Application of RIPK1 O-GlcNAc glycosylation inhibitor in preparing a drug for enhancing the sensitivity of renal cell carcinoma to sunitinib

Abstract

The present application provides an application of an RIPK1 O-GlcNAc glycosylation inhibitor in the preparation of a drug for enhancing the sensitivity of sunitinib to renal cell carcinoma. The present application discovers that RIPK1 O-GlcNAc glycosylation, especially the O-GlcNAc glycosylation of serine at positions 331, 440, and 669, reduces the sensitivity of renal cell carcinoma to sunitinib. Therefore, by inhibiting RIPK1 O-GlcNAc glycosylation or knocking down the expression of O-GlcNAc transferase, the sensitivity of renal cell carcinoma to sunitinib can be increased. Therefore, the O-GlcNAc glycosylation level of RIPK1 can be used as a biomarker for predicting the sensitivity of renal cell carcinoma to sunitinib; by using an RIPK1 O-GlcNAc glycosylation inhibitor to reduce the RIPK1 O-GlcNAc glycosylation level, especially the O-GlcNAc glycosylation of serine at positions 331, 440, and 669, the sensitivity of renal cell carcinoma to sunitinib can be increased, and the therapeutic effect of sunitinib can be improved.

Description

Technical Field

[0001] This application relates to the field of biomedical technologies, and particularly to the use of a RIPK1 O-GlcNAc glycosylation inhibitor in the preparation of a drug for enhancing the sensitivity of renal cell carcinoma to sunitinib. Background Art

[0002] In the past two decades, renal cell carcinoma (RCC) has been one of the most common malignant tumors globally, with an increasing incidence. Clear cell renal cell carcinoma (ccRCC) is the most common histological type, accounting for approximately 75% of RCC cases. Despite the progress made in the early diagnosis of renal cell carcinoma, approximately 30% of renal cell carcinoma patients have distant metastases at the time of diagnosis, and approximately 30% of patients with localized and locally advanced renal cell carcinoma develop metastatic renal cell carcinoma (mRCC) after surgical treatment.

[0003] Tyrosine kinase inhibitors (TKIs) are promising drugs for the treatment of mRCC, which have greatly extended the survival of patients. However, most mRCC patients develop resistance to sunitinib after 6 - 11 months of treatment. Summary of the Invention

[0004] Embodiments of this application provide the use of a RIPK1 O-GlcNAc glycosylation inhibitor in the preparation of a drug for enhancing the sensitivity of renal cell carcinoma to sunitinib to solve the problems existing in the related technologies. The technical solutions are as follows:

[0005] In a first aspect, embodiments of this application provide the use of a reagent for detecting the O-GlcNAc glycosylation level of biomarker RIPK1 in the preparation of a product for predicting the resistance of renal cell carcinoma to sunitinib.

[0006] In one embodiment, the O-GlcNAc glycosylation level of one or more of Ser331, Ser440, or Ser669 in biomarker RIPK1 is detected.

[0007] In a second aspect, embodiments of this application provide the use of a RIPK1 O-GlcNAc glycosylation inhibitor in the preparation of a drug for enhancing the sensitivity of renal cell carcinoma to sunitinib.

[0008] In one embodiment, the inhibitor inhibits the O-GlcNAc glycosylation of one or more of Ser331, Ser440, or Ser669 in RIPK1.

[0009] In one embodiment, the inhibitor directly inhibits the O-GlcNAc glycosylation of RIPK1;

[0010] and / or;

[0011] Inhibiting the O-GlcNAc glycosylation of RIPK1 by inhibiting the production or expression of O-GlcNAc transferase.

[0012] In one embodiment, the inhibitor is OSMI-1 and / or a lentiviral vector that interferes with O-GlcNAc transferase.

[0013] In one embodiment, the sequence of the lentiviral vector that interferes with O-GlcNAc transferase is 5’-GGAGACAAGAGCCAGACAATA-3’.

[0014] In a third aspect, an embodiment of the present application provides a pharmaceutical composition for treating renal cell carcinoma, which is characterized by comprising sunitinib and any one of the above-mentioned inhibitors.

[0015] In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable excipient.

[0016] In one embodiment, the dosage form of the drug includes tablets, capsules, oral liquids, granules, suspensions, injections, powder injections, dripping pills, sustained-release agents, controlled-release agents or targeted preparations.

[0017] The advantages or beneficial effects in the above technical solutions at least include:

[0018] 1. The present application has specifically studied the resistance of renal cell carcinoma to sunitinib and found that the O-GlcNAc glycosylation of receptor-interacting serine / threonine kinase 1 (RIPK1), especially the O-GlcNAc glycosylation of serine at positions 331, 440, and 669, reduces the sensitivity of renal cell carcinoma to sunitinib. It is found in the present application that the total O-GlcNAc glycosylation level is significantly up-regulated in sunitinib-resistant patients, and by inhibiting the O-GlcNAc glycosylation of RIPK1 or knocking down the expression of O-GlcNAc transferase, the sensitivity of renal cell carcinoma to sunitinib is increased. Therefore, the O-GlcNAc glycosylation level of RIPK1 can be used as a biomarker to predict the sensitivity of RCC to sunitinib, thereby providing a reference for the selection of clinical RCC treatment regimens.

[0019] 2. The pharmaceutical composition for treating renal cell carcinoma in the present application reduces the O-GlcNAc glycosylation level of RIPK1, especially the OO-GlcNAc glycosylation of serine at positions 331, 440, and 669, through an RIPK1 O-GlcNAc glycosylation inhibitor, thereby increasing the sensitivity of RCC to sunitinib and improving the therapeutic effect of sunitinib.

[0020] The above summary is for the purpose of the specification only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of the present application will be readily apparent by reference to the drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In the drawings, unless otherwise specified, the same reference numerals throughout the several views denote the same or similar components or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings depict only some embodiments disclosed in accordance with the present application and should not be regarded as limiting the scope of the present application.

[0022] Figure 1 Schematic diagram for establishing a sunitinib-resistant xenograft model;

[0023] Figure 2 Principal component analysis diagram for untargeted metabolic analysis of sunitinib-sensitive and -resistant tumors;

[0024] Figure 3 Expression quantity diagram of metabolites related to the HBP pathway of glucose metabolism;

[0025] Figure 4 Western blot diagram of O-GlcNAc glycosylation in sunitinib-sensitive and -resistant renal cancer tissues;

[0026] Figure 5 Immunohistochemistry (IHC) detection diagram in sunitinib-sensitive and -resistant renal cancer tissues;

[0027] Figure 6 IC50 determination results in sunitinib-sensitive and -resistant renal cancer cell lines;

[0028] Figure 7 Crystal violet staining diagram of sunitinib-sensitive and -resistant renal cancer cells treated with sunitinib or control;

[0029] Figure 8 Total O-GlcNAc glycosylation western blot and content diagram in sunitinib-sensitive and -resistant renal cancer cells;

[0030] Figure 9 Representative bright-field and crystal violet staining diagrams of Caki-1, 786O, and ACHN cells exposed to sunitinib (with or without OSMI-1); where A is the representative bright-field diagram of Caki-1, 786O, and ACHN cells exposed to sunitinib (with or without OSMI-1); B is the crystal violet staining image of Caki-1, 786O, and ACHN cells exposed to sunitinib (with or without OSMI-1);

[0031] Figure 10 Figure showing the apoptosis rates of Caki-1, 786O, and ACHN cells exposed to sunitinib (with or without OSMI-1) detected by CCK8;

[0032] Figure 11 Figure for analyzing the apoptosis of 786O and ACHN cells exposed to sunitinib with or without OSMI-1 by flow cytometry;

[0033] Figure 12 Figure showing the levels of apoptotic markers activated Caspase-3, activated Caspase8, and activated PARP detected by Western blotting in Caki-1, 786O, and ACHN cells in sunitinib with or without OSMI-1; where A is without OSMI-1; B is Caki-1, 786O, and ACHN cells with OSMI-1;

[0034] Figure 13 Figure for in vivo xenograft experiment, showing the tumor situation in mice; where A is the appearance figure; B is the volume change figure; C is the weight comparison figure.

[0035] Figure 14 Figure for IHC of tumors in mice in in vivo xenograft experiment;

[0036] Figure 15 Figure for the results of TUNEL detection of tumors in mice in in vivo xenograft experiment;

[0037] Figure 16 Figure for the results of IHC detection of two key enzymes in sunitinib-sensitive and -resistant RCC tissues;

[0038] Figure 17 Figure for Western blotting of OGT in sunitinib-sensitive and -resistant RCC cells;

[0039] Figure 18 Figure for IF staining of OGT in sunitinib-sensitive and -resistant RCC cells;

[0040] Figure 19 Figure for Western blotting of OGT protein expression in RCC cells after sunitinib treatment;

[0041] Figure 20 Figure for survival analysis based on OGT in the TCGA-KIRC dataset;

[0042] Figure 21 Figure for the results of downregulating OGT expression by shRNA; where A is the Western blotting figure of O-GlcNA; B is the sunitinib sensitivity figure; C is the sunitinib-induced apoptosis figure; D is the Western blotting figure of the expression of apoptotic markers;

[0043] Figure 22 For the in vivo xenograft experiment with OGT knockout, graphs showing the tumor conditions in mice; among which, A is the external view graph; B is the graph of volume change; C is the graph of weight comparison;

[0044] Figure 23 IHC staining graph for the in vivo xenograft experiment with OGT knockout;

[0045] Figure 24 Graph of the result of exogenous CoIP when the OGT plasmid labeled with HA was transfected into 293T cells with or without the RIPK1 plasmid labeled with Myc;

[0046] Figure 25 Graph of the result of endogenous CoIP determination using the anti-RIPK1 antibody;

[0047] Figure 26 Graph of the 3D model predicted by the HDOCK software for the interaction between OGT / RIPK1;

[0048] Figure 27 Graph of Western blot of O-GlcNAc glycosylation when the RIPK1 plasmid labeled with Flag and the OGT plasmid labeled with HA at different concentrations were co-transfected into 293T cells;

[0049] Figure 28 Graph of the result of CoIP determination; among which, A is the graph of the result determined using the anti-O-GlcNAc antibody; B is the graph of the result of CoIP determination using the anti-Flag antibody; C is the graph of the result of K.CoIP determination using the anti-O-GlcNAc antibody;

[0050] Figure 29 Schematic diagram for constructing the full-length and truncated OGT plasmids labeled with HA;

[0051] Figure 30 Graph of the result of CoIP determination using the anti-Flag antibody for the co-transfection of 293T cells with the full-length and truncated genes of HA-labeled OGT and Flag-labeled RIPK1;

[0052] Figure 31 Schematic diagram for constructing the full-length and truncated OGT plasmids labeled with Myc;

[0053] Figure 32 Graph of the result of CoIP determination using the anti-HA antibody for the co-transfection of 293T cells with the full-length and truncated fragments of Myc-labeled RIPK1 and HA-labeled OGT;

[0054] Figure 33 Schematic diagram for predicting the potential O-GlcNAc glycosylation sites of RIPK1 in the online database YinOYang;

[0055] Figure 34 For sequence verification of the constructed RIPK1 plasmids for Sanger sequencing WT 、RIPK1 S331A 、RIPK1 S440A and RIPK1 S669A as well as RIPK1 3A ;

[0056] Figure 35 Results of CoIP assay for O-GlcNAc sites of RIPK1, where A is verification of potential O-GlcNAc glycosylation sites of RIPK1 with anti-Flag antibody; B is verification of potential O-GlcNAc glycosylation sites of RIPK1 with anti-O-GlcNac antibody

[0057] Figure 36 CoIP assay was used to verify potential O-GlcNAc glycosylation sites of RIPK1 with OSMI-1 and TMG

[0058] Figure 37 Results of CoIP assay, where A is for verification of potential O-GlcNAc glycosylation sites of RIPK1 in Caki-1 cells stably expressing RIPK1 WT or RIPK1 3A ; B is crystal violet staining of Caki-1 cells stably expressing RIPK1 WT or RIPK1 3A after sunitinib treatment

[0059] Figure 38 Graph of CCK8 assay results of Caki-1 cells stably expressing RIPK1 WT or RIPK1 3A after sunitinib treatment

[0060] Figure 39 Western blot detection graph of apoptosis markers in Caki-1 cells stably expressing RIPK1 WT or RIPK1 S331A after sunitinib treatment

[0061] Figure 40 CoIP assay for the stability of RIPK1 / FADD / Caspase-8 complex in sunitinib-sensitive and -resistant renal cell carcinoma cells

[0062] Figure 41Effect of OSMI-1 inhibitor and OGT knockout on the formation of RIPK1 / FADD / Caspase-8 complex. Among them, A shows the effect of inhibiting O-GlcNAc glycosylation with OSMI-1 on the formation of RIPK1 / FADD / Caspase-8 complex; B shows the effect of inhibiting O-GlcNAc glycosylation with shOGT on the formation of RIPK1 / FADD / Caspase-8 complex.

[0063] Figure 42 For stably expressing RIPK1 WT or RIPK1 3A Effect of RIPK1-O-GlcNAc glycosylation on the formation of RIPK1 / FADD / Caspase-8 complex in Caki-1 cells;

[0064] Figure 43 To detect whether NF-κB is activated by sunitinib treatment through Western blot;

[0065] Figure 44 IF assay results of sunitinib-induced nuclear localization of P65;

[0066] Figure 45 Schematic diagram of O-GlcNAc glycosylation regulation of RIPK1-dependent apoptosis. Detailed implementation mode

[0067] In the following, only some exemplary embodiments are simply described. As those skilled in the art can recognize, the described embodiments can be modified in various different ways without departing from the spirit or scope of the present application. Therefore, the drawings and descriptions are considered to be exemplary in nature rather than restrictive.

[0068] Example 1

[0069] I. Materials and methods

[0070] 1. Materials

[0071] 1.1. Cell culture

[0072] RCC cell lines (Caki-1, 786O and ACHN) and human embryonic kidney cell line 293T cells were purchased from the American Type Culture Collection (ATCC). Caki-1, ACHN and 293T cells were cultured in DMEM medium (Gibco). 786O cells were cultured in RPMI 1640 medium (Gibco). All media were supplemented with 10% fetal bovine serum (Haixing, Suzhou) and 1% penicillin / streptomycin (New Cell&Molecular Biotech, Suzhou) to maintain cell growth.

[0073] 1.2. Antibodies, inhibitors, and chemicals

[0074] The following commercial antibodies, inhibitors, and chemicals were used in this study: anti-O-GlcNAc (#12938, CST; #59623, Santa Cruz; #PTM-951RM, PTM-BIO), anti-caspase-3 (#14220, CST), anti-cleaved caspase-3 (#25128-1-AP, ProteinTech), anti-caspase-8 (#9746, CST), anti-cleaved caspase-8 (#9496, CST), anti-PARP (#9542, CST), anti-OGT (#24083, CST; #ab96718, Abcam), anti-OGA (#ab105217, Abcam), anti-RIPK1 (#3493, CST), anti-HA-tag (#3724, CST), anti-Myc-tag (#2278, CST), anti-Flag-tag (#66008-4, ProteinTech), anti-FADD (#271748, Santa Cruz), anti-ubiquitination (#10201-2-AP, ProteinTech), anti-CYLD (#8462, CST), anti-A20 (#5630, CST), anti-cIAP1 (#7065, CST), anti-cIAP2 (#3031, CST), anti-GAPDH (#60004-1, ProteinTech), HA agarose (#A2095, Sigma-Aldrich), Myc agarose (#A7470, Sigma-Aldrich), Flag agarose (#A2220, Sigma-Aldrich), Sunitinib (#7781, Selleck), Z-VAD-FMK (#7023, Selleck), Necrostatin-1 (#8037, Selleck), Ferrostatin-1 (#7243, Selleck), OSMI-1 (#9835, Selleck), Thiamet G (HY12588, MedChemExpress), CHX (#7418, Selleck), and MG132 (#2619, Selleck).

[0075] 2. Method

[0076] 2.1. Establishment of drug-resistant cell lines

[0077] Sunitinib-sensitive and sunitinib-resistant renal cell carcinoma cell lines were cultivated by the stepwise selection method. The RCC cell lines were cultured in medium containing sunitinib for 2 weeks and then in medium without sunitinib for 1 week; the concentration of sunitinib was gradually increased (2 - 10 μM) in each round; the whole culture process lasted about 10 months, and finally, sunitinib-resistant RCC cell lines were obtained and named Caki-1-R, 786O-R, and ACHN-R, respectively. The sensitivity of these cell lines was evaluated by testing the IC50 value of sunitinib..

[0078] 2.2. Construction of sunitinib-sensitive and sunitinib-resistant renal cell carcinoma models in mice

[0079] Approximately 5.0×10 6 / 100 μl of Caki-1 cells were subcutaneously injected into the upper back of 6-week-old BALB / c nude mice; when the volume of the first-generation tumor reached 200 mm 3 , the mice were given sunitinib or saline according to the standard protocol (40 mg / kg / day, orally for 4 weeks, and oral administration was suspended for 2 weeks). After the treatment process was completed, the first-generation tumor was surgically separated and cut into 1 mm 3 small pieces. Subsequently, these 1 mm 3 tumor pieces were transplanted into the second-generation mice, and saline or sunitinib treatment was carried out. Finally, the third-generation mice were sacrificed, and sunitinib-sensitive and drug-resistant renal cell carcinoma tissues were surgically separated for further experiments. The construction schematic diagram is as shown in Figure 1 .

[0080] 2.3. Plasmids

[0081] The sequences encoding full-length human RIPK1, truncated RIPK1 (RIPK1 WT , RIPK1 DD , RIPK1 △DD , RIPK1 KD , RIPK1 △KD and RIPK1 ID ) and mutant RIPK1 S331A , RIPK1 S440A , RIPK1 S669A and RIPK1 3A were subcloned into the pcDNA3-Flag or pcDNA3-Myc vector.

[0082] The sequences encoding full-length human OGT and truncated OGT (OGT WT , OGT TPR, OGT TPR-NLS , OGT △TPR and OGT △CAT ), and the sequences of mutant OGT (OGT K852R and OGT K908R ) were subcloned into the pcDNA3-HA vector.

[0083] Using pcDNA3-RIPK1 as a template, wild-type RIPK1 and mutant RIPK1 were subcloned into a lentiviral vector containing an N-terminal Flag tag to obtain lentiviral vectors expressing wild-type RIPK1 and mutant RIPK1. All products obtained by DNA cloning were verified by Sanger sequencing (Tsingke Biotch, Beijing); lentiviral shRNAs for OGT were purchased from IGE Biotechnology (Guangzhou, China).

[0084] 2.4. Liquid chromatography-tandem mass spectrometry (LC-MS / MS)

[0085] Untargeted metabolomic analysis was performed by LC-MS / MS technology of Shanghai Applied Protein Technology Co., Ltd. to identify metabolic changes in sunitinib-sensitive and sunitinib-resistant renal cell carcinoma tumors; immunoprecipitation was performed using an anti-OGT antibody or IgG in renal cell carcinoma cells Caki-1 and Caki-1-R to identify OGT-binding proteins; proteins enriched with the anti-OGT antibody were separated by polyacrylamide gel electrophoresis and silver stained, and the bound proteins were analyzed by mass spectrometry.

[0086] 2.5. Cell transfection and lentiviral infection

[0087] Plasmids were transfected into RCC cell lines using Lipofectamine 3000 transfection reagent (Invitrogen) according to the instructions. Plasmids were transfected into 293T cells using PEI transfection reagent (Polysciences) according to the instructions. For lentiviral infection, 293T cells were transfected with packaging vectors (pMD2.G and psPAX2) and GFP shRNA or OGT shRNA, and then the infected viral particles were collected. The viral particles were added to the medium together with 10 μg / ml of polyethylenimine (Beyotime) and co-incubated with the cells for 8 hours. Thereafter, puromycin was used to screen for stably expressing cell lines and control cells.

[0088] 2.6. Western blotting

[0089] RCC cells were lysed with EBC buffer (50 mM Tris, 120 mM NaCl, 0.5% NP-40) or RIPA lysis buffer (Cell Signaling Technology) with protease / phosphatase inhibitor mixture (New Cell&Molecular Biotech, Suzhou). Equal amounts of protein extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. After separation, the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes, and the PVDF membranes were blocked with TBST buffer containing 5% bovine serum albumin (BSA) to prevent non-specific binding. Then the blocked PVDF membranes were incubated with specific primary antibodies overnight at 4 °C, and secondary antibodies labeled with horseradish peroxidase (HRP) were used. Finally, the protein bands were visualized using the GelView 6000Pro imaging system from BLT Company (China).

[0090] 2.7. Flow cytometry

[0091] Flow cytometry analysis was used to evaluate the role of O-GlcNAc glycosylation or OGT in sunitinib-induced apoptosis of RCC cells. The role of O-GlcNAc glycosylation was explored by inhibiting global O-GlcNAc glycosylation using the small molecule inhibitor OSMI-1. In addition, to explore the role of OGT, RCC cells were transfected with shNC or shOGT and collected after treatment with sunitinib for 48 hours. Subsequently, after washing with PBS, they were first stained with Annexin V-FITC, then with PI, and finally the apoptosis rate of RCC cells was detected using a FACS flow cytometer (Becton Dickinson).

[0092] 2.8. Immunohistochemistry

[0093] Immunohistochemistry was performed on renal cell carcinoma tissues to detect and show specific proteins. First, 4-μm thick RCC tissue sections were dewaxed and dehydrated in preparation for antibody staining. The tissue sections were treated with 3% hydrogen peroxide for 10 minutes at room temperature to inhibit endogenous peroxidase activity. Then, the sections were blocked with 5% BSA for 30 minutes at room temperature to reduce non-specific binding of antibodies. Subsequently, the sections were incubated with rabbit or mouse antibodies specific for the target protein overnight at 4 °C. The next day, the sections were washed three times with PBS to remove unbound primary antibodies, and then the sections were incubated with secondary antibodies for 1 hour at 37 °C. After washing, the tissue sections were stained with 3,3'-diaminobenzidine substrate, and images of the stained tissue sections were obtained using an upright microscope (Olympus, Japan).

[0094] 2.9. Immunofluorescence and confocal microscopy

[0095] First, RCC cells were fixed with 4% paraformaldehyde for 15 minutes. After fixation, the cells were treated with 0.1% Triton X-100 for 15 minutes at room temperature to increase cell membrane permeability. To avoid non-specific binding, the cells were blocked with 5% BSA for 1 hour at room temperature. Then, the cells were incubated with specific antibodies overnight at 4°C. The next day, the cells were washed three times with PBS to remove unbound primary antibodies. Subsequently, the cells were incubated with the corresponding secondary conjugated antibodies for 1 hour at room temperature. Finally, the nuclei were stained with DAPI, and the stained cells were imaged using an Olympus (Tokyo, Japan) confocal microscope.

[0096] 2.10. Co-immunoprecipitation (CoIP) assay

[0097] Approximately 1×10^7 cells were collected and lysed using EBC buffer containing protease / phosphatase mixture; 5% of the cell lysate was reserved as a positive control. For endogenous CoIP detection, the remaining cell lysate was incubated with the first antibody overnight at 4°C. Subsequently, the lysate was incubated with protein A / G immunomagnetic beads for 6 hours at 4°C. For exogenous CoIP antibodies, the remaining cell lysate was co-incubated with anti-HA affinity gel (Sigma-Aldrich, Germany), anti-Flag affinity gel (Yeasen, Shanghai), or anti-Myc affinity gel (Sigma-Aldrich, Germany). After incubation, the immune complexes were washed three times with NETN buffer (20 mM Tris, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40) to remove non-specifically bound proteins and ensure the specificity of subsequent steps. Then, under standard denaturing conditions, the proteins were eluted from the beads with loading buffer, and the purified proteins were detected by Western blotting.

[0098] 2.11. Xenograft tumor model

[0099] 5×10 6 Caki-1 cells were subcutaneously injected into the upper back of nude mice (BALB / c, 6 weeks old, n = 6 per group). The mice were orally administered sunitinib or saline according to a standard protocol (40 mg / kg / day) while the tumor volume reached 200 mm 3 ; 4 - 6 weeks later, the mice were sacrificed and the tumor tissues were excised for analysis.

[0100] 2.12. Statistics

[0101] The Student's t-test was used to compare the differences in gene expression between the two groups; analysis of variance was used to compare the differences in gene expression among multiple groups; the log-rank test was used to evaluate survival differences; all statistical tests were two-sided tests.

[0102] II. Conclusions

[0103] 1.1. O-GlcNAc glycosylation was significantly upregulated in sunitinib-resistant renal cell carcinoma

[0104] Untargeted metabolic analysis of sunitinib-sensitive and sunitinib-resistant tumors was performed using liquid chromatography-tandem mass spectrometry (LC-MS / MS). The results of principal component analysis (PCA) were as Figure 2 shown:

[0105] From Figure 2 the results of the principal component analysis showed significant changes in the metabolic profiles between sunitinib-sensitive and sunitinib-resistant tumors.

[0106] Regarding the metabolic changes of glucose, as Figure 3 shown, in sunitinib-resistant tumors, the abundances of two key metabolites, D-glucosamine 6-phosphate (GlcN-6-P) and uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), in the hexosamine biosynthetic pathway (HBP) were significantly increased; and UDP-GlcNAc produced by HBP is the donor substrate for O-GlcNAc glycosylation modification; indicating that the level of O-GlcNAc glycosylation may be increased in sunitinib-resistant tumors.

[0107] Detection was performed using Western and immunohistochemistry (IHC), and the results were as Figure 4 and Figure 5 shown. From Figure 4 and Figure 5 it was shown that the total O-GlcNAc glycosylation level in the sunitinib-resistant RCC group was significantly upregulated compared with that in sunitinib-sensitive RCC tissues.

[0108] Sunitinib-resistant RCC cells (Caki-1-R, ACHN-R, and 786O-R) established by repeated induction of sunitinib treatment, compared with sunitinib-sensitive RCC cells (Caki-1, ACHN, and 786O), had significantly increased sunitinib IC50 values in sunitinib-resistant RCC cells (Caki-1-R, ACHN-R, and 786O-R), as Figure 6 and Figure 7 shown.

[0109] Simultaneously detect the total O-GlcNAc glycosylation levels in sunitinib-sensitive and -resistant RCC cells. The total O-GlcNAc glycosylation level was significantly upregulated in resistant RCC cells (Caki-1-R, ACHN-R, and 786O-R) compared to sensitive RCC cells. Figure 8 .

[0110] Therefore, it can be known from the above detection that O-GlcNAc glycosylation is significantly upregulated in sunitinib-resistant renal cell carcinoma.

[0111] 2.2 Inhibition of O-GlcNAc glycosylation increases sunitinib-induced apoptosis of renal cell carcinoma cells

[0112] Use the small molecule inhibitor OSMI-1 to inhibit global O-GlcNAc glycosylation. The results are shown in Figure 9 and Figure 10 . From Figure 9 A Representative bright-field images of Caki-1, 786O, and ACHN cells exposed to sunitinib (with or without OSMI-1) and Figure 9 B Crystal violet staining images of Caki-1, 786O, and ACHN cells exposed to sunitinib (with or without OSMI-1), and Figure 10 The apoptosis rates of Caki-1, 786O, and ACHN cells exposed to sunitinib (with or without OSMI-1) were detected using CCK8. It can be seen that inhibition of O-GlcNAc glycosylation by OSMI-1 significantly increased sunitinib-induced RCC cell death.

[0113] Flow cytometry showed that inhibition of O-GlcNAc glycosylation significantly increased sunitinib-induced apoptosis of RCC cells. Figure 11 .

[0114] After sunitinib treatment, Caki-1, 786O, and ACHN cells were treated with sunitinib with or without OSMI-1; the levels of apoptotic markers including activated Caspase-3, activated Caspase8, and activated PARP were detected by Western blotting. The results are shown in Figure 12 . From Figure 12 it can be seen that inhibition of O-GlcNAc glycosylation significantly increased the expression of apoptotic markers including activated Caspase-3, activated Caspase8, and activated PARP.

[0115] Further in vivo xenograft experiments were performed. 1×10 6Caki-1 cells were subcutaneously injected into each nude mouse (n = 5); the mice were orally administered sunitinib, saline, or a combination of OSMI-1 and sunitinib according to a standard protocol (40 mg / kg / day). After 4 weeks, the mice were sacrificed and the tumor tissues were excised for imaging analysis. The tumor conditions were as Figure 13 shown, where Figure 13 A is the appearance diagram; Figure 13 B is the volume change diagram; Figure 13 C is the weight comparison diagram; IHC was as Figure 14 shown; the TUNEL detection results were as Figure 15 shown.

[0116] It can be seen from Figure 13 that the inhibition of O-GlcNAc glycosylation by OSMI-1 significantly improved the efficacy of sunitinib in the treatment of renal cell carcinoma; the combination of sunitinib and OSMI-1 inhibitor group significantly inhibited tumor growth. Compared with the sunitinib group, the tumor volume was smaller and the weight was lighter.

[0117] Figure 14 The IHC analysis of showed that the activated Caspase-3 in the combination of sunitinib and OSMI-1 inhibitor group was significantly increased compared with the sunitinib group. Figure 15 The TUNEL detection results of further showed that the proportion of apoptotic cells in the combination of sunitinib and OSMI-1 inhibitor group was the highest.

[0118] Through the above-mentioned use effects of the OSMI-1 inhibitor, it can be shown that the inhibition of O-GlcNAc glycosylation can increase the apoptosis of renal cell carcinoma cells induced by sunitinib.

[0119] 2.3. OGT knockout increases the sensitivity of sunitinib in patients with renal cell carcinoma

[0120] O-GlcNAc transferase (OGT) and GlcNAcase (OGA) control the dynamic cycle of O-GlcN acylation modification; IHC detection of the above two key enzymes in sunitinib-sensitive and -resistant RCC cells was as Figure 16 shown. It can be seen from Figure 16 that in sunitinib-resistant renal cell carcinoma, OGT rather than OGA is upregulated. It was also determined by Western blot that OGT was upregulated in sunitinib-resistant RCC cells compared with sunitinib-sensitive RCC cells, Figure 17 . By immunofluorescence (IF) detection, OGT was also significantly upregulated in sunitinib-resistant RCC cells, Figure 18 .

[0121] Subsequently, sunitinib was continuously administered to RCC cells sensitive to sunitinib, and the expression level of OGT in renal cell carcinoma cells after 6 h, 12 h, and 24 h of sunitinib treatment was determined by Western blot. The results are as Figure 19 shown. As Figure 19 can be seen, the expression of OGT in renal cell carcinoma cells increased with the increase in sunitinib treatment time.

[0122] The prognostic outcome of OGT in renal cell carcinoma tissues was evaluated using Gene Expression Profiling Interactive Analysis (GEPIA), as Figure 20 shown. The results showed that the survival time of RCC patients with high OGT expression was shorter than that of patients with low OGT expression.

[0123] For RCC cells, the expression of OGT was downregulated by shRNA, and the results are as Figure 21 shown, where Figure 21 A is the Western blot of O-GlcNA; Figure 21 B is the sunitinib sensitivity graph; Figure 21 C is the sunitinib-induced apoptosis graph; Figure 21 D is the Western blot of the expression of apoptosis markers.

[0124] Figure 21 The results showed that Western blot determined the total O-GlcNAc glycosylation level, and OGT knockout significantly reduced the total O-GlcNAc glycosylation in RCC cells; OGT knockout significantly increased the sensitivity of RCC cells to sunitinib; flow cytometry showed that OGT downregulation significantly increased sunitinib-induced apoptosis of RCC cells; in addition, the expression of activated Caspase-3, activated Caspase8, and activated PARP in shOGT RCC cells significantly increased after sunitinib treatment.

[0125] Furthermore, in vivo xenograft experiments were carried out; 1×10 6 shNC or shOGT Caki-1 cells were subcutaneously injected into each nude mouse (n = 5); the mice were orally administered sunitinib, normal saline, or a combination of OSMI-1 and sunitinib according to the standard protocol (40 mg / kg / day). After 4 weeks, the mice were sacrificed and the tumor tissues were excised for imaging analysis. The tumor conditions are as Figure 22 shown, where Figure 22 A is the appearance graph; Figure 22 B is the volume change graph; Figure 22 C is the weight comparison graph; IHC is as Figure 23 shown. Obviously, OGT knockout significantly improved the efficacy of sunitinib; IHC detection showed that the expression of activated Caspase-3 in shOGT RCC tissues significantly increased.

[0126] Therefore, the important role of OGT-mediated O-GlcNAc glycosylation in sunitinib sensitivity, and the knockout of OGT improved the therapeutic effect of sunitinib.

[0127] 2.4. OGT specifically interacts with RIPK1 and promotes RIPK1 O-GlcNAc glycosylation

[0128] HA-tagged OGT plasmid was transfected into 293T cells with or without Myc-tagged RIPK1 plasmid for exogenous CoIP assay, and the results are as Figure 24 shown. Exogenous CoIP assay in 293T confirmed the direct interaction between OGT and RIPK1 ( Figure 4 D-E).

[0129] Endogenous CoIP assay was performed using anti-RIPK1 antibody, and the results are as Figure 25 shown. Endogenous CoIP assay in RCC cells showed that OGT specifically interacts with RIPK1, and this interaction was enhanced in sunitinib-resistant RCC Caki-1-R. The 3D model of OGT / RIPK1 interaction was predicted using HDOCK software as Figure 26 shown. It indicates the direct interaction between OGT and RIPK1 in sunitinib-sensitive and sunitinib-resistant RCC cells.

[0130] Flag-tagged RIPK1 plasmid and different concentrations of HA-tagged OGT plasmid were co-transfected into 293T cells, and the total O-GlcNAc glycosylation level was detected by Western blotting, as Figure 27 shown; the results showed that OGT significantly induced the total O-GlcNAc glycosylation level and RIPK1 O-GlcN acylation.

[0131] CoIP assay was further performed using anti-O-GlcNAc antibody or anti-Flag-RIPK1. As Figure 28 shown in A, OGT significantly promoted RIPK1 O-GlcNAc glycosylation; HA-tagged OGT (WT or mutant) and Flag-tagged RIPK1 plasmid were transfected into 293T cells, and CoIP assay was performed using anti-Flag antibody. As Figure 28 shown in B, O-GlcN glycosylated RIPK1 depends on the enzymatic activity of OGT; consistent with the above results, K.CoIP assay was performed using anti-O-GlcNAc antibody. Compared with sunitinib-sensitive RCC cells, the RIPK1 O-GlcNAc glycosylation in sunitinib-resistant RCC cells was significantly increased, as Figure 28 shown in C.

[0132] Therefore, OGT specifically interacts with RIPK1 and promotes RIPK1 O-GlcNAc glycosylation.

[0133] 2.5. RIPK1 O-GlcNAc glycosylation at Ser 331 、Ser 440 and Ser 669 sensitivity to sunitinib

[0134] Construct full-length and truncated HA-tagged OGT plasmids as shown in Figure 29 (named HA-OGT WT 、HA-OGT TPR 、HA-OGT TPR-NLS 、HA-OGT △TPR and HA-OGT △CAT ) based on their domain sequences.

[0135] Co-transfect the plasmid with the Flag-tagged RIPK1 plasmid into 293T cells for CoIP assay, and the results are shown in Figure 30 ; indicating that the tetratricopeptide repeat (TPR) domain of OGT directly interacts with RIPK1.

[0136] Similarly, construct full-length and truncated Myc-tagged RIPK1 as shown in Figure 31 (named Myc-RIPK1 WT 、Myc-RIPK1 DD 、Myc-RIPK1 △DD 、Myc-RIPK1 KD 、Myc-RAPK1 △KD and Myc-RINK1 ID ) based on their domain sequences. Figure 32 CoIP analysis of

[0137] showed that the intermediate (ID) domain of RIPK1 is crucial for interaction with OGT. Figure 33 The potential sites of O-GlcNAc glycosylation on RIPK1 were predicted using the online database YinOYang (Gupta and Brunak, 2002). As shown in

[0138] the results of WT , it is very likely that Ser331, Ser440 and Ser669 of RIPK1 are modified by O-GlcNAc glycosylation. S331A Construct Flag-tagged wild-type RIPK1 (RIPK1 S440A ), single-site mutant RIPK1 S669Aand the three - point mutant RIP K1 (RIPK1 3A ) plasmid, which was verified by Sanger sequencing, Figure 34 .

[0139] As Figure 35 shown, compared with RIPK1 WT , the single mutations (RIPK1 S331A , RIPK1 S440A and RIPK1 S669A ) moderately reduced the O - GlcNAc glycosylation signal of RIPK1, while RIPK1 3A significantly reduced the O - GlcNAc glycosylation modification signal.

[0140] Similarly, as Figure 36 shown, OSMI - 1 could reduce the O - GlcNAc glycosylation of RIPK1 WT , TMG could increase the O - GlcNAc glycosylation, while they could not affect the O - Glc acylation of RIPK1 3A , indicating that Ser 331, Ser 440 and Ser 669 are the key O - GlcN acylation sites on RIPK1.

[0141] By stably expressing RIPK1 WT or RIPK1 3A in Caki - 1 cells, Figure 37 it was shown that compared with RIPK1 WT , RIPK1 3A significantly reduced the O - GlcNAc glycosylation modification signal in Caki - 1 cells. Compared with RIPK1 WT , RIPK1 3A significantly increased sunitinib - induced apoptosis of RCC cells, as Figure 38 shown, indicating that the O - GlcNAc glycosylation of RIPK1 at Ser 331, Ser 440 and Ser 669 reduced sunitinib sensitivity. Figure 39 Western blotting also showed that after sunitinib treatment, RIPK13A significantly increased activated Caspase - 3, Caspase - 8 and PARP in RCC cells.

[0142] Therefore, OGT specifically interacts with RIPK1 through its TPR domain and promotes the O - GlcNAc glycosylation of RIPK1 at Ser 331, Ser440 and Ser 669, inhibiting sunitinib - induced apoptosis of RCC cells.

[0143] 2.6. O-GlcNAc glycosylation affects the formation of the RIPK1 / FADD / Caspase-8 complex and NF-κB activation

[0144] Immunoprecipitate FADD in sunitinib-sensitive and -resistant RCC cells after sunitinib treatment, and detect RIPK1 and Caspase-8, as Figure 40 shown, compared with sunitinib-sensitive RCC cells, the RIPK1 / FADD / Caspase-8 complex in sunitinib-resistant RCC cells was significantly reduced.

[0145] Figure 41 Regarding the effects of the OSMI-1 inhibitor and OGT knockout on the formation of the RIPK1 / FADD / Caspase-8 complex, as can be seen from Figure 41 A, inhibition of O-GlcNAc glycosylation by the OSMI-1 inhibitor led to the formation of the RIPK1 / FADD / Caspase-8 complex in RCC cells after sunitinib treatment, indicating that O-GlcNAc glycosylation regulates sunitinib sensitivity by affecting the RIPK1 / ADD / SCaspase-8 complex; Figure 41 as can be seen from B, inhibition of O-GlcNAc glycosylation by OGT knockout significantly induced the formation of the RIPK1 / FADD / Caspase-8 complex in RCC cells.

[0146] Moreover, as Figure 42 shown, the RIPK1 / FADD / Caspase-8 complex was significantly increased in Caki-1 cells stably expressing RIPK1 3A , indicating that RIPK1 O-GlcNAc glycosylation at Ser 331, Ser 440, and Ser 669 inhibited the formation of the RIPK1 / ADD / CCaspase-8 complex.

[0147] Next, detect whether NF-κB was activated after sunitinib treatment by Western blotting, as Figure 43 shown, p-P65 was significantly induced in RCC cells after sunitinib treatment. p-p65 is an indicator of p65 activation and serves as the main transcription factor of the NF-κB signaling pathway, indicating that the NF-κB pathway was activated to activate the transcription of target genes, thereby maintaining cell survival after sunitinib treatment. Further Figure 44 The IF assay shown indicated that sunitinib treatment significantly induced the nuclear localization of P65, while inhibition of O-GlcNAc glycosylation by OSMI-1 significantly inhibited sunitinib-induced nuclear localization of P65, as Figure 45 .

[0148] In summary, the present application specifically studied the resistance of renal cell carcinoma to sunitinib and found that the O-GlcNAc glycosylation of receptor-interacting serine / threonine kinase 1 (RIPK1), especially the O-GlcNAc glycosylation of serine at positions 331, 440, and 669, reduces the sensitivity of renal cell carcinoma to sunitinib. By inhibiting the O-GlcNAc glycosylation of RIPK1 or knocking down the expression of O-GlcNA transferase, the sensitivity of renal cell carcinoma to sunitinib is increased. Therefore, the level of O-GlcNAc glycosylation of RIPK1 can be used as a biomarker to predict the sensitivity of RCC to sunitinib, thereby providing a reference for the selection of clinical RCC treatment regimens. And by using an RIPK1 O-GlcNAc glycosylation inhibitor to reduce the level of RIPK1 O-GlcNAc glycosylation, especially the O-GlcNAc glycosylation of serine at positions 331, 440, and 669, the sensitivity of RCC to sunitinib is increased and the therapeutic effect of sunitinib is improved.

[0149] In the description of this specification, the descriptions referring to terms "one embodiment", "some embodiments", "example", "specific example", or "some examples", etc. mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. Moreover, the specific features, structures, materials, or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, without conflict, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0150] In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include at least one of such features. In the description of the present application, "a plurality" means two or more unless otherwise specifically defined.

[0151] The above is only the specific implementation manner of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art can easily think of various changes or substitutions within the technical scope disclosed by the present application, and these should all be covered by the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. Use of a reagent for detecting the O-GlcNAc glycosylation level of the biomarker RIPK1 in the preparation of a product for predicting the resistance of renal cell carcinoma to sunitinib, characterized in that: Detect the O-GlcNAc glycosylation levels of more than two Ser331, Ser440 or Ser669 in the biomarker RIPK1.

2. Use of an inhibitor of RIPK1 O-GlcNAc glycosylation in the preparation of a drug for enhancing the sensitivity of renal cell carcinoma to sunitinib, characterized in that: The inhibitor inhibits O-GlcNAcylation of two or more of Ser331, Ser440 or Ser669 in RIPK1.

3. The use according to claim 2, characterized in that: The inhibitor directly inhibits O-GlcNAc glycosylation of RIPK1; and / or; Inhibition of O-GlcNAcylation of RIPK1 by inhibiting the production or expression of O-GlcNAc transferase.

4. The use according to claim 3, characterized in that: The inhibitor is OSMI-1 and / or a lentiviral vector interfering with O-GlcNAc transferase.

5. The use according to claim 4, characterized in that: The sequence of the lentiviral vector interfering with O-GlcNA transferase is 5'-GGAGACAAGAGCCAGACAATA-3'.

6. A pharmaceutical composition for treating renal cell carcinoma, characterized in that: The invention comprises sunitinib and the inhibitor according to any one of claims 2 to 5.

7. A pharmaceutical composition for treating renal cell carcinoma according to claim 6, characterized in that: The pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable excipient.

8. A pharmaceutical composition for treating renal cell carcinoma according to claim 6, characterized in that: The dosage form of the drug includes tablets, capsules, oral liquids, granules, suspensions, injections, powder injections, dripping pills, sustained-release preparations, controlled-release preparations or targeted preparations.

Images