Use of USP7 inhibitors in the preparation of products for treating lung cancer

Abstract

The present invention discloses the use of USP7 inhibitors in the preparation of products for the treatment of lung cancer, wherein the lung cancer is non-small cell lung cancer (NSCLC) that is resistant to KRAS inhibitors; such non-small cell lung cancers include squamous cell carcinoma, lung adenocarcinoma, and large cell lung cancer. The present invention provides the use of USP7 inhibitors in the preparation of products for the treatment of lung cancer, and through a large number of cell and animal level experiments, it is confirmed that whether used alone or in combination with KRAS inhibitors, USP7 inhibitors can effectively inhibit NSCLC cell proliferation and overcome KRAS inhibitor resistance in KRAS-G12C mutant NSCLC.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of a USP7 inhibitor in preparing a product for treating lung cancer. Background Art

[0002] The RAS family of proteins (KRAS, NRAS, and HRAS) are small GTPases that cycle between inactive (GDP-bound) and active (GTP-bound) states in normal cells, acting as molecular switches. RAS mutations disrupt the guanine exchange cycle, locking RAS in the active, GTP-bound state. This continuously activates downstream signaling pathways, promotes cell growth and proliferation, and can lead to cell carcinogenesis.

[0003] RAS mutation or amplification is one of the most common abnormalities in human cancers, with KRAS mutations causing 85% of all RAS mutations. There are multiple KRAS mutation subtypes, primarily concentrated at codons 12 / 13 and 61. Common mutation subtypes include G12A, G12C, G12D, G12S, G12V, G12R, and G13D, or KRAS wild-type amplification. The distribution of KRAS mutations varies across cancers, with G12C mutations occurring in 41% of lung adenocarcinomas (LUADs), while G12D and G12V are the two most common mutant alleles in colorectal cancer (CRC) and pancreatic cancer (PDAC). KRAS mutations predominate in NSCLC, accounting for 78% of all RAS mutations found in these tumors.

[0004] Because KRAS mutations are prevalent in multiple diseases, they have become a sought-after target in cancer drug development. In 2013, the Shokat research team at the University of California, Los Angeles, discovered that the cysteine ​​residue introduced into the KRAS G12C mutant creates a binding pocket (S-IIP) that readily forms covalent bonds with small molecules, locking mutant KRAS in an inactive state. This important discovery spurred the development of the mutant-selective KRAS G12C inhibitors, Sotorasib (AMG510) and Adagrasib (Mirati). KRAS G12C inhibitors (Adagrasib and Sotorasi) have shown clinical promise in lung cancers harboring KRAS G12C mutations. However, because KRAS G12C inhibitors target a single target, cancer cells may acquire drug resistance by promoting overexpression of the target protein or new mutations in the target protein. Therefore, combining KRAS G12C inhibitors with other targeted agents is expected to mitigate the development of drug resistance. At the same time, G12C only accounts for a small part of KRAS mutations. Other mutations such as KRAS G12D and KRAS G12V still have huge treatment needs. The development of a single small molecule regulator that effectively targets multiple types of KRAS mutants is of great clinical significance.

[0005] Ubiquitination is a post-translational modification that covalently links the carboxyl group at the C-terminus of the ubiquitin molecule to the N-terminus or amino group of a lysine residue on a substrate protein through a cascade reaction catalyzed by ubiquitin ligases. Substrate ubiquitination can alter its function, localization, stability, and protein-protein interactions. Deubiquitination is the opposite of ubiquitination. Catalyzed by deubiquitinases (DUBs), ubiquitin molecules on substrate proteins are removed, preventing them from being degraded by the proteasome. Inhibiting deubiquitinases can lead to selective protein degradation and may affect other "undruggable" targets. Summary of the Invention

[0006] The present invention aims to address at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes the use of a USP7 inhibitor in the preparation of a product for treating lung cancer. The USP7 inhibitor, when used alone, can promote the regression of KRAS inhibitor-resistant non-small cell lung cancer (NSCLC).

[0007] The present invention also proposes the use of a USP7 inhibitor in combination with a KRAS inhibitor in the preparation of a product for treating lung cancer.

[0008] According to a first aspect of the present invention, there is provided a use of a USP7 inhibitor in the preparation of a product for treating lung cancer, wherein the lung cancer is non-small cell lung cancer resistant to KRAS inhibitors.

[0009] The USP7 is a widely expressed deubiquitinating enzyme that can remove ubiquitin and protect substrate proteins from degradation.

[0010] In some embodiments of the present invention, the non-small cell lung cancer includes squamous cell carcinoma, lung adenocarcinoma and large cell lung cancer.

[0011] In some embodiments of the present invention, the USP inhibitor comprises at least one of (1) to (5):

[0012] (1) Compounds that specifically inhibit USP7 expression;

[0013] (2) interfering molecules that specifically interfere with USP7 expression;

[0014] (3) antibodies or ligands that specifically bind to the USP7 protein;

[0015] (4) Gene editing reagents for specific knockout of USP7;

[0016] (5) Compounds that specifically inhibit USP7 activity.

[0017] In some embodiments of the present invention, the compound that specifically inhibits USP7 activity includes at least one of GNE-6776, HBX41108, and P22077.

[0018] In some embodiments of the present invention, the interfering molecule that specifically interferes with USP7 expression includes siRNA or shRNA.

[0019] In some embodiments of the present invention, the shRNA comprises the nucleotide sequences shown in SEQ ID NOs: 7 to 9.

[0020] In some embodiments of the present invention, the gene editing reagent for specifically knocking out USP7 includes an sgRNA fragment, and the target gene of the sgRNA fragment is the USP7 gene.

[0021] In some embodiments of the present invention, the sgRNA fragment includes the nucleotide sequence shown in SEQ ID NO: 3 to 5.

[0022] In some embodiments of the present invention, the gene editing reagent for specifically knocking out USP7 also includes Cas9 protein.

[0023] In some embodiments of the present invention, the product for treating lung cancer comprises a USP7 inhibitor and a KRAS inhibitor.

[0024] In some embodiments of the invention, the KRAS inhibitor includes at least one of AMG-510 and MRTX849.

[0025] According to a second aspect of the present invention, a use of a USP7 inhibitor in combination with a KRAS inhibitor in the preparation of a product for treating lung cancer is proposed, wherein the lung cancer is non-small cell lung cancer resistant to KRAS inhibitors.

[0026] In some embodiments of the invention, the KRAS inhibitor includes at least one of AMG-510 and MRTX849.

[0027] In some embodiments of the present invention, the non-small cell lung cancer includes squamous cell carcinoma, lung adenocarcinoma and large cell lung cancer.

[0028] In some embodiments of the present invention, the USP inhibitor comprises at least one of (1) to (5):

[0029] (1) Compounds that specifically inhibit USP7 expression;

[0030] (2) interfering molecules that specifically interfere with USP7 expression;

[0031] (3) antibodies or ligands that specifically bind to the USP7 protein;

[0032] (4) Gene editing reagents for specific knockout of USP7;

[0033] (5) Compounds that specifically inhibit USP7 activity.

[0034] In some embodiments of the present invention, the compound that specifically inhibits USP7 activity includes at least one of GNE-6776, HBX41108, and P22077.

[0035] In some embodiments of the present invention, the interfering molecule that specifically interferes with USP7 expression includes siRNA or shRNA.

[0036] In some embodiments of the present invention, the shRNA comprises the nucleotide sequence shown in SEQ ID NO:X.

[0037] In some embodiments of the present invention, the gene editing reagent for specifically knocking out USP7 includes an sgRNA fragment, and the target gene of the sgRNA fragment is the USP7 gene.

[0038] In some embodiments of the present invention, the sgRNA fragment comprises a nucleotide sequence as shown in SEQ ID NO: X.

[0039] In some embodiments of the present invention, the gene editing reagent for specifically knocking out USP7 also includes Cas9 protein.

[0040] The present invention has at least the following beneficial effects:

[0041] The present invention provides the use of a USP7 inhibitor in the preparation of a product for treating lung cancer, and confirms through a large number of cell and animal level experiments that whether the USP7 inhibitor is used alone or in combination with a KRAS inhibitor, it can effectively inhibit NSCLC cell proliferation and overcome KRAS inhibitor resistance in KRAS-G12C mutant NSCLC.

[0042] The present invention provides the use of a USP7 inhibitor combined with a KRAS inhibitor in the preparation of a product for treating lung cancer. The USP inhibitor can target and inhibit USP7, thereby specifically degrading wild-type or mutant KRAS proteins, and then promoting NSCLC tumor regression. Among them, the combination of USP7 inhibitors and KRAS inhibitors demonstrates the synergistic effect of drug combinations, providing a new targeting approach for overcoming the drug resistance of KRAS inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0044] Figure 1 Figure 1 shows the results of identifying KRAS-interacting proteins in A549 cells by immunoprecipitation and mass spectrometry (IP-Mass) in Example 1 of the present invention; Figure A shows the silver staining results, and Figure B shows a volcano plot showing the statistical distribution of KRAS-interacting proteins, with KRAS represented by pink dots, significantly increased proteins represented by cyan dots, and deubiquitinating enzymes represented by blue dots;

[0045] Figure 2 Figures 1 and 2 show the immunoprecipitation results and fluorescence complementation results of lung cancer A549 cells in Example 1 of the present invention; Figure A shows the co-IP results of USP7 enriched by KRAS, Figure B shows the co-IP results of KRAS enriched by USP7, and Figure C shows the results of fluorescence complementation assay of the binding between USP7 and KRAS in living cells. Scale bar: 100 μm.

[0046] Figure 3 This is a diagram showing the immunoprecipitation results of Example 1 of the present invention verifying the binding of the USP7 TRAF domain to KRAS in HEK293T cells;

[0047] Figure 4 The results of the GST pull-down test and the ITC test for verifying the binding of the USP7 TRAF domain to KRAS in Example 1 of the present invention are shown;

[0048] Figure 5 Figure 1 is a graph showing the KRAS expression detection results after overexpression (gain) or knockout / inhibition (loss) of USP7 in lung cancer cells according to Example 1 of the present invention; Figure A shows the detection results of exogenous KRAS protein expression levels in HEK293T cells after overexpressing exogenous USP7 in a concentration gradient; Figure B shows, from left to right, the detection results of endogenous KRAS protein expression levels in A549 cell lines, H358 cell lines, and H1299 cell lines after overexpressing exogenous USP7 in a concentration gradient; Figure C shows the detection results of KRAS at the protein and mRNA levels after knocking out USP7 in A549 cells by CRISPR-Cas9 technology, and the bar graph on the right shows the numerical grayscale statistics of the corresponding changes; Figure D shows the detection results of KRAS protein expression levels in A549 cells after administration of three different USP7 inhibitors, and the bar graph on the right shows the numerical grayscale statistics of the corresponding changes;

[0049] Figure 6 Figures 1 and 2 show the results of a complementation test on USP7 KO lung cancer cells A549 and the half-life detection results of KRAS protein in Example 1 of the present invention; Figure A shows the detection results of KRAS protein expression levels after USP7 KO cells are complemented with exogenous USP7 WT or USP7 C223 mutant (deubiquitinase activity deficiency), and the results on the lower side show the grayscale statistics of the corresponding changes; Figure B shows the degradation of KRAS protein in A549 cells overexpressing exogenous USP7 WT after cycloheximide for 0 h, 12 h, and 24 h; Figure C shows the degradation of KRAS protein in USP7 KO cells after cycloheximide for 0 h, 12 h, and 24 h;

[0050] Figure 7 Figures 1 and 2 show the KRAS ubiquitination results after overexpressing USP7 or inhibiting / knocking down USP7 expression in Example 1 of the present invention; Figure A shows the KRAS ubiquitination detection results in HEK293T cells after overexpressing USP7 WT or USP7-C223S, and the cells were treated with 10 μM MG132 for 4 hours during the experiment; Figure B shows the KRAS ubiquitination detection results in A549 cells after treatment with the USP7 inhibitor GNE677640 (40 μM); Figure C shows the KRAS ubiquitination detection results in A549 cells after transfection with shRNA to knock down USP7 expression;

[0051] Figure 8 This is a graph showing the detection results of in vitro deubiquitination of KRAS by the recombinant USP7 TRAF-CD protein according to Example 1 of the present invention; wherein, the in vitro incubation time of the USP7 TRAF-CD protein and KRAS is 2 hours;

[0052] Figure 9 Figures 1 and 2 show the results of an in vitro screening test for E2 enzymes that promote KRAS ubiquitination according to Example 1 of the present invention; Figure A shows the results of screening for ubiquitination enzymes using an in vitro E2 enzyme screening kit; Figure B shows the in vitro verification of the cleavage effect of USP7 on a multimer formed by the E1 enzyme UBA1, the E2 enzyme UBE2N / 2V2 complex, and the E3 enzyme WDR76;

[0053] Figure 10 This is a schematic diagram of the working model of USP7 stabilizing KRAS by deubiquitination, obtained through the experimental results of Example 1 of the present invention;

[0054] Figure 11 This is a graph showing the detection results of seven ubiquitin chains in HEK293T cells overexpressing USP7 in Example 1 of the present invention;

[0055] Figure 12 Figures 1 and 2 show the results of ubiquitination detection of endogenous KRAS K48 and K63 ubiquitin chains in lung cancer cells that inhibit USP7 or overexpress USP7 in Example 1 of the present invention; Figure A shows the detection results of endogenous KRAS K48 and K63 ubiquitin chains in A549 cells after treatment with a USP7 inhibitor; Figure B shows the detection results of KRAS ubiquitin chains formed by Ub K48R mutations and K63 mutations in HEK293T cells after overexpressing exogenous USP7;

[0056] Figure 13 Graph showing the mass spectrometry results of HEK293T cells overexpressing exogenous USP7 in Example 1 of the present invention; the left side is a schematic diagram of the mass spectrometry process, and the right side is a mass spectrometry profile;

[0057] Figure 14 This is a graph showing the results of KRASK147 site K48 ubiquitination detection in HEK293T cells overexpressing exogenous USP7-WT or USP7-C223S in Example 1 of the present invention;

[0058] Figure 15 This figure shows the results of detecting protein levels and phosphorylation levels of the KRAS downstream signaling pathway in A549 cells after treatment with the USP inhibitor GNE6776 (40 μM) in Example 1 of the present invention; the left side shows the grayscale statistics of the corresponding changes. EGF (20 ng / mL) treatment is used to activate the signaling pathway;

[0059] Figure 16 This is a graph showing the results of a colony formation test after treatment of three lung cancer cells with the USP7 inhibitor GNE6776 (40 μM) in Example 1 of the present invention;

[0060] Figure 17Figures 1 and 2 show the cell viability and proliferation results of lung cancer cells treated with USP7 inhibitors in Example 1 of the present invention; Figure A shows the cell viability results of KRAS-WT and KRAS-KO cells treated with different concentrations of the USP7 inhibitor HBX41,108; Figure B shows the growth and proliferation of A549 and H1395 cells treated with different concentrations of the USP7 inhibitor;

[0061] Figure 18 Figures 1 and 2 show the analysis results of USP7 and KRAS expression in lung cancer patient tissue microarrays according to Example 1 of the present invention; Figure A shows immunohistochemical staining of lung cancer tumor tissue and adjacent paracancerous tissue; Figures B and C show statistical graphs of USP7 and KRAS expression levels in adjacent paracancerous tissue and tumor tissue;

[0062] Figure 19 Figures 1 and 2 show the analysis results of USP7 and KRAS expression in lung cancer patient tissue microarrays and the prognosis analysis results of USP7 and KRAS in lung cancer patients, respectively. Figure A shows the correlation analysis between USP7 and KRAS expression in tumor tissues, Figure B shows the correlation analysis between USP7 and KRAS expression in adjacent paracancerous tissues, Figure C shows the Kaplan-Meier survival analysis of USP7 and lung cancer patient survival rate, and Figure D shows the Kaplan-Meier survival analysis of KRAS and lung cancer patient survival rate.

[0063] Figure 20 Figures 1 and 2 show the results of interaction testing between USP7 and different states or mutant forms of KRAS according to Example 1 of the present invention; Figure A is a hypothetical schematic diagram of the relationship between USP7 and different states of KRAS; Figure B shows the results of an in vitro GST-pulldown assay; and Figure C shows the results of a co-IP assay between USP7 and six KRAS mutants.

[0064] Figure 21 Figure 1 shows the results of detecting the GTP-bound KRAS protein level in A549 cells with USP7 knockout / inhibition / overexpression according to Example 1 of the present invention; Figure A shows A549 cells with USP7 knockout, Figure B shows A549 cells treated with a USP7 inhibitor, and Figure C shows A549 cells overexpressing USP7 treated with EGF (20 ng / mL) to activate the signaling pathway;

[0065] Figure 22 This is a graph showing the results of administering a USP7 inhibitor and detecting six KRAS G12 mutant proteins in Example 1 of the present invention;

[0066] Figure 23Figure 1 shows the results of the deubiquitination experiment in Example 1 of the present invention; Figure A shows the in vitro deubiquitination assay of GDP-KRAS or GTP-KRAS deubiquitinated by recombinant full-length USP7, and Figure B shows the ubiquitination of six KRASG12 mutants in cells overexpressing USP7;

[0067] Figure 24 Figures 2A and 2B show the growth and proliferation results of conventional H358 cells and drug-resistant H358 cells after treatment with different drugs in Example 2 of the present invention; Figure A shows the changes in growth activity of wild-type H358 cells after drug administration, Figure B shows the changes in growth activity of drug-resistant H358 cells after drug administration, and Figure C shows the results of a colony formation assay of drug-resistant H358 cells after drug administration;

[0068] Figure 25 Figures 3 show the test results of Example 3 of the present invention demonstrating the therapeutic effect of a USP7 inhibitor and a KRAS inhibitor combined on drug-resistant NSCLC in animals. Figure A is a schematic diagram of drug treatment; Figure B shows tumor morphology eight days after dosing; Figure C shows a statistical graph of tumor weight eight days after dosing; and Figures D and E are curves of NSCLC tumor growth and body weight changes in mice, respectively.

[0069] Figure 26 Schematic diagram of the mechanism by which the USP7 inhibitors of the present invention exert therapeutic effects on NSCLC. DETAILED DESCRIPTION

[0070] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0071] Example 1 Verification of the therapeutic effect of USP7 inhibitors on NSCLC at the cellular level

[0072] This example verifies the therapeutic effect of USP7 inhibitors on NSCLC at the cellular level. The specific experimental methods and experimental results are as follows:

[0073] 1. Direct interaction between the deubiquitinating enzyme USP7 and KRAS

[0074] 1) In the non-small cell lung cancer A549 cell line, intracellular overexpression was achieved by transfection with a Flag-tagged KRAS expression plasmid. Immunoprecipitation (IP) was performed using Flag-tag-specific affinity chromatography (Flag-beads) to capture the Flag-tagged KRAS protein and its interacting proteins.

[0075] 2) The sample captured in step 1) (Flag-tagged KRAS protein and its interacting proteins) is subjected to mass spectrometry (i.e., immunoprecipitation-mass spectrometry, IP-MS). The IP-MS experimental method includes:

[0076] In A549 cells, Lipo3000 was used to overexpress Flag-vector (pFLAG-CMV-2, purchased from Sigma, Cat. No. E7398) and Flag-KRAS (KRAS fragment was inserted into the pFLAG-CMV-2 plasmid backbone, and the expressed KRAS fragment sequence is shown in SEQ ID NO: 1). After 48 hours, the cells were collected and lysed, and immunoprecipitated using Flag-M2-beads (purchased from Millipore, Cat# A2220). The next day, the beads were washed three times, a portion was used for immunoblotting, and the remaining beads were subjected to SDS-PAGE, silver staining, gel cutting, proteolysis, desalting, lyophilization, mass spectrometry analysis, library search, and then data analysis and confirmation of the target protein. The results are shown below. Figure 1 shown.

[0077] Amino acid sequence of the overexpressed KRAS fragment: MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQRVEDAFYTLVREIRQYRLKKISKEEKTPGCVKIKKCIIM (SEQ ID NO: 1).

[0078] Figure 1 The results showed that USP7 is a deubiquitinating enzyme that interacts with KRAS protein. The statistical results of KRAS interacting proteins and USP7 interacting proteins captured by IP-MS are shown in Table 1. As can be seen from Table 1, the detection of USP7 interacting protein group shows that KRAS protein is also enriched. Figure 1 Table 1 preliminarily demonstrated the binding activity between KRAS protein and USP7.

[0079] Table 1 Statistics of IP-MS analysis results

[0080]

[0081] 3) In A549 cells, the endogenous specific binding of KRAS and USP7 was verified by co-immunoprecipitation (co-IP) assay (see the previous IP-MS assay for the co-IP assay method). The results were as follows: Figure 2 A and Figure 2 B. The overexpression of Flag-USP7 also used the pFLAG-CMV-2 plasmid backbone and inserted the USP7 fragment. The sequence of the expressed USP7 fragment is shown in SEQ ID NO: 2.

[0082]

[0083] 4) USP7 and KRAS colocalization and interaction fluorescence signals were observed in HEK293T cells by bimolecular fluorescence complementation (BiFC) assay ( Figure 2 C), indicating that KRAS is a direct target of USP7; the BiFC assay method is as follows: molecular cloning technology was used to construct the target protein bimolecular fluorescence complementation vectors VN173-KRAS and VC155-USP7. VN173 and VC155 were used as control groups and transfected into HEK293T cells, respectively. After 24 hours, fluorescence microscopy was used to observe the interaction between USP7 protein and KRAS protein in living cells.

[0084] 5) USP7 contains an N-terminal TRAF substrate binding domain, a catalytic domain CD domain and five ubiquitin-like UBL domains. Co-IP experiments found that deletion of the TRAF domain blocked the binding of USP7 to K RAS protein in HEK293T cells ( Figure 3 ), indicating that the TRAF domain is crucial for the interaction between the two.

[0085] 6) To detect the USP7-KRAS interaction, an in vitro GST pull-down assay was performed using the following steps:

[0086] 5 μg of KRAS protein (sequence shown in SEQ ID NO: 1) was incubated with GST or GST-USP7-TRAF (5 μg) in GST pull-down buffer (20 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1 mM MgCl2, 0.1% NP-40, 10% glycerol, and 1% BSA) on ice for 30 minutes; then, 10 μL of GST-beads (purchased from Millipore, Cat#70541-5) was added to the mixture and reacted at 4°C for 2 hours; the GST-agarose beads were pulled down with GST, washed five times with GST pull-down buffer, and 50 μL of 2× SDS sample buffer was added to elute the protein; the proteins in the eluate were separated by SDS-PAGE, and the interaction was detected by Coomassie brilliant blue staining and immunoblotting, and the results were shown as follows. Figure 4 A; the amino acid sequence of the USP7-TRAF portion of the GST-USP7-TRAF is shown in SEQ ID NO: 2;

[0087] The dissociation constant (Kd) of USP7-TRAF and KRAS4A was determined by isothermal titration calorimetry (ITC). USP7-TRAF and KRAS4A proteins were diluted in the same buffer (20 mM HEPES, 150 mM NaCl, 5 mM MgCl2, pH 7.5). 200 μL of USP7-TRAF (0.2 mM) was added to the sample cell, and the injection needle contained 20 μL (2 mM) of KRAS4A protein. At 25°C, a reference power of 5 μcal / s was used, with the first injection of 0.4 μL of sample and the subsequent 19 injections of 2 μL of sample. The thermal effect was then normalized to the amount of injected protein, and the data were analyzed using Microcal-ITC200 analysis software. The results are shown in Figure 2. Figure 4 As shown in B; Figure 4 It can be seen that there is binding activity between the USP7 TRAF domain and the KRAS4A protein, and the affinity between the two tested by the ITC experiment is 121μM.

[0088] 2. USP7 stabilizes KRAS protein levels

[0089] Previous studies have shown that the stability of KRAS is strictly controlled by multiple E3 ubiquitin ligases. USP7 is a deubiquitinating enzyme. This example tested whether it plays a role in regulating the stability of KRAS.

[0090] 1) Flag-USP7 and Myc-KRAS plasmids with increasing concentrations were co-transfected into HEK293T cells, and proteins from HEK293T cells were collected for immunoblotting and precipitation. The results were as follows: Figure 5 As shown in A; Figure 5 As shown in Figure 1, exogenous KRAS increased in a concentration-dependent manner. The Myc plasmid backbone was pCMV-Myc, purchased from Youbao Biotechnology, catalog number VT1064; the expressed KRAS sequence is shown in SEQ ID NO: 1.

[0091] 2) Two KRAS mutant NSCLC cell lines, A549 (KRAS G12S) and H358 (KRAS G12C), and wild-type H1299 (KRAS WT) were selected for overexpression of USP7. Protein samples of cells overexpressing USP7 were collected for immunoblotting and precipitation. The results were as follows: Figure 5 As shown in B; Figure 5 As shown in Figure B, overexpression of USP7 led to a concentration-dependent increase in endogenous KRAS in the above three cell lines.

[0092] 3) A549 cells were selected for USP7 gene knockout (KO) using the CRISPR-Cas9 editing system. The sgRNA sequences targeting USP7 were as follows:

[0093] sgRNA1: 5'-GCACAAAACACGGAGGGCTAAGG-3' (SEQ ID NO: 3),

[0094] sgRNA2: 5'-AGCACTAGCATTAATCGACATGG-3' (SEQ ID NO: 4),

[0095] sgRNA3: 5'-AAAATCAAAAGGCTATGTAGAGG-3' (SEQ ID NO: 5);

[0096] Any of the three sgRNAs above can be used. All USP7 KO cells used below are A549;

[0097] KO A549 cell samples were collected to detect changes in KRAS protein and mRNA levels. The results were as follows: Figure 5 As shown in C; Figure 5 C shows that USP7 knockout significantly reduced the endogenous KRAS protein level in A549 cells, but had no effect on KRAS mRNA level, indicating that USP7 may regulate KRAS protein stability through post-translational modification.

[0098] 4) A549 cells were treated with USP7 inhibitors (GNE-6776, HBX41,108, P22077) and the KRAS protein levels in the treated cells were detected. Figure 5 D shown; Figure 5 D shows that USP7 inhibitors lead to decreased KRAS protein levels.

[0099] 5) Wild-type USP7 WT and the deubiquitinase site inactivated mutant USP7-C223S were re-transfected into the USP7-KO cells prepared in step 3) to detect the difference in the regulation of compensatory USP7 on the restoration of KRAS protein levels. The results were as follows: Figure 6 As shown in A; Figure 6

[0100] 6) Cycloheximide (CHX, a protein synthesis inhibitor) was administered to A549 cells overexpressing USP7 and knocking out USP7, and the KRAS protein degradation rate and half-life were measured. The results were as follows: Figure 6 B and Figure 6 As shown in C; Figure 6 B~ Figure 6 C shows that USP7 overexpression prolongs the half-life of endogenous KRAS, while USP7-KO accelerates the degradation of KRAS.

[0101] These results indicate that USP7 stabilizes KRAS expression in NSCLC cell lines, and this function is related to its deubiquitinase activity.

[0102] 3. USP7 cleaves KRAS K147 site K48 chain ubiquitination

[0103] In order to investigate whether USP7 has the function of regulating KRAS by deubiquitination, this example established a series of exogenous, endogenous and in vitro ubiquitination test systems to monitor changes in KRAS ubiquitination levels.

[0104] 1) Ubiquitination assay was performed in HEK293T cells to detect the effect of overexpression of exogenous wild-type USP7 (WT) or USP7-C223S on KRAS ubiquitination. The results are as follows Figure 7 As shown in A; Figure 7 As shown in Figure 1, overexpression of USP7 can reduce the ubiquitination level of KRAS, while the USP7-C223S mutant lacking deubiquitinase activity has no effect on the ubiquitination of KRAS.

[0105] 2) Ubiquitination assays were performed in A549 cells to detect the effects of 24 h treatment with USP7 inhibitor GNE677640 (40 μM) and shRNA knockdown of USP7 on endogenous KRAS ubiquitination levels. The results were as follows: Figure 7 B and Figure 7 C; the sequences of the shRNAs used are as follows (all three shRNAs can be used individually):

[0106] shRNA1: 5'-CCTGGATTTGTGGTTACGTTATTCAAGAGATAACGTAACCACAAA TCCAGGTTTTTT-3' (SEQ ID NO: 7);

[0107] shRNA2: 5'-GCAGAGAAAGGTGTGAAAATTTCAAGAGAATTTCACACCTTTCTCT GCTTTTTT-3' (SEQ ID NO: 8);

[0108] shRNA3: 5'-GCGATTACAAGAAGAGAAATTCAAGAGATTTCTCTTCTTGTAATC GCTTTTTT-3' (SEQ ID NO: 9);

[0109] Depend on Figure 7 B and Figure 7 C shows that in endogenous experiments, whether knocking down USP7 (KD) by shRNA or inhibiting the deubiquitinase activity of USP7 using GNE-6776, the ubiquitination level of endogenous KRAS was significantly increased.

[0110] 3) In an in vitro experiment, the TRAF-CD domain of USP7 (sequence shown in SEQ ID NO: 2) was purified from Escherichia coli for deubiquitination assay, and the recombinant USP7 TRAF-CD protein was used to detect deubiquitination of KRAS purified from HEK 293T cells by immunoblotting; the purified KRAS was specifically obtained by co-transfection of Flag-KRAS and HA-Ub (plasmid pRK5-HA-Ubiquitin-WT purchased from Addgene, catalog number Plasmid #17608) into HEK293T cells and simultaneous treatment with MG132 to obtain ubiquitinated KRAS. The obtained KRAS was further mixed with the TRAF-CD domain of USP7 and reacted at 37°C for 2 hours. The ubiquitination level of KRAS was detected by immunoblotting, and the results were as follows: Figure 8 shown by Figure 8 It can be seen that as the concentration of co-incubated USP7 (TRAF+CD) increases, the ubiquitination level of KR AS gradually decreases.

[0111] 4) Since the E2 enzyme involved in KRAS polyubiquitination is currently unknown, this example used an in vitro E2 enzyme screening kit (Youbi Biotechnology, Cat#UBK-982) to observe that KRAS can form monoubiquitination or diubiquitination in the presence of UbcH2, UbcH3, UbcH5a, UbcH5b, or UbcH5c, while forming polyubiquitination in the presence of UbcH6 or the UBE2N / 2V2 complex (screening results are shown in Figure 2). Figure 9 A); accordingly, in vitro ubiquitination assay was performed by combining KRAS expressed and purified from E. coli with E1 (UBC1), E2 (UbcH5b), E3 (WDR76), ATP, Mg 2+Ub was first ubiquitinated in a test tube, and then USP7 was added for separation by SDS-PAGE and the KRAS ubiquitination level was detected by Coomassie staining. The results confirmed that 100 nM full-length USP7 (sequence shown in SEQ ID NO: 2) could completely cleave the multimeric ubiquitinated band formed by the E1 enzyme UBA1, the E2 enzyme UBE2N / 2V2 complex and the E3 enzyme W DR76 at 37°C (as shown in Figure 2). Figure 9 B).

[0112] The above results indicate that USP7 can specifically cleave the polyubiquitin chains formed by KRAS. The schematic diagram of the working model of USP7 stabilizing KRAS by deubiquitination is shown in the figure below. Figure 10 shown.

[0113] 5) To explore the clear molecular mechanism by which USP7 deubiquitinates KRAS and affects its stability, this example detected seven types of ubiquitin chains formed by KRAS in HEK293T cells overexpressing exogenous USP7 through chain selectivity assays and ubiquitination site identification. The expression plasmids of the seven HA-UB mutants used were purchased from Addgene, and their names and catalog numbers are pRK5-HA-Ubiquitin-K6 / Plasmid#121151, pRK5-HA-Ubiquitin-K11 / Plasmid#22901, pRK5-HA-Ubiquitin-K27 / Plasmid#22902, pRK5-HA-Ubiquitin-K29 / Plasmid#22903, and p RK5-HA-Ubiquitin-K33 / Plasmid#17607, pRK5-HA-Ubiquitin-K48 / Plasmid#17605, pRK5-HA-Ubiquitin-K63 / Plasmid#17606; the results were as follows: Figure 11 shown by Figure 11 It can be seen that USP7 can deubiquitinate the K48 chains formed by KRAS and has a weak effect on the K63 ubiquitin chains.

[0114] To improve the accuracy of the conclusion, we further used specific antibodies to capture K48 and anti-K63 chains to detect changes in endogenous KRAS ubiquitin chains in A549 cells. The results are as follows Figure 12 As shown in A; Figure 12 As shown in Figure 4, GNE-6776 treatment inhibited USP7 activity, leading to enhanced ubiquitination of the endogenously formed K48 chain of KRAS, while no effect was detected on the ubiquitination of the K63 chain.

[0115] At the same time, ubiquitination assay was performed on HEK293T cells to detect the effect of overexpression of exogenous USP7 on KRAS ubiquitin chains formed by Ub K48R mutation and K63R mutation. The results were as follows Figure 12 As shown in B; Figure 12 B shows that USP 7 loses its cleavage function on K48R mutant ubiquitin chains, but still has cleavage function after K63R mutation. Figure 11 and Figure 12 Both indicate that USP7 deubiquitinates KRAS K48-type polyubiquitin chains. HA-UB-K48R and HA-UB-K63R utilize the aforementioned pRK5-HA-Ubiquitin-K6 backbone. The amino acid sequence of the K48R fragment is MQIFVKTLT GKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGRQLEDGRTLSDYNIQKESTLHL VLRLRGG (SEQ ID NO: 10), and the amino acid sequence of the K63R fragment is MQIFVKTLTGKTITLEVEP SDTIENVKAKIQDKEGIPPDQQRLIFAGRQLEDGRTLSDYNIQRESTLHLVLRLRGG (SEQ ID NO: 11).

[0116] 6) Mass spectrometry was used to detect the effect of overexpression of exogenous USP7 on ubiquitination of KRAS at different lysine sites in HEK293T cells. Figure 13 shown by Figure 13 It can be seen that the KRAS deubiquitination site of USP7 is lysine 147; further, the ubiquitination assay was used in HEK293T cells to detect the effect of overexpression of exogenous wild-type USP7 (WT) or USP7-C223S on the ubiquitination of KRAS K147 site K48. The results are as follows Figure 14 shown by Figure 14It can be seen that USP7 significantly removes KRAS K147 site K48 chain ubiquitination. Among them, Flag-K147R and Flag-K147 both use the aforementioned pFLAG-CMV-2 plasmid backbone. The amino acid sequence of the K147R fragment is MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSARTRQRVEDAFYTLVREIRQYRLKKISKEEKTPGCVKIKKCIIM (SEQ ID NO: 12), and the amino acid sequence of the K147 fragment is MTEYRLVV VGAGGVGRSALTIQLIQNHFVDEYDPTIEDSYRRQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTRSFEDIHHYREQIRRVRDSEDVPMVLVGNRCDLPSRTVDTRQAQDLARSYGIPFIETSARTRQRVEDAFYTLVREIRQYRLRRISREERTPGCVRIRRCIIM (SEQ ID NO: 13).

[0117] Based on the above results, it can be seen that USP7 exerts a deubiquitination effect by cleaving the K48 ubiquitin chain at the K147 site of KRAS, thereby stabilizing the KRAS protein level.

[0118] 4. USP7 regulates KRAS to promote the occurrence and development of NSCLC

[0119] 1) A549 cells were treated with the USP7 inhibitor GNE6776 (40 μM) and the protein levels and phosphorylation levels of the KRAS downstream signaling pathway were detected. The results were as follows Figure 15 shown by Figure 15 It can be seen that in A549 cells, GNE-6776 treatment inhibited the deubiquitinase activity of USP7 and significantly reduced the phosphorylation levels of KRAS downstream effector molecules ERK and AK T, indicating that KRAS-dependent tumor growth signal transduction can be reduced by inhibiting USP7.

[0120] 2) A549, H358, and H1299 cells were treated with GNE6776 (40 μM) and the effect on cell proliferation was assessed by colony formation assay. The colony formation assay included the following steps:

[0121] The cells to be treated were seeded in a 6-well plate at a density of 1000 cells per well. After the cells were stabilized for 24 hours, GNE6776 was added at a final concentration of 40 μM according to the experimental requirements. After culturing for 20 days, the culture medium was discarded, the cells were washed twice with PBS solution, and then fixed with acetic acid / methanol solution for 15 minutes; then 0.1% crystal violet solution was added to stain the cells, and then the excess stain was slowly washed away with running water, and the cells were allowed to air dry; finally, the stained cells were observed and the relevant data were compiled; the results are shown in Figure 2. Figure 16 shown by Figure 16 It can be seen that USP7 inhibitors can significantly inhibit the proliferation of three NSCLC cell lines including A549, H358 and H1299.

[0122] 3) In order to illustrate the connection between USP7 promoting lung cancer growth and KRAS and KRAS ubiquitination levels, we constructed KRAS knockout (KO) A549 cells, and on this basis, stably transformed KRAS WT or KRAS-K147R A549 cells, and compared the effects of USP7 overexpression on the growth of the three lung cancer cells. The experimental results showed that in KRAS-deficient A549 cells, the effect of USP7 overexpression on cell proliferation decreased. In A549 cells supplemented with KRAS WT, USP7 overexpression restored the promoting effect on cell proliferation. In A549 cells supplemented with KRAS K147R, USP7 overexpression did not affect the growth of lung cancer cells. The results are as follows. Figure 17 As shown in A. Figure 17 A shows that USP7 can regulate the stability of KRAS protein and its downstream signaling pathway by removing ubiquitination at the KRAS K147 site to promote the proliferation of lung cancer cells.

[0123] 4) A comparative analysis was conducted between A549 cells with KRAS-G12C mutation and H1395 cells driven by BRAF-G469C mutation. The growth and proliferation changes of A549 cells and H1395 cells after treatment with different concentrations of GNE-6776 for 24 hours were detected. The results are as follows: Figure 17 As shown in B; Figure 17 As shown in Figure 2, under the condition of 40 μM GNE6776, the growth of A549 cells was almost completely inhibited, while H1395 cells had no significant effect, indicating that USP7 inhibitors may be more effective in KRAS-driven lung cancer.

[0124] 5) To investigate the relevance of these findings to clinical data, immunohistochemistry (IHC) staining was performed to analyze USP7 and KRAS expression in tissue microarrays (TMAs) of NSCLC patient samples. The analysis included 80 adjacent normal tissue samples and 94 cancerous tissue samples. The specific methods were as follows:

[0125] NSCLC patient sample tissue microarrays were purchased from Shanghai Xinchao Biotechnology Co., Ltd., and immunohistochemical staining of USP7 and KRAS was performed by the company to obtain the protein expression levels of USP7 and KRAS in patient tumors and adjacent normal tissues, as well as the correlation between lung cancer and USP7 and KRAS expression. After screening and removing some case tissue data, the microarray had a total of 174 spots (chip ID: HLugA180Su07), including 80 adjacent normal tissues and 94 patient tissues. USP7 antibody information: CST Cat#4833, used at a dilution of 1:100; KRAS antibody information: Abcam Cat#275875, used at a dilution of 1:100. The results are shown in the figure below. Figure 18 and Figure 19 As shown;

[0126] Depend on Figure 18 It can be seen that the expression of USP7 and KRAS in NSCLC tissue samples was significantly stronger than that in adjacent tissue samples, with high expression levels in 46% (44 / 94) and 76% (72 / 94) of NSCLC tissues respectively. Figure 19 It can be seen that correlation analysis showed that there was a significant positive correlation between USP7 and KRAS protein levels in lung cancer tissues (R = 0.372, P = 0.001, Figure 19 A), while this correlation was relatively weak in adjacent tissues ( Figure 19 B); In addition, survival analysis of cases matched with the chip showed that the survival rate of patients with high expression of USP7 or high expression of KRAS was significantly lower than that of patients with low expression levels ( Figure 19 C and Figure 19 D).

[0127] The above results indicate that USP7 promotes lung cancer cell proliferation by regulating KRAS and its downstream signaling pathways. The USP7-KRAS axis may play a key role in the occurrence and progression of lung cancer. Treatment targeting this axis may constitute a new treatment strategy for NSCL C.

[0128] 5. USP7 broadly regulates the stability of wild-type and G12 mutant KRAS proteins

[0129] Mutations at sites like G12 in KRAS place it in a continuously activated state bound to GTP, which is one of the key factors driving carcinogenesis. This example uses experiments to determine whether USP7 has a universal regulatory effect on KRAS or a specific regulatory effect on a certain mutant. The working mechanism is shown in the following diagram: Figure 20 As shown in A.

[0130] 1) In vitro enrichment of GDP-bound KRAS protein and GTP-bound KRAS protein, and the interaction between the TRAF domain of USP7 and the two states of KRAS was verified by GST-pull-down assay. The results are as follows Figure 20 As shown in B; Figure 20 As shown in Figure 2, the TRAF domain of USP7 interacts with both states of KRAS, and the interaction efficiency is basically the same, indicating that the regulation of USP7 on KRAS mutants is similar to that on the wild type. Consistently, the co-IP results of USP7 deubiquitinating six KRAS G12 mutants are shown in Figure 2. Figure 20 As shown in C, Figure 20 C demonstrates direct binding between USP7 and six KRAS mutants: G12A, G12C, G12D, G12S, G12V, and G12R.

[0131] 2) In terms of protein stability regulation, USP7-KO or GNE6776 treatment reduced the levels of total KRAS and GTP-bound KRAS proteins ( Figure 21 A and Figure 21 B), while USP7 overexpression had the opposite effect ( Figure 21 C). Consistently, the stability of oncogenic KRAS mutants including G12A, G12C, G12D, G12S, and G12V was also attenuated by another USP7 inhibitor, HBX41,108, in HEK293T cells ( Figure 22 ).

[0132] 3) In terms of deubiquitination function, in vitro deubiquitination assays were performed in HEK293T cells to determine the deubiquitination of GDP-KRAS or GTP-KRAS by recombinant full-length USP7. Figure 23 As shown in A; Figure 23 As shown in A, USP7 has a cleavage effect on both GDP-bound and GTP-bound KRAS. Within 10 minutes, the cleavage efficiency of USP7 on GTP-bound KRAS is slightly higher than that on GDP-bound KRAS. Similarly, in vitro deubiquitination assays were performed on different KRAS G12 mutants, and the results were as follows: Figure 23 As shown in B; Figure 23 B shows that USP7 not only has a deubiquitination effect on wild-type KRAS, but also has a deubiquitination effect on various KRAS mutants.

[0133] These results indicate that USP7 has a general regulatory effect on the stability of KRAS protein by removing K48 chain ubiquitination, and USP7 is a potential target for the development of pan-KRAS inhibitors.

[0134] Example 2 Verification of the therapeutic effect of the combined use of USP7 inhibitors and KRAS inhibitors on drug-resistant NSCLC at the cellular level

[0135] This example verifies the therapeutic effect of the combined use of USP7 inhibitors and KRAS inhibitors on drug-resistant NSCL C at the cellular level. The specific experimental methods and experimental results are as follows:

[0136] 1) Construction of AMG510-resistant lung cancer cell line (H358-AMGR):

[0137] First, H358 cells were seeded in a 10-cm culture dish with an initial confluence of 50% to 70%, and cultured using RPMI1640 medium containing 10% fetal bovine serum; AMG510 was added to the culture system with an initial concentration of 1 nmol / L, and fresh medium containing the drug was replaced every three days to maintain cell growth; when the cells reached confluence, the drug concentration was gradually increased at semi-logarithmic intervals until the concentration of AMG510 reached 1 mmol / L; through this screening process, a drug-resistant cell line was successfully obtained, named H358-AMGR, and the corresponding concentration of drug (1 mmol / L AMG510) was continuously used in subsequent culture to maintain these drug-resistant cell lines.

[0138] 2) Using the drug-resistant cell line H358-AMGR constructed in step 1), five control experiments were then set up in the ordinary H358 cell line and the above-mentioned drug-resistant cell line, including single treatment with AMG510, two USP7 inhibitors G NE6776 and HBX41,108, and the combination of AMG510 and any USP7 inhibitor. The effects of the five different drug treatments on tumor cell growth and proliferation were studied by cell viability assays (MTS assay) and colony formation assays;

[0139] ①MTS assay to detect cell proliferation:

[0140] The MTS test uses CellTiter The AQueous One Solution Cell Proliferation Assay Kit (Promega, Cat# G3580) uses MTS, a new-generation tetrazolium blue salt compound that can be reduced to a colored formazan product by various dehydrogenases in the mitochondria of living cells. The color intensity of the product is highly correlated with the viable cell count of certain sensitive cell lines within a certain range. Five control groups were set up: AMG510 alone, GNE6776 alone, HBX41,108 alone, AMG510 + GNE6776, and AMG510 + HBX41,108. Within each group, a gradient of dosing (including no dosing) was included. H358-WT cells and H358-AMGR were seeded in 96-well plates at an average density of 5000 cells / well. The absorbance of cells at different drug gradients was measured 24 hours after drug administration. The non-drug group was used as the baseline. Cell viability = absorbance after different drug gradient treatments / absorbance of the non-drug group * 100%. The data were used to draw a bar graph. The results are shown in Figure 2. Figure 24 A and Figure 24 As shown in B;

[0141] Depend on Figure 24 A and Figure 24 B shows that AMG510 inhibited the survival and proliferation of WT-H358 cells in a dose-dependent manner, but had no inhibitory effect on the growth of drug-resistant H358-AMGR cells, indicating the successful establishment of AMG510-resistant cells; GNE-6776, as a USP7 inhibitor, showed a significant inhibitory effect on the growth of H358-WT cells and H358-AMGR cells when used alone, indicating that USP7 inhibitors can overcome AMG510 resistance and inhibit cancer cell proliferation at the cellular level; and, compared with the use of GNE-6776 alone, the growth inhibition effect of H358-WT cells and H358-AMGR cells was more significant when GNE-6776 and AMG510 were used in combination. Another USP7 inhibitor HBX41,108 also has a similar tumor inhibitory effect.

[0142] ②Colony formation test:

[0143] In H358-AMGR cells, AMG510, GNE6776 and AMG510+GNE6776 were administered respectively, and the cell proliferation changes were detected by colony formation assay. The specific method of colony formation assay is described in Example 1. The results are shown in FIG. Figure 24 As shown in C;

[0144] Depend on Figure 24C shows that in H358-AMGR cells, GNE6776 alone or in combination with AMG510 has an inhibitory effect on cell proliferation, and the combined use produces a synergistic effect.

[0145] Therefore, the combination of USP7 inhibitor and AMG510 showed synergistic efficacy against KRAS G12C mutant NSCLC cells and corresponding AMG510-resistant cells.

[0146] Example 3 Animal-level verification of the therapeutic effect of the combined use of USP7 inhibitors and KRAS inhibitors on drug-resistant NSCLC

[0147] This example verifies the therapeutic effect of the combined use of USP7 inhibitors and KRAS inhibitors on drug-resistant NSCL C at the animal level. The specific test methods and test results are as follows:

[0148] 1) Establishment of a KRAS inhibitor-resistant NSCLC nude mouse model:

[0149] The H358-AMGR drug-resistant cells prepared in Example 2 were cultured at a rate of 1×10 7 The cells were injected subcutaneously into immunodeficient (NOD / SCID) mice. Tumor formation was observed starting two weeks after injection. Thereafter, tumor growth was monitored every two days, and the weight and size of the tumor were recorded. When the tumor grew to 200 mm 3 Afterwards, AMG510 (30 mg / kg), GNE6776 (20 mg / kg) and AMG510 + GNE6776 were administered alone, respectively. The schematic diagram of drug administration is shown in the figure below. Figure 25 As shown in A; the test solution for the blank control group consisted of water for injection, 1% dimethyl sulfoxide (DMSO) and 2% polyethylene glycol 300 (PEG300). The administration cycle was once a day, and the possible systemic toxicity during the treatment was indirectly evaluated by observing the changes in the weight of the mice. After one week of continuous administration, the size, weight and volume of the mouse tumors were measured. The tumor volume of the mice was calculated according to the following formula: tumor volume = 0.5 × long diameter × short diameter squared, and a tumor growth curve was drawn. At the end of the experiment, the mice were killed with carbon dioxide. The results are shown in Figure 25 B~ Figure 25 As shown in E.

[0150] Depend on Figure 25It can be seen that AMG510 treatment alone has partial inhibitory activity on mouse tumors, with an average reduction of 43% in tumor volume. GNE6776 treatment alone shows good tumor inhibitory effect, with an average reduction of 60% in tumor volume. A more obvious tumor inhibitory effect was observed when AMG510 was combined with GNE6776, with an average reduction of 77% in tumor volume.

[0151] The growth of drug-resistant tumors in mice was also inhibited to a certain extent after using AMG510 alone. The reason is that when the drug-resistant cell lines constructed in vitro are used in in vivo models, they may show different drug resistance performances due to the complex tumor microenvironment in the body. The immune cells and stromal cells in the in vivo environment may affect the efficacy of the drug. Figure 25 The results actually reflect the synergistic effect of USP7 inhibitors combined with KRAS inhibitors.

[0152] Based on the results of Examples 2 and 3, it can be seen that treatment with USP7 inhibitors, whether used alone or in combination with AMG510, can inhibit NSCLC cell proliferation and overcome AMG510 resistance in KRAS-G12C mutant NSCLC.

[0153] The present invention provides an application of a USP7 inhibitor in the preparation of a product for treating lung cancer. The working mechanism of the application is shown in FIG. Figure 26 shown.

[0154] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. Use of a USP7 inhibitor in the preparation of a product for treating lung cancer, characterized in that: The lung cancer is non-small cell lung cancer with KRAS inhibitor resistance; The USP7 inhibitors include GNE-6776 and / or HBX41108; The KRAS inhibitors include AMG-510.

2. The use according to claim 1, characterized in that The non-small cell lung cancer includes squamous cell carcinoma, lung adenocarcinoma and large cell lung cancer.

3. Use of a USP7 inhibitor in combination with a KRAS inhibitor in the preparation of a product for treating lung cancer, characterized in that: The lung cancer is non-small cell lung cancer with KRAS inhibitor resistance; The USP7 inhibitors include GNE-6776 and / or HBX41108; The KRAS inhibitors include AMG-510.

4. The use according to claim 3, characterized in that The non-small cell lung cancer includes squamous cell carcinoma, lung adenocarcinoma and large cell lung cancer.

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