyloric region. The morbidity and mortality of gastric cancer are extremely high. The main reason for the high incidence of gastric cancer is the diversity of its pathogenic factors, such as Helicobacter pylori infection, high nitrate and nitrite diet, family genetic factors, chemical exposure, smoking, drinking and other factors. The main reason for the high mortality rate of gastric cancer is the limited treatment methods and effective methods for gastric cancer. At present, there are five main treatment methods for gastric cancer: surgery, chemotherapy, immunotherapy, molecular targeted therapy and radiotherapy. However, these treatment methods and means are not ideal for improving the overall survival rate of patients. Therefore, there is an urgent need to find some effective means and methods to treat gastric cancer.

[0003] In the 20th century, scientists proposed the concept of "synthetic lethality". The so-called "synthetic lethality" means that in a cell, when a gene mutates and causes loss of function, the cell will not die. If two genes mutate at the same time and cause loss of function, the cell will die. The concept of "synthetic lethality" makes it possible for drugs to specifically kill tumor cells, because the occurrence of tumors is usually caused by mutations in some genes with relatively important functions. For non-tumor cells, the gene is normal. Therefore, some drugs can be specially designed and developed to specifically kill tumor cells without obvious killing effects on normal cells. PARP inhibitors are the most representative drugs developed based on the concept of "synthetic lethality". In some tumors, such as breast cancer or ovarian cancer, there are usually BRCA1 / 2 gene mutations, which will lead to impaired homologous recombination repair. For such tumors, if PARP inhibitors are used to prevent base excision repair, the spontaneous DNA single-strand breaks in the tumor cells will become DNA double-strand breaks, and eventually die due to homologous recombination repair defects. This is the principle of synthetic lethality of PARP inhibitors. Currently, four PARP inhibitors have been approved by the FDA for the treatment of tumors such as breast cancer, ovarian cancer, pancreatic cancer and prostate cancer.

[0004] In the DNA homologous recombination repair signaling pathway, if one of the genes mutates or loses its function, homologous recombination repair will be defective. Therefore, in later studies, scientists found that PARP inhibitors have a synthetic lethal effect not only in tumors with BRCA1 / 2 mutations or other homologous recombination-related gene mutations, but also in tumors without homologous recombination repair gene mutations, as long as the function of genes related to homologous recombination repair is inhibited, PARP inhibitors can also produce a significant synthetic lethal effect on tumor cells. This has greatly expanded the scope of application of PARP inhibitors and provided new treatments for many previously difficult-to-treat tumors. Therefore, finding new drugs combined with PARP inhibitors to treat tumors has become a research hotspot.

[0005] Protein kinase inhibitors are a class of drugs that inhibit protein phosphorylation. Currently, there are 52 kinase inhibitors approved by the FDA, of which 46 are used to treat tumors, such as Sorafenib, which acts on RAF kinase, mainly used to treat renal cell carcinoma; Gefitinib, which acts on EGFR-TK kinase, mainly used to treat non-small cell lung cancer. Compared with traditional chemotherapy drugs, kinase inhibitor targeted drugs have the advantages of higher safety, less toxic side effects, and more obvious therapeutic effects. Therefore, kinase inhibitors are considered to be one of the most promising targeted drugs in tumor treatment.

[0006] Although PARP inhibitors and kinase inhibitors have achieved good therapeutic effects in tumor treatment, single kinase inhibitors or PARP inhibitors have no significant effect on the treatment of gastric cancer. Summary of the invention

[0007] The purpose of the present invention is to provide the use of an IRAK1 inhibitor in combination with a PARP inhibitor in the preparation of an anti-tumor medicament. The use of an IRAK1 inhibitor in combination with a PARP inhibitor can produce an obvious synergistic lethal effect, which is helpful for the treatment of tumors.

[0008] The present invention provides the use of an IRAK1 inhibitor combined with a PARP inhibitor in the preparation of an anti-tumor medicament.

[0009] Preferably, the IRAK1 inhibitor includes an IRAK1 / 4 inhibitor.

[0010] Preferably, the PARP inhibitor includes Olaparib or Niparib.

[0011] Preferably, the anti-tumor comprises anti-IRAK1 highly expressed tumor cells.

[0012] Preferably, the tumor cells with high expression of IRAK1 include gastric cancer cells and cervical cancer cells.

[0013] The present invention also provides a pharmaceutical composition for resisting tumor cells with high IRAK1 expression, comprising an IRAK1 inhibitor and Olaparib, or an IRAK1 inhibitor and Niparib.

[0014] Preferably, in the pharmaceutical composition, the mass ratio of the IRAK1 inhibitor to Olaparib is 5:1 to 5:6, and the mass ratio of the IRAK1 inhibitor to Niparib is 1:10.

[0015] The present invention also provides a drug for resisting tumor cells with high expression of IRAK1, comprising an IRAK1 inhibitor, Olaparib and Niparib.

[0016] Preferably, it also includes pharmaceutically acceptable excipients.

[0017] Beneficial effects: The present invention provides the use of IRAK1 inhibitors in combination with PARP inhibitors in the preparation of anti-tumor agents. In tumors with high expression of IRAK1, IRAK1 can promote the occurrence of NBS1 phosphorylation, thereby improving the efficiency of DNA homologous recombination repair, resulting in insensitivity to PARP inhibitor treatment; if IRAK1 enzyme activity is inhibited, NBS1 phosphorylation will be reduced, and the efficiency of homologous recombination repair will decrease. At this time, the use of combined PRAP inhibitors has an obvious synthetic lethal effect. The present invention has found a new treatment plan for tumors with high expression of IRAK1. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0019] Figure 1 It indicated that IRAK1 inhibitors and PARP inhibitors were screened through kinase inhibitor libraries and biological databases and showed synthetic lethality in gastric cancer cells;

[0020] Figure 2 It showed that IRAK1 / 4 inhibitors and PARP inhibitors had synthetic lethal effects in various gastric cancer cell lines;

[0021] Figure 3 It indicates that IRAK1 / 4 inhibitors and PARP inhibitors have synthetic lethal effects in HeLa cells;

[0022] Figure 4It indicates that IRAK1 inhibitors and PARP inhibitors do not have synthetic lethal effects in non-cancerous gastric epithelial cells;

[0023] Figure 5 It indicates that IRAK1 / 4 inhibitors and PARP inhibitors have synergistic lethal effects in vivo;

[0024] Figure 6 It was shown that knocking out IRAK1 would lead to mild genomic instability;

[0025] Figure 7 This indicates that knocking out IRAK1 or inhibiting its enzymatic activity impairs DNA damage repair;

[0026] Figure 8 It indicated that knockout of IRAK1 resulted in impaired RAD51 recruitment and decreased RPA2 (S4-S8) phosphorylation;

[0027] Fig. 9 This indicates that knocking out IRAK1 does not affect the recruitment of 53BP1;

[0028] Fig.10 It indicates that IRAK1 will be phosphorylated after DNA damage;

[0029] Fig.11 It indicates that whole protein phosphomic mass spectrometry revealed that IRAK1 regulated the phosphorylation of NBS1;

[0030] Fig.12 It indicates that IRAK1 accumulates in the nucleus after DNA damage;

[0031] Fig.13 This indicates that IRAK1 regulates the phosphorylation of NBS1;

[0032] Fig.14 This indicates that IRAK1 does not affect the expression and localization distribution of NBS1 at the protein level;

[0033] Fig.15 This indicates that IRAK1 interacts with NBS1, and the interaction between phosphorylated IRAK1 and NBS1 is stronger;

[0034] Fig.16 Schematic diagram showing how IRAK1 regulates sensitivity to PARP inhibitors. DETAILED DESCRIPTION

[0035] The present invention provides an application of an IRAK1 inhibitor combined with a PARP inhibitor in the preparation of an anti-tumor medicament.

[0036] The IRAK1 inhibitor of the present invention preferably includes an IRAK1 / 4 inhibitor. In the present invention, since there is currently no selective inhibitor of IRAK1, only a selective inhibitor of IRAK4 and an IRAK1 / 4 inhibitor that can simultaneously inhibit the enzyme activity of IRAK1 and IRAK4, an IRAK1 / 4 inhibitor is used in the embodiment of the present invention for combined use with a PARP inhibitor to produce a synthetic lethal effect. In the embodiment of the present invention, an inhibitor specific to IRAK4 is used in combination with Olaparib, but it is found that there is no synthetic lethal effect between the IRAK4 inhibitor and Olaparib, indicating that the synthetic lethal effect of the IRAK1 / 4 inhibitor and the PARP inhibitor is mainly caused by the inhibition of the enzyme activity of IRAK1, so IRAK1 can be used as a potential target for the treatment of gastric cancer.

[0037] The PARP inhibitor of the present invention includes Olaparib or Niraparib. The present invention does not specifically limit the sources of the IRAK1 inhibitor and the PARP inhibitor, and conventional commercial reagents in the art can be used.

[0038] The anti-tumor agents of the present invention preferably include tumor cells with high expression of IRAK1, more preferably gastric cancer cells and cervical cancer cells. For example, in the embodiments of the present invention, various gastric cancer cell lines, such as SGC7901, SNU1 and MKN45, were used to confirm that both IRAK1 / 4 inhibitors and Olaparib had synthetic lethal effects; at the same time, experiments were also conducted using cervical cancer cell line HeLa cells, and it was found that the sensitivity of Olaparib was enhanced regardless of whether IRAK1 was knocked out or IRAK1 enzyme activity was inhibited, confirming that in non-gastric cancer cell lines with high expression of IRAK1, IRAK1 / 4 inhibitors and Olaparib also had synthetic lethal effects; at the same time, IRAK1 / 4 inhibitors and Niraparib also had similar synthetic lethal effects.

[0039] The present invention also provides a pharmaceutical composition for resisting tumor cells with high IRAK1 expression, comprising an IRAK1 inhibitor and Olaparib, or an IRAK1 inhibitor and Niraparib.

[0040] In the present invention, since there is no selective inhibitor of IRAK1, the so-called IRAK1 inhibitor actually refers to an IRAK1 / 4 inhibitor, and in the pharmaceutical composition, the mass ratio of the IRAK1 / 4 inhibitor to Olaparib is preferably 5:1 to 5:6, and the mass ratio of the IRAK1 inhibitor to Niparib is preferably 1:10.

[0041] The present invention also provides a drug for resisting tumor cells with high expression of IRAK1, comprising an IRAK1 inhibitor and Olaparib, or an IRAK1 inhibitor and Niraparib.

[0042] The medicine of the present invention preferably also includes a pharmaceutically acceptable excipient.

[0043] In tumors with high expression of IRAK1, IRAK1 can promote the phosphorylation of NBS1, thereby increasing the efficiency of DNA homologous recombination repair, resulting in insensitivity to PARP inhibitor treatment; if the IRAK1 enzyme activity is inhibited, NBS1 phosphorylation will be reduced and the efficiency of homologous recombination repair will be reduced. At this time, the combined use of PRAP inhibitors has a significant synthetic lethal effect.

[0044] To further illustrate the present invention, the application of the IRAK1 inhibitor combined with the PARP inhibitor provided by the present invention in the preparation of an anti-tumor agent is described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0045] In the examples of the present invention, the reagents and consumables used were conventionally obtained unless otherwise specified:

[0046] 1. Cell lines, plasmids, strains, and experimental animals

[0047] Cell lines: HEK293T cells and HeLa cells were cultured in our laboratory. The gastric cancer cell line SGC7901 was donated by Professor Han Wenling of the Department of Immunology, Peking University. The gastric cancer cell lines MGC803, SNU1, MKN45 and the non-cancerous gastric epithelial cell line GES-1 were donated by the laboratory of Peking University Cancer Hospital.

[0048] Plasmids: The SFB / MYC / GST-tagged plasmids used in this study were constructed by the Gateway system.

[0049] Strains: The competent E. coli TOP10, BL21 and DH5α used in this study were prepared and maintained in our laboratory.

[0050] Experimental animals: The experimental mice used in this study were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. The experimental animals were kept in a clean or SPF environment, and all animal experiments complied with the regulations of the Ethics Committee of Peking University Health Science Center.

[0051] 2. The antibodies used in the present invention are shown in Table 1:

[0052] Table 1 Antibodies used in the present invention

[0053]

[0054]

[0055] 3. PCR Primers

[0056] The primers used in the present invention are shown in Table 2:

[0057] Table 2 PCR primers used in the present invention

[0058]

[0059]

[0060] In the embodiments of the present invention, each experiment was repeated three times or more, and the experimental data were analyzed using a t-test. When p < 0.05, it was considered statistically significant. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001.

[0061] Example 1

[0062] 1. Experiment and Methods

[0063] 1. Plasmid construction

[0064] The plasmids of SFB tag, Myc tag and GST tag used in the present invention were constructed by Gateway cloning system. TM BP Clonase TM Enzyme Mix (Cat. No. 11789013) and Gateway TM LR Clonase TM The enzyme mixture (Cat. No.: 11791043) was operated according to the instructions of the kit.

[0065] 2. Plasmid extraction

[0066] Plasmid small-scale and large-scale extractions were performed according to the kit instructions of Tiangen Biotechnology Company.

[0067] 3. Cell culture, recovery and cryopreservation

[0068] 1) Cell recovery and culture

[0069] Take out the cells from liquid nitrogen or -80℃ freezer, thaw in 37℃ water bath, centrifuge, add culture medium to resuspend the cells, then transfer to culture dish, culture in 37℃ constant temperature incubator, subculture every 36-48h. HEK293T cells and HeLa cells use DMEM culture medium, gastric cancer cell lines use 1640 culture medium.

[0070] 2) Cell cryopreservation

[0071] Verse or sterile PBS, trypsinize, add trypsin to the culture medium, pipette and transfer the cells to a centrifuge tube, centrifuge, discard the supernatant, add cell freezing solution to resuspend the cells and transfer to a cryopreservation tube. Place at 4℃ for 30min, -20℃ for 2h, -80℃ for 24-48h, and freeze in liquid nitrogen for long term.

[0072] 4. Plasmid transfection

[0073] Inoculate cells and transfect when the cell confluence reaches 60% to 80%; prepare the transfection system according to the size of the culture dish. For example, taking a 6cm cell culture dish as an example, add 500μL of OPTI-MEM culture medium to the EP tube, then add 5μg of plasmid, mix gently, then add 15μL of transfection reagent, mix thoroughly and let stand for 15 minutes, add the mixture of plasmid DNA and transfection reagent dropwise to the cell culture dish, mix gently, and continue to culture in the incubator. Replace the new culture medium 4 to 6 hours after transfection, and collect the cells 36 to 48 hours after transfection for subsequent experiments.

[0074] 5. Preparation of SFB tag stable expression cell lines

[0075] HEK293T cells were plated, and the plasmid with SFB tag was transfected when the cell growth confluence reached 60% to 80%. The medium was changed 4 to 6 hours after transfection, and the culture medium with a final concentration of 2 μg / ml puromycin was added 36 to 48 hours later. After culturing for 6 to 8 days, density gradient dilution was performed to dilute the cells to a final concentration of 1 cell / 100 μL. The diluted cells were transferred to 96-well plates for culture. When the cells grew to a certain density, they were transferred to 24-well plates, 12-well plates, 6-well plates and 10 cm culture dishes in turn for growth, and the stably expressing cell lines were identified by Western blot and immunofluorescence.

[0076] 6. Construction of CRISPR-Cas9 vector and preparation of gene knockout cells

[0077] 1) The gRNAs of the IRAK1 gene (Table 2) were scored using the http: / / tools.genome-engineering.org website, and the two gRNAs with the highest scores and the lowest off-target rates were screened out.

[0078] 2) Synthesize primers and construct plasmids.

[0079] 3) After sequencing is correct, perform plasmid extraction, transfection, puromycin selection, gradient dilution to obtain monoclonal cell lines, and confirm the gene knockout cell lines through Western blot and T-vector ligation sequencing.

[0080] 7. Western blot

[0081] Collect cells, centrifuge at 400g for 5 min at 4°C, discard the supernatant, and wash the cells once with pre-cooled PBS; discard the supernatant, add cell lysis buffer RIPA or NETN and protease inhibitors to the cell pellet, mix thoroughly, and rotate on a turntable at 4°C for 20 min; centrifuge at 13400g for 10 min at 4°C, aspirate the supernatant, quantify the total cell protein using the Broadford method, add SDS loading buffer to the protein sample, and heat at 100°C for 10 min; perform electrophoresis, transfer to the membrane, and block with 5% skim milk or 5% BSA at room temperature for 1 h; add primary antibody and incubate at 4°C overnight; wash the membrane, add secondary antibody, incubate at room temperature for 1 h, wash the membrane, expose to light, and analyze the experimental results.

[0082] 8. Co-immunoprecipitation

[0083] 1) HEK293T cells were plated and transfected with two plasmids with different labels when the cell confluence reached 60% to 80%. The medium was changed 4 to 6 hours after transfection and the cells were collected 36 to 48 hours later.

[0084] 2) Collect the cells, centrifuge at 400g for 5 min at 4°C, discard the supernatant, and wash the cells once with PBS;

[0085] 3) Add 600-800 μL NETN lysis buffer and lyse on a rotating disk at 4°C for 20 min;

[0086] 4) Centrifuge at 13400 g for 10 min at 4°C;

[0087] 5) Transfer the supernatant to a new EP tube, mix well, take 40-50 μL as input, add SDS protein loading buffer, and heat at 100°C for 5-10 min; add 20-30 μL S-protein Agarose to the remaining sample and incubate on a rotating disk at 4°C for 2-3 h;

[0088] 6) Centrifuge at 400 g for 1 min at 4°C, discard the supernatant, add pre-cooled NETN lysis buffer to wash S-beads 3 to 5 times, and discard the supernatant;

[0089] 8) Add 20 μL 2× SDS protein loading buffer and heat at 100°C for 10 min;

[0090] 9) Electrophoresis, transfer, blocking with 5% skim milk at room temperature for 1 hour, incubation with primary antibody at 4°C overnight; washing the membrane three times with TBST, 10 minutes each time, incubation with HRP-labeled secondary antibody at room temperature for 1 hour, washing the membrane three times with TBST, exposure, and analysis of the results.

[0091] 9. Tandem affinity purification coupled to mass spectrometry analysis

[0092] 1) Select cells with the best stable expression status, expand to 20-30 plates, irradiate with IR or add 10 μmol / L CPT for 4-6 hours, collect cells, centrifuge, wash cells once with PBS, and discard the supernatant;

[0093] 2) Add 12-15 ml NETN to lyse the cells, lyse at 4°C for 20 min, centrifuge, transfer the supernatant (soluble) to a centrifuge tube, add 100 μL Strepavidin-sepharose, and incubate at 4°C on a rotating disk for 2-4 h;

[0094] 3) Transfer the precipitate (chromatin) to an EP tube and wash with PBS 2 to 3 times;

[0095] 4) Add Nuclease, sonicate, incubate in a 37°C water bath for 5 min, centrifuge, collect the supernatant in an EP tube, add 50 μL Strepavidin-sepharose, and incubate on a 4°C turntable for 2-4 h;

[0096] 5) Centrifuge, discard the supernatant, wash NETN four times, add Biotin at a final concentration of 5 mg / ml to competitively bind to the protein on Strepavidin-sepharose, and incubate at 4°C overnight;

[0097] 6) Centrifuge, add 60 μL S-beads to the supernatant, and allow to bind for 3 to 4 hours;

[0098] 7) Centrifuge and wash S-beads with NETN 4 to 6 times, discard the supernatant; add 25 μL 2× loading buffer and heat at 100°C for 10 min;

[0099] 8) Electrophoresis, Coomassie Brilliant Blue staining, decolorization, photography, cutting out the target band, and mass spectrometry detection.

[0100] 10. Immunofluorescence experiment

[0101] 1) Add coverslips to the cell culture dish, plate the cells, and perform corresponding treatments after the cells adhere to the wall;

[0102] 2) Discard the culture medium, wash the cells with PBS, and fix them with paraformaldehyde for 15 min;

[0103] 3) Wash the cells with PBS and treat with 0.25% Triton-X100 perforation solution for 5 min;

[0104] 4) Wash the cells with PBS and block with 2% BSA for 5 min at room temperature;

[0105] 5) Add primary antibody and incubate at room temperature for 1 hour; wash cells with PBS, add secondary antibody and incubate at room temperature in the dark for 30 minutes;

[0106] 7) Wash the cells with PBS, stain the nucleus with DAPI, seal the slides, and analyze the experimental results under a confocal microscope.

[0107] 11. Comet Experiment

[0108] This study used the alkaline comet assay, and the experimental steps are as follows:

[0109] 1) Weigh low melting point agarose and dissolve it in PBS (W / V 1%), then dissolve it in a 65℃ water bath;

[0110] 2) Weigh agarose with normal melting point and dissolve it in PBS (W / V is 0.5%), heat it in a microwave oven to dissolve, take 180 μL of the dissolved agarose solution and pour it evenly on the glass slide, cover it with a glass slide, and wait for the agarose gel to solidify;

[0111] 3) Digest the cells and make a single-cell suspension. Remove the coverslip in step 2 and mix the single-cell suspension with an equal volume of 1% LMA (Low Melting Agarose). After pipetting evenly, quickly pipette 180 μL of the mixture and drop it onto the first layer of agarose. Add a coverslip and wait for the agarose gel to solidify.

[0112] 4) Place the gel slide in a dish, remove the cover glass, and add pre-cooled cell lysis buffer (2.5 mol / L NaCl, 0.1 mol / L EDTA- 2 Na, 10mmol / L Tris-HCl, pH 10.0, 10% DMSO, 1% Triton -X100), lysed at 4°C for 2h or overnight;

[0113] 5) Take out the slide and wash it with double distilled water; add electrophoresis buffer (300mmol / L NaOH, 1mmol / L EDTA- 2 Na, pH 13.0) unwind for 0.5-1h;

[0114] 6) Electrophoresis at 20 V, 200 mA in an ice bath for 30 min; neutralization with neutralizing solution (0.4 mol / L Tris-HCl, pH 7.50); ​​dropwise addition of 100 μL PI for staining in the dark for 10 min; washing with PBS, and analysis of the results under a fluorescence microscope.

[0115] 12. Flow cytometry for cell cycle detection

[0116] 1) Digest the cells with trypsin, centrifuge, and wash with PBS; resuspend the cells in 250 μL PBS, add 750 μL pre-cooled anhydrous ethanol dropwise, mix gently by pipetting, and place at 4°C to fix overnight;

[0117] 3) Centrifuge, discard the supernatant, add 1 ml of pre-cooled PBS to resuspend the cells, and centrifuge at 4°C, 500g for 5 min;

[0118] 4) Discard the supernatant, add 120 μL pre-cooled PBS, 80 μL 0.25% Triton-X100 punching solution and 100 μg / ml RNase, resuspend the cells, and incubate in a 37°C water bath for 30 min;

[0119] 5) Add PI to a final concentration of 50 μg / ml and incubate at room temperature in the dark for 30 min;

[0120] 6) Add pre-cooled PBS to resuspend the cells, filter through a 300-mesh sieve, and analyze the results on a flow cytometer.

[0121] 13. Clone formation experiment

[0122] 1) Trypsinize cells, centrifuge at room temperature;

[0123] 2) Add fresh culture medium, resuspend the cells, count, and serially dilute to a cell density of 1000 cells / ml;

[0124] 3) Plating: add 1 ml of the gradient diluted cells to each 6 cm culture dish, then add 3 ml of fresh culture medium, shake thoroughly to make the cells evenly distributed, and culture for 8 to 12 hours;

[0125] 4) Perform corresponding treatments, such as IR irradiation, CPT and PARP inhibitors and other drug treatments, replace fresh culture medium every 3-4 days, and culture for 9-15 days; if combined drug treatment is used, add kinase inhibitors 24 hours in advance, then add PARP inhibitors for 24 hours, wash cells three times with PBS, then add culture medium containing kinase inhibitors for 48 hours, wash cells three times with PBS, and add fresh culture medium to continue culturing for 7-11 days;

[0126] 5) Discard the culture medium, stain with Coomassie Brilliant Blue for 30 min, decolorize with water, air dry, take photos and count the number of clones. Relative survival rate = number of experimental cell clones in the treatment group / number of experimental cell clones in the non-treatment group × 100%.

[0127] 14. Ionizing Radiation Experiment

[0128] Plate the cells 8 to 12 hours in advance, place the cells in an X-ray irradiator, set the radiation dose, and irradiate. After irradiation, collect the cells or continue culturing for the required time according to the experimental purpose.

[0129] 15. CCK8 (Cell Counting Kit-8) screening of kinase inhibitors and PARP inhibitors for synthetic lethality experiments

[0130] 1) Digest the cells and centrifuge; discard the supernatant, add culture medium to resuspend the cells, and count;

[0131] 2) Dilute the cells to 2×10 4 / ml; divided into four groups, added with 5μmol / L Olaparib, 10μmol / L kinase inhibitor, 5μmol / L Olaparib + 10μmol / L kinase inhibitor and control group, the control group was added with an equal volume of DMSO;

[0132] 3) Add 200 μL of PBS to the edge wells of the 96-well plate, add 100 μL of diluted cells to the center well, and plate six replicate wells for each group of cells. Three replicate wells are used as the OD measurement value at the 0h time point, and three replicate wells are used as the OD measurement value at the 96h time point;

[0133] 4) Add 10 μL CCK8; culture for 2 h, and measure OD 450 The absorbance value is taken as the measurement value at the 0h time point;

[0134] 5) The remaining cells were cultured for 96 h, then 10 μL CCK8 was added, incubated for 2 h, and the OD was measured. 450 The absorbance value was used to analyze the results of synthetic lethality.

[0135] 16. Whole-protein phosphorylation mass spectrometry analysis

[0136] After HeLa cells and HeLa-IRAK1-KO cells were treated with radiation / Olaparib / CPT, the cells were collected in cryotubes, snap-frozen in liquid nitrogen, and then transferred to dry ice and sent to Hangzhou Jingjie Biotechnology Co., Ltd. for whole-protein phosphorylation mass spectrometry analysis.

[0137] 17. Proximity ligation assay (PLA)

[0138] The operation was performed according to the instructions of Duolink In Situ PLA (Cat. No. DUO92001) kit.

[0139] 2. Results and Analysis

[0140] 1. Through the kinase inhibitor library and biological database, IRAK1 inhibitors and PARP inhibitors were screened to have synthetic lethal effects in gastric cancer cells

[0141] The PARP inhibitor Olaparib was used to screen the combination of 240 kinase inhibitors in the gastric cancer cell line MGC803. Figure 1A). Screening results showed that multiple kinase inhibitors have synthetic lethal effects with Olaparib ( Figure 1 In Figure B), the top ten kinase inhibitors in terms of synthetic lethality are Src inhibitors, ULK1 inhibitors, IGF-1R inhibitors, ATM inhibitors, ERK inhibitors, PKD1 inhibitors, PAK4 inhibitors, ROCK1 inhibitors, IRAK1 / 4 inhibitors and MTOR inhibitors.

[0142] In order to distinguish whether it is IRAK1 enzyme activity or IRAK4 enzyme activity or both that play a role, and thus have a synthetic lethal effect with PARP inhibitors, IRAK4-specific inhibitors were used in combination with Olaparib, and it was found that there was no synthetic lethal effect between IRAK4 inhibitors and Olaparib ( Figure 1 This indicates that the synthetic lethality of IRAK1 / 4 inhibitors and PARP inhibitors is mainly due to the inhibition of IRAK1 enzyme activity.

[0143] The five candidate genes were further screened through biological databases to confirm the final candidates. First, the five candidate genes were analyzed through the human proteomics database, and it was found that in immunohistochemical staining, the expression difference of IRAK1 protein in normal gastric tissue and gastric cancer tissue was the largest, and the expression level was also the highest; while the expression of PKD1 and MTOR in normal gastric tissue was higher than that in gastric cancer tissue; the expression difference of PAK4 and ROCK1 in normal gastric tissue and gastric cancer was not much ( Figure 1 (D, E) Based on the screening results of the kinase inhibitor library and the biological database, the present invention believes that IRAK1 can be used as a potential target for gastric cancer treatment.

[0144] 2. IRAK1 inhibitors and PARP inhibitors have synthetic lethal effects in various gastric cancer cells

[0145] Experimental exploration was conducted in gastric cancer cell lines SGC7901, SNU1 and MKN45. The experimental results showed that IRAK1 / 4 inhibitors and Olaparib both had synthetic lethal effects in gastric cancer cell lines SGC7901, SNU1 and MKN45 ( Figure 2 These experimental results exclude cell-specific effects and indicate that IRAK1 / 4 inhibitors and Olaparib have synthetic lethal effects in a variety of gastric cancer cells.

[0146] Since CCK8 experiments mainly observe the proliferation state of cells in a short period of time, in order to further explore the long-term synergistic lethal effect of IRAK1 / 4 inhibitors and PARP inhibitors in vitro, the present invention conducted a monoclonal formation experiment in cells SGC7901 and MGC803. The experimental results suggest that in gastric cancer cells SGC7901, IRAK1 / 4 inhibitors and Olaparib have a very obvious synthetic lethal effect ( Figure 2 (B, C).

[0147] In order to exclude the synergistic lethal effect caused by the off-target effect of IRAK1 / 4 inhibitors, IRAK1 KO cells were prepared by CRISPR-Cas9 technology ( Figure 3 In IRAK1 KO cells, the addition of Olaparib also has a significant lethal effect ( Figure 2 DF). It is proved that knocking out IRAK1 or inhibiting its enzyme activity can enhance the sensitivity of Olaparib and produce a good synthetic lethal effect. Similar experimental results were also found in gastric cancer cells MGC803, that is, IRAK1 / 4 inhibitors and Olaparib have a good synergistic lethal effect ( Figure 2 (G, H).

[0148] 3. IRAK1 inhibitors and PARP inhibitors also have synthetic lethal effects in other tumor cells with high expression of IRAK1

[0149] The same experiment was also performed in HeLa cells, and the results showed that the sensitivity of Olaparib increased regardless of whether IRAK1 was knocked out or IRAK1 enzyme activity was inhibited ( Figure 3 (A, B).

[0150] The alkaline comet assay was used to observe the damage to cells caused by the combination of the two. The results showed that in HeLa cells, when the IRAK1 / 4 inhibitor was used alone, the cells did not produce obvious tailing; when Olaparib was used alone, the cells produced a certain amount of tailing; when the two were used in combination, the cells produced obvious tailing ( Figure 3 Middle C) confirmed that the combination of IRAK1 / 4 inhibitors and PARP inhibitors caused more severe DNA damage in cells.

[0151] In addition, knocking out IRAK1 will also cause cells to produce a certain amount of tailing, which indicates that knocking out IRAK1 may cause a certain amount of spontaneous DNA damage. After adding Olaparib treatment to IRAK1 KO cells, the tailing phenomenon of the comet assay showed a significant increase.

[0152] 4. IRAK1 / 4 inhibitors and PARP inhibitors do not have synthetic lethal effects in non-cancerous gastric epithelial cells

[0153] The non-cancerous gastric epithelial cell line GES-1 was selected for monoclonal formation experiments. The experimental results showed that in GES-1 cells, the combination of IRAK1 / 4 inhibitors and Olaparib did not produce a significant synergistic lethal effect ( Figure 4 A and B in the figure indicate that the combination of IRAK1 / 4 inhibitors and PARP inhibitors only selectively kills tumor cells, while the killing effect on non-tumor cells is not enhanced by the combination. In order to further explore whether the combination of IRAK1 / 4 inhibitors and Olaparib will cause DNA damage to cells in the short term, alkaline comet electrophoresis experiments were performed. The experimental results showed that when IRAK1 / 4 inhibitors were combined with Olaparib, the tail length produced by cells was not stronger than the tail length produced by using a single drug ( Figure 4 This indicates that the combination of IRAK1 / 4 inhibitors and Olaparib does not produce obvious toxic side effects on non-tumor cells, and the toxic side effects of the two inhibitors are not stronger than those of a single drug. This suggests that the combination of IRAK1 / 4 inhibitors and Olaparib can be used as one of the candidate treatments for tumors with high IRAK1 expression.

[0154] 5. Nude mouse tumor formation experiments confirmed that IRAK1 / 4 inhibitors and PARP inhibitors have synergistic lethal effects in vivo

[0155] Niraparib is another PARP inhibitor approved by the FDA for marketing. It has a stronger effect on inhibiting the activity of PARP family enzymes. We used Niraparib inhibitor in in vivo animal experiments. We first inoculated gastric cancer cells MGC803 subcutaneously in Balb / c nude mice. When the tumor grew to a certain size, the mice were randomly divided into 4 groups according to the tumor size and mouse weight: control group, IRAK1 / 4 inhibitor group (5 mg / kg), Niraparib (50 mg / kg) group and IRAK1 / 4 inhibitor (5 mg / kg) + Niraparib (50 mg / kg) group. The control group was given DMSO, and the experimental groups were given IRAK1 inhibitor, Niraparib and the combination of the two drugs, respectively. The drugs were intraperitoneally administered once every two days. The size of the tumor and the changes in the weight of the mice were measured at the same time. After the tumor grew to a certain size, the mice were killed, the tumors were removed, and data analysis was performed.

[0156] The experimental results showed that after a certain period of drug treatment, the tumor volume of the Niraparib group was reduced compared with the control group, and the tumor volume of the IRAK1 / 4 inhibitor group was also reduced, while the tumor volume of the IRAK1 inhibitor and Niraparib combination group was significantly reduced ( Figure 5 In addition, the tumor weight in the drug combination group was significantly smaller than that in the control group, IRAK1 / 4 inhibitor group, and Niraparib group ( Figure 5 During the entire treatment period, there was no significant difference in the weight changes of mice in the control group, IRAK1 / 4 inhibitor group, Niraparib group, and drug combination group ( Figure 5 This indicates that IRAK1 / 4 inhibitors, Niraparib, or the combination of the two drugs have no obvious toxic side effects on mice, suggesting that IRAK1 inhibitors and PARP inhibitors can be used as candidate drugs for the treatment of gastric cancer.

[0157] 6. Knockout of IRAK1 leads to mild genomic instability

[0158] When the alkaline comet electrophoresis experiment was used to observe the DNA damage of cells after drug treatment, it was found that compared with the control group, the cells that knocked out IRAK1 in HeLa cells and SGC7901 cells could produce obvious tailing ( Figure 6 This indicates that knocking out IRAK1 will cause some spontaneous DNA damage in cells.

[0159] The changes of γ-H2AX at the protein level were examined and it was found that after knocking out IRAK1, γ-H2AX increased to a certain extent at the protein level ( Figure 6 C), which further confirmed that knocking out IRAK1 would cause cells to have slight spontaneous genomic instability. In the experiment, after knocking out IRAK1, compared with the control group cells, the number of micronuclei in the KO group cells increased, and a very small number of cells also had mitotic disorders, inconsistent nuclear size or multinucleation ( Figure 6 These experimental results suggest that IRAK1 may play some previously unreported functions in maintaining genome stability.

[0160] 7. Knocking out IRAK1 or inhibiting its enzyme activity will lead to impaired cellular DNA repair

[0161] When DNA is damaged, damage repair proteins will be recruited to the DNA breaks. If immunofluorescence staining is performed using antibodies against the corresponding proteins, aggregation of fluorescent bright spots can be observed in the cell nucleus. These fluorescent bright spots are called Foci. Therefore, Foci represent the recruitment of damage repair proteins at the damage site.

[0162] Based on the previous experimental results, it can be seen that knocking out IRAK1 will lead to genomic instability, and γ-H2AX will increase at the protein level, which suggests that IRAK1 may play an important role in DNA damage repair. Next, the present invention further explores it through experiments. First, HeLa cells were divided into three groups: WT group, IRAK1 / 4i group and IRAK1-KO group, and 3Gy radiation (IR) treatment was given to each group. Then the cells were allowed to recover for 24 hours, and then the changes in γ-H2AX foci were detected by immunofluorescence to explore whether knocking out IRAK1 or inhibiting its enzyme activity would affect DNA repair. The experimental results showed that knocking out IRAK1 or inhibiting its enzyme activity would significantly increase γ-H2AX foci ( Figure 7 A and B in the middle). Flow cytometry was also used to detect changes in the cell cycle. The experimental results showed that there was no significant change in the cell cycle after knocking out IRAK1 ( Figure 7 This suggests that the changes in γ-H2AX foci are not caused by the cell cycle, but are caused by impaired DNA repair after knocking out IRAK1 or inhibiting its enzymatic activity.

[0163] 8. Knockout of IRAK1 results in impaired RAD51 recruitment and reduced RPA2 (S4-S8) phosphorylation

[0164] Detect more foci changes of DNA damage repair-related proteins. WT group cells and IRAK1-KO group cells were treated with 10Gy of radiation (IR), and the cells were allowed to recover for 3 hours. Then, the changes of DNA damage repair-related protein foci were detected by immunofluorescence. It was found that the recruitment of RAD51 was significantly impaired after knocking out IRAK1 ( Figure 8 A and B), and the changes in the cell cycle were also detected, and it was found that knocking out IRAK1 had no significant effect on the cell cycle ( Figure 8 C), which indicates that the impaired recruitment of RAD51 foci is not caused by the cell cycle, but by the knockout of IRAK1. In addition, immunofluorescence and Western blot showed that the phosphorylation of RPA2 (S4-S8) was significantly weakened after the knockout of IRAK1 ( Figure 8 These experimental results demonstrate that knocking out IRAK1 or inhibiting its enzymatic activity leads to impaired DNA repair, which is mainly caused by RAD51-mediated homologous recombination repair.

[0165] 9. Knockout of IRAK1 does not affect the recruitment of 53BP1

[0166] The recruitment changes of 53BP1 foci were detected under the condition of radiation treatment. Through immunofluorescence experiments, it was found that knocking out IRAK1 had no obvious effect on the recruitment of 53BP1 ( Fig. 9 This indicates that IRAK1 is not involved in the regulation of non-homologous end joining.

[0167] In summary, IRAK1 is involved in the regulation of DNA damage repair, and knocking out IRAK1 or inhibiting its enzymatic activity will lead to impaired DNA repair.

[0168] 10. IRAK1 is phosphorylated after DNA damage

[0169] Since IRAK1 is a kinase, the present invention first explored whether IRAK1 could be activated after treatment with PARP inhibitors, CPT and ionizing radiation. The experimental results showed that when PARP inhibitors, CPT and ionizing radiation caused DNA damage, IRAK1 would be phosphorylated ( Fig.10 Therefore, HeLa cells and HeLa-IRAK1 KO cells were treated with DNA damage and then sent to Hangzhou Jingjie Biotechnology Co., Ltd. for whole protein phosphorylation mass spectrometry analysis ( Fig.10 (C, D).

[0170] 11. Whole-protein phosphomic mass spectrometry reveals that IRAK1 regulates the phosphorylation of NBS1

[0171] After mass spectrometry results were classified based on GO database and KEGG pathway analysis, it was found that knocking out IRAK1 would lead to downregulation of the DNA homologous recombination repair signaling pathway ( Fig.11 This is consistent with the previous observation that knocking out IRAK1 or inhibiting its enzymatic activity will lead to an increase in γ-H2AX foci, impaired RAD51 recruitment, reduced RPA2 phosphorylation and unaffected 53BP1 recruitment, indicating that IRAK1 is indeed involved in the regulation of DNA damage repair, and mainly regulates the signaling pathway related to homologous recombination repair.

[0172] In the results of whole protein phosphorylation mass spectrometry, many molecules related to DNA damage repair and DNA replication were found. In particular, when IRAK1 was knocked out, the phosphorylation of NBS1 (NBN) S432 and S343 sites was significantly reduced ( Fig.11 (B). NBS1 is a member of the MRN complex (MRE11-RAD50-NBS1), which is involved in the perception and response of DNA damage, DNA double-strand break (DSB) repair, DNA recombination, maintenance of telomere integrity, control of cell cycle checkpoints and meiosis. This suggests that IRAK1 is likely to participate in the regulation of DNA damage repair by regulating the phosphorylation of NBS1.

[0173] 12. After DNA damage, IRAK1 accumulates in the cell nucleus

[0174] The results of whole protein phosphorylation mass spectrometry suggest that IRAK1 may regulate the phosphorylation of NBS1. Since NBS1 is mainly localized in the nucleus, while IRAK1 is mainly localized in the cytoplasm, and there are also literature reports that IRAK1 can be localized in the nucleus, the localization of IRAK1 was analyzed by immunofluorescence, and it was found that most of IRAK1 was localized in the cytoplasm, and a small part was localized in the nucleus ( Fig.12 After radiation treatment, the fraction of IRAK1 localized in the cell nucleus increased significantly ( Fig.12 ), which indicates that after DNA damage occurs, IRAK1 in the cytoplasm may enter the nucleus and regulate the phosphorylation of NBS1.

[0175] 13. Experimental results confirm that IRAK1 regulates the phosphorylation of NBS1 and ATM

[0176] Western blot was used to verify the phosphorylation changes of NBS1 in HeLa cells, and it was found that after 1 h of IR treatment, knocking out IRAK1 or inhibiting its enzyme activity would lead to a significant decrease in the phosphorylation of the NBS1 S343 site ( Fig.13 In order to exclude cell line specificity, the same experiment was also performed in gastric cancer cell MGC803. The experimental results further confirmed that inhibition of IRAK1 activity would lead to a significant decrease in the phosphorylation of NBS1 S343 and S432 sites ( Fig.13 (middle B).

[0177] Inhibition of IRAK1 activity also leads to decreased phosphorylation of ATM S1981 site ( Fig.14 To further prove that IRAK1 regulates the phosphorylation of NBS1, immunofluorescence experiments were performed in HeLa cells and gastric cancer cell SGC7901, and the same conclusion was obtained. Knocking out IRAK1 or inhibiting its enzyme activity would significantly reduce the phosphorylation of NBS1 S343 site ( Fig.13 C and D). The same results were obtained under the condition of Olaparib treatment. Knocking out IRAK1 or inhibiting its enzyme activity led to a significant decrease in the phosphorylation of NBS1 S343 and S432 sites ( Fig.13 Middle E).

[0178] Finally, in cells overexpressing IRAK1, phosphorylation of NBS1 S343, S432, and ATM S1981 was enhanced, whereas this phenomenon was not detected in cells overexpressing an IRAK1 enzyme-deficient mutant ( Fig.13These experimental results fully confirmed that IRAK1 regulates the phosphorylation of NBS1 S343, S432 and ATM S1981 sites, thereby regulating DNA homologous recombination repair.

[0179] 14. IRAK1 does not affect the expression and localization of NBS1 at the protein level

[0180] The mechanism by which IRAK1 affects the phosphorylation of NBS1 was further explored. Since the expression level and localization distribution of proteins will affect the changes in phosphorylation, in the results of whole-protein phosphorylation mass spectrometry, the most obvious reduction in phosphorylation sites is the S549 and S551 sites of SRRM1, and SRRM1 is a molecule related to transcriptional regulation. Therefore, the present invention first analyzed ATM and MRN complexes at the mRNA level and found that inhibiting IRAK1 enzyme activity would cause a slight increase in the mRNA level of ATM, but the mRNA level of the MRN complex did not change significantly; in IRAK1 KO cells, the mRNA levels of ATM and RAD50 did not change significantly, while the mRNA levels of NBS1 and MRE11 decreased ( Fig.14 Next, the expression and localization of the MRN complex were detected at the protein level. It was found that knocking out IRAK1 or inhibiting its enzyme activity had no significant effect on the protein expression level of the MRN complex, and there was no significant difference in the distribution of the MRN complex in the cytoplasm and the nucleus (Figure 14B). In addition, the soluble part and the chromatin part of the MRN complex were separated. The results showed that regardless of the presence or absence of radiation treatment, knocking out IRAK1 or inhibiting its enzyme activity did not affect the localization distribution of the MRN complex ( Fig.14 These experimental results indicate that IRAK1 does not regulate the phosphorylation of NBS1 by affecting the expression and localization of the MRN complex.

[0181] 15. IRAK1 interacts with NBS1, and the interaction between IRAK1 and NBS1 is stronger after DNA damage

[0182] First, affinity tandem mass spectrometry analysis was performed on NBS1, and the mass spectrometry results showed that it contained 8 IRAK1 peptides ( Fig.15 This suggests that NBS1 and IRAK1 may interact. Next, pull-down experiments were performed in cells stably expressing SFB-IRAK1, and it was found that IRAK1 and NBS1 interacted with each other, and the interaction was stronger after DNA damage occurred ( Fig.15 (middle B).

[0183] Proximity ligation assay (PLA) is an in vitro protein analysis technique that can study protein localization, quantification, interaction, modification and function. The principle is to use a pair of affinity probes to dually recognize the target molecule, and then generate an amplifiable detection signal, converting the detection of protein into the detection of DNA, and realizing the analysis of trace proteins with extremely high detection sensitivity and specificity. In order to further confirm whether NBS1 and IRAK1 really interact, PLA experiments were performed in cell lines with stable expression of SFB-IRAK1. The experimental results showed that NBS1 and IRAK1 co-localized, and the co-localization of the two increased after DNA damage occurred ( Fig.15 C). The above experimental results show that there is an interaction between IRAK1 and NBS1. After DNA damage, the interaction between the two is stronger. In tumors with high expression of IRAK1, IRAK1 can promote the phosphorylation of NBS1, which will recruit more repair proteins RPA and RAD51 to the damaged DNA, improve the efficiency of homologous recombination repair, and make tumor cells insensitive to PARP inhibitors. If IRAK1 / 4 inhibitors are used to inhibit IRAK1 enzyme activity, NBS1 phosphorylation is reduced, the recruitment of repair proteins RPA and RAD51 is impaired, and the efficiency of homologous recombination repair is reduced, thereby increasing the sensitivity of tumor cells to PARP inhibitors ( Fig.16 ).

[0184] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of IRAK1 inhibitor combined with PARP inhibitor in the preparation of medicaments against tumor cells with high expression of IRAK1; The IRAK1 inhibitor is an IRAK1 / 4 inhibitor; The PARP inhibitor is Olaparib or Niraparib; The tumor cells with high expression of IRAK1 are gastric cancer cells; The mass ratio of the IRAK1 / 4 inhibitor to Olaparib is 5:1 to 5:6; or the mass ratio of the IRAK1 / 4 inhibitor to Niraparib is 1:

10.

2. A pharmaceutical composition for resisting tumor cells with high expression of IRAK1, characterized in that: Including IRAK1 / 4 inhibitors and Olaparib, or IRAK1 / 4 inhibitors and Niraparib; In the pharmaceutical composition, the mass ratio of the IRAK1 / 4 inhibitor to Olaparib is 5:1-5:6, and the mass ratio of the IRAK1 / 4 inhibitor to Niraparib is 1:

10.

3. A drug for inhibiting tumor cells with high expression of IRAK1, characterized in that: Including IRAK1 / 4 inhibitors and Olaparib, or IRAK1 / 4 inhibitors and Niraparib; The mass ratio of the IRAK1 / 4 inhibitor to Olaparib is 5:1 to 5:6; The mass ratio of the IRAK1 / 4 inhibitor to Niraparib is 1:

10.

4. The drug according to claim 3, characterized in that Pharmaceutically acceptable excipients are also included.

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