Use of aurkb as a diagnostic and therapeutic target for gastrointestinal stromal tumors

Abstract

The present application relates to the application of AURKB as a diagnostic and therapeutic target for gastrointestinal stromal tumor. Specifically, the research of the present application reveals the key role of AURKB in gastrointestinal stromal tumor, verifies that the administration of AURKB and / or ATAD2 gene knockout or inhibitor can produce the effect of inhibiting gastrointestinal stromal tumor, thereby providing the application of AURKB inhibitor, ATAD2 inhibitor, or the combination of the two in the preparation of a drug for treating GIST. The present application also provides the application of AURKB as a diagnostic target for gastrointestinal stromal tumor. The present application provides a new target and a new method for diagnosing and treating gastrointestinal stromal tumor, especially advanced (primary high-risk or metastasis) and drug-resistant gastrointestinal stromal tumor, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of AURKB as a target for the diagnosis and treatment of gastrointestinal stromal tumors. Background Technology

[0002] Gastrointestinal stromal tumors (GISTs) are mesenchymal tumors originating from the interstitial cells of Cajal (ICCs) in the gastrointestinal tract. They account for approximately 1-3% of all gastrointestinal malignancies, but constitute the majority of mesenchymal tumors of the digestive tract. The risk of GISTs is classified based on tumor location, size, mitotic figures, and whether the tumor has ruptured. Patients with high-risk or distantly metastatic GISTs have a poorer prognosis.

[0003] Currently, surgical resection is the preferred treatment for localized GIST. For advanced or metastatic patients, targeted therapies such as imatinib, sunitinib, regorafenib, and ripretinib have become standard treatments. However, there is a high probability of developing drug resistance in patients with gastrointestinal stromal tumors receiving targeted therapy, leading to treatment failure. Second-line, third-line, and fourth-line targeted therapies all present with secondary resistance issues.

[0004] Therefore, there is an urgent need in this field to find new targets related to gastrointestinal stromal tumors (GISTs) in order to develop new methods for treating GISTs. Summary of the Invention

[0005] The purpose of this invention is to provide AURKB as a target for the diagnosis and treatment of gastrointestinal stromal tumors.

[0006] In a first aspect of the invention, the use of an Aurora kinase B (AURKB) inhibitor in the preparation of a medicament for treating gastrointestinal stromal tumors is provided.

[0007] In another preferred embodiment, the AURKB inhibitor is an inhibitor of the AURKB gene, RNA, or its encoded protein.

[0008] In another preferred embodiment, the AURKB inhibitor includes: an agent that reduces the expression level of the AURKB gene, an agent that knocks out the AURKB gene, an agent that reduces the amount of AURKB protein, and / or an agent that reduces the activity of the AURKB protein.

[0009] In another preferred embodiment, the AURKB inhibitor is selected from the group consisting of small molecule compounds, antibodies, microRNA, siRNA, shRNA, antisense oligonucleotides (ASO), nucleic acid aptamers, gene editors, or combinations thereof.

[0010] In another preferred embodiment, the AURKB inhibitor is a small molecule compound, including: ilorasertib, ZM447439, VX680, AZD1152, AZD2811, or a combination thereof.

[0011] In another preferred embodiment, the AURKB inhibitor is a gene editor for knocking out or silencing the AURKB gene, including: CRISPR / Cas9, TALEN, ZFN, and base editor.

[0012] In another preferred embodiment, the sequence of the sgRNA of the CRISPR / Cas9 system is as follows:

[0013] (1) Forward: CACCGTCTAGAGTATGCCCCCCGCG (SEQ ID NO:1), Reverse: AAACCGCGGGGGGCATACTCTAGAC (SEQ ID NO:2); or

[0014] (2) Forward: CACCGAATCTGCTCTTAGGGCTCAA (SEQ ID NO:3), Reverse: AAACTTGAGCCCTAAGAGCAGATTC (SEQ ID NO:4).

[0015] In another preferred embodiment, the AURKB inhibitor can reduce the number or activity of AURKB protein by at least 20%, preferably at least 50%, and more preferably at least 80%.

[0016] In another preferred embodiment, the gastrointestinal stromal tumor includes: a gastrointestinal stromal tumor with high AURKB expression, a gastrointestinal stromal tumor with high ATAD2 expression, or a combination thereof.

[0017] In another preferred embodiment, the gastrointestinal stromal tumor includes: advanced gastrointestinal stromal tumor, high-risk gastrointestinal stromal tumor, metastatic gastrointestinal stromal tumor, drug-resistant gastrointestinal stromal tumor, and refractory gastrointestinal stromal tumor.

[0018] In another preferred embodiment, the drug-resistant gastrointestinal stromal tumor refers to a tumor resistant to drugs selected from the group consisting of: imatinib, sunitinib, regorafenib, riplatinib, and afatinib.

[0019] In a second aspect of the invention, the use of an ATAD2 inhibitor in the preparation of a medicament for treating gastrointestinal stromal tumors is provided.

[0020] In another preferred embodiment, the ATAD2 inhibitor is an inhibitor of the ATAD2 gene, RNA, or its encoded protein.

[0021] In another preferred embodiment, the ATAD2 inhibitor comprises: an agent that reduces the expression level of the ATAD2 gene, an agent that knocks out the ATAD2 gene, an agent that reduces the amount of ATAD2 protein, and / or an agent that reduces the activity of the ATAD2 protein.

[0022] In another preferred embodiment, the ATAD2 inhibitor is selected from the group consisting of small molecule compounds, antibodies, microRNA, siRNA, shRNA, antisense oligonucleotides (ASO), nucleic acid aptamers, gene editors, or combinations thereof.

[0023] In another preferred embodiment, the ATAD2 inhibitor is a small molecule compound, including: BAY-850, AM879, AZ13824374, GSK8814, and theophylline derivatives.

[0024] In another preferred embodiment, the ATAD2 inhibitor is a gene editor for knocking out or silencing the ATAD2 gene, including: CRISPR / Cas9, TALEN, ZFN, and base editor.

[0025] In another preferred embodiment, the sequence of the sgRNA of the CRISPR / Cas9 system is as follows:

[0026] (1) Forward: CACCGGTACGATTTGATAGTGTTGG (SEQ ID NO:5), Reverse: AAACCCAACACTATCAAATCGTACC (SEQ ID NO:6); or

[0027] (2) Forward: CACCGTTGCCAATGAGTGCAGTCAA (SEQ ID NO:7), Reverse: AAACTTGACTGCACTCATTGGCAAC (SEQ ID NO:8).

[0028] In another preferred embodiment, the ATAD2 inhibitor can reduce the amount or activity of ATAD2 protein by at least 30%, preferably at least 50%, and more preferably at least 80%.

[0029] In another preferred embodiment, the gastrointestinal stromal tumor includes: a gastrointestinal stromal tumor with high AURKB expression, a gastrointestinal stromal tumor with high ATAD2 expression, or a combination thereof.

[0030] In another preferred embodiment, the gastrointestinal stromal tumor includes: advanced gastrointestinal stromal tumor, high-risk gastrointestinal stromal tumor, metastatic gastrointestinal stromal tumor, drug-resistant gastrointestinal stromal tumor, and refractory gastrointestinal stromal tumor.

[0031] In a third aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0032] (a) An AURKB inhibitor as the primary active ingredient;

[0033] (b) An ATAD2 inhibitor as a second active ingredient; and

[0034] (c) Pharmaceutically acceptable carriers.

[0035] In another preferred embodiment, the pharmaceutical composition includes: injection, tablet, powder, granule, capsule, lyophilized form, solution, and syrup.

[0036] In another preferred embodiment, the pharmaceutically acceptable carrier includes, but is not limited to: solvents, dispersion media, coatings, antibacterial or antifungal agents, isotonic agents and absorption delay agents, buffered saline, physiological saline, phosphate buffer, citrate buffer, acetate buffer, bicarbonate buffer, sucrose solution, salt solution, polysorbate solution, or combinations thereof.

[0037] In another preferred embodiment, the pharmaceutically acceptable carrier is a delivery carrier, including but not limited to liposomes, nanoparticles, etc.

[0038] In a fourth aspect of the invention, a medicine box is provided, the medicine box comprising:

[0039] (a) A first pharmaceutical composition comprising (a1) an AURKB inhibitor as a first active ingredient; and (a2) a pharmaceutically acceptable carrier;

[0040] and (b) a second pharmaceutical composition comprising (b1) an ATAD2 inhibitor as a second active ingredient; and (a2) a pharmaceutically acceptable carrier.

[0041] In another preferred embodiment, the first pharmaceutical composition and the second pharmaceutical composition are independent of each other.

[0042] In a fifth aspect of the invention, there is provided the use of an AURKB detection reagent for preparing a kit for diagnosing gastrointestinal stromal tumors.

[0043] In another preferred embodiment, the AURKB detection reagent includes a reagent for detecting the AURKB gene, mRNA, cDNA, or protein.

[0044] In another preferred embodiment, the AURKB detection reagent includes a reagent for detecting the expression level of AURKB, preferably, the expression level includes protein expression level or mRNA expression level.

[0045] In another preferred embodiment, the detection reagent includes antibodies, primers, probes, sequencing libraries, nucleic acid chips (such as DNA chips) or protein chips.

[0046] In another preferred embodiment, the detection reagent is a nucleic acid reagent for detecting AURKB mRNA or cDNA.

[0047] In another preferred embodiment, the protein comprises a full-length protein or a protein fragment.

[0048] In another preferred embodiment, the kit contains one or more detection reagents selected from the group consisting of:

[0049] (i) AURKB-specific antibodies, AURKB-specific binding molecules; and / or

[0050] (ii) Primers or primer pairs, probes or chips for specific amplification of AURKB mRNA or AURKB cDNA.

[0051] In another preferred embodiment, the detection reagent is coupled with or carries a detectable marker.

[0052] In another preferred embodiment, the detectable marker is selected from the group consisting of chromophores, chemiluminescent groups, fluorophores, isotopes, or enzymes.

[0053] In another preferred embodiment, the specific antibody for AURKB is a monoclonal antibody or a polyclonal antibody.

[0054] In another preferred embodiment, the expression level of AURKB protein in the sample is detected by RT-PCR or immunohistochemistry.

[0055] In another preferred embodiment, the detection includes testing a sample of the subject's tumor tissue.

[0056] In another preferred embodiment, an upregulated AURKB expression level in the sample indicates that the subject has a gastrointestinal stromal tumor; preferably, it indicates that the subject has a high-risk and / or metastatic gastrointestinal stromal tumor.

[0057] In another preferred embodiment, the kit further includes detection reagents selected from the group consisting of:

[0058] (Y1) A reagent for detecting the ATAD2 gene, mRNA, cDNA, or protein; and / or

[0059] (Y2) A reagent used to detect the FOXM1 gene, mRNA, cDNA, or protein.

[0060] In another preferred embodiment, when the expression levels of AURKB and ATAD2 in the sample are both upregulated, it indicates that the subject has a gastrointestinal stromal tumor; preferably, it indicates that the subject has a high-risk and / or metastatic gastrointestinal stromal tumor.

[0061] In a sixth aspect of the invention, a method for screening candidate compounds for treating gastrointestinal stromal tumors is provided, the method comprising the steps of:

[0062] (a) In the test group, the test compound was added to the cell culture system, and the expression level (E1) and / or activity (A1) of AURKB in the cells of the test group were observed; in the control group, the test compound was not added to the same cell culture system, and the expression level (E0) and / or activity (A0) of AURKB in the cells of the control group were observed.

[0063] If the expression level (E1) and / or activity (A1) of AURKB in the test group is significantly lower than that in the control group, it indicates that the test compound is a candidate compound for the treatment of gastrointestinal stromal tumors that inhibits the expression and / or activity of AURKB.

[0064] In another preferred embodiment, the expression level of AURKB is obtained by qPCR.

[0065] In another preferred embodiment, the method further includes the step of:

[0066] (b) For the candidate compounds obtained in step (a), further test whether they have a downregulating effect on AURKB expression.

[0067] In another preferred embodiment, the method includes step (c): applying the candidate compound identified in step (a) to a mammalian model and determining its effect on the mammal.

[0068] In another preferred embodiment, the mammal is a mammal suffering from gastrointestinal stromal tumors.

[0069] In another preferred embodiment, “significantly lower than” means E1 / E0 ≤ 1 / 2, more preferably ≤ 1 / 3, and even more preferably ≤ 1 / 4.

[0070] In another preferred embodiment, “significantly lower than” means A1 / A0 ≤ 1 / 2, more preferably ≤ 1 / 3, and even more preferably ≤ 1 / 4.

[0071] In another preferred embodiment, the cells include gastrointestinal stromal tumor cells, including: GIST-T1, GISTT-T1 / 820, GIST430, and GIST430 / 654 cell lines.

[0072] In another preferred embodiment, the cells are cells cultured in vitro.

[0073] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0074] In a seventh aspect of the invention, a method for treating gastrointestinal stromal tumors is provided, comprising the step of administering an AURKB inhibitor to a subject in need.

[0075] In another preferred embodiment, the object includes a human or a non-human mammal.

[0076] In another preferred embodiment, the subject is a patient with a gastrointestinal stromal tumor.

[0077] In another preferred embodiment, the gastrointestinal stromal tumor includes high-risk, metastatic, and / or drug-resistant gastrointestinal stromal tumors.

[0078] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0079] Figure 1The integrated screening of GIST patient samples using whole transcriptome sequencing, whole-genome CRISPR screening, and small molecule compound library screening is shown. (A) is a schematic diagram of the screening; (B) a volcano plot shows differentially expressed genes in metastatic vs. low-risk GIST samples. The two vertical dashed lines in the figure represent gene expression differences of 4 and -4 folds in metastatic vs. low-risk GIST patient samples, respectively, and the horizontal dashed line represents a corrected p-value of 0.05. The color and size of the dots in the figure reflect the magnitude of the corrected p-value; (C) a heatmap shows the expression of 212 genes with gene expression differences greater than 4 or less than -4 folds in metastatic vs. low-risk GIST patient samples. Genes related to cell proliferation are marked; (D) a circle plot shows the results of GO analysis for the 212 genes in (C). The scatter plot in the outer circle represents genes enriched in this pathway and their log2FCs, with yellow indicating downregulation; (E) Pierman correlation analysis shows the correlation between CRISPR score and βscore in GIST430 and GIST430 / 654 cells; (F) The scatter plot shows the different sensitivities of GIST430 / 654 to 4919 small molecule compounds in the MCE Online Bioactive Compound Library. Aurora kinase family inhibitor ilorasertib and ATAD2 inhibitor BAY-850 are marked in red. Core small molecule drugs with inhibition rates greater than 0.9 are marked in blue. Sunitinib, afatinib, and retrinib were used as positive controls for drug screening. Horizontal lines indicate drug inhibition rates equal to 0.9; (G) The pie chart shows the pathway enrichment results for core small molecule drugs with inhibition rates greater than 0.9.

[0080] Figure 2 The integrated screening identified AURKB as a potential target for high-risk and metastatic GIST. (A) The UpSet plot shows the overlapping genes among the three screenings. The numbers above the bars represent the number of overlapping genes between different screenings, and the numbers on the left side of the graph represent the total number of genes included in different screenings; (B) A scatter plot of CRISPR values ​​ranking genes with expression levels higher than 4-fold in metastatic vs. low-risk GIST patient samples. AURKB is highlighted in red; (C) Transcriptome expression levels of AURKB in GIST patient samples of different risk levels; (D) AURKB expression levels in different metastatic lesions of the same GIST patient; (E) Immunohistochemical characterization of AURKB protein expression levels in GIST patient samples of different risk grades. GAPDH was used as an internal control; (F) Results of AURKB immunohistochemistry in a cohort of 315 formalin-fixed paraffin-embedded surgical clinical GIST samples. Scale bar: 30 micrometers; (G) Statistical analysis of plot (F).

[0081] Figure 3The results show that GIST cells depend on AURKB. (A) Scatter plot of CRISPR values ​​ranked in the Hemming ML et al., 2022 GIST430 / 654 CRISPR screening for genes with expression levels higher than 4-fold in metastatic vs. low-risk GIST patient samples. AURKB is highlighted in red. (B) Scatter plot of Z-values ​​ranked in the DepMap GIST-T1 RNAi database for genes with expression levels higher than 4-fold in metastatic vs. low-risk GIST patient samples. AURKB is highlighted in red. (C) DepMap contains AURKB RNAi screening data for 710 tumor cell lines. GIST-T1 is highlighted in red. (D) Pearson correlation analysis showed a significant positive correlation between AURKB and MKI67 transcriptional expression levels in 105 GIST patient samples.

[0082] Figure 4 The results show that AURKB knockout inhibits tumor growth and proliferation. (A) CellTiter-Glo viability assays showed that lentivirus-mediated AURKB knockout reduced the viability of GIST-T1 and GIST430 / 654 cells; (B) Flow cytometry showed that AURKB knockout led to cell cycle arrest in the G2 / M phase; (C) Crystal violet staining showed that AURKB knockout reduced the proliferation capacity of GIST-T1 cells; (D) SA-β-Gal staining showed that AURKB knockout increased the proportion of senescent cells; (EJ) AURKB knockout inhibited the tumorigenicity of GIST430 / 654 (EG) and GIST-T1 (HJ) cells in nude mice. (K) The left image shows hematoxylin and eosin staining from tumors (F) and (I), and the right image shows immunohistochemical staining with KI67.

[0083] Figure 5 The results show that AURKB knockout inhibits tumor progression. (A) Immunoblotting validates the knockout efficiency of AURKB in multiple GIST cell lines. GAPDH was used as an internal control. (B) CellTiter-Glo viability assay shows that lentivirus-mediated AURKB knockout reduces the cell viability of GIST-T1 / 820 and GIST430 cells. (C) Flow cytometry shows that AURKB knockout leads to cell cycle arrest in the G2 / M phase. (D) Crystal violet staining shows that AURKB knockout reduces the proliferation capacity of GIST-T1 / 820 cells. (E) SA-β-Gal staining shows that AURKB knockout increases the proportion of senescent cells. Scale bar: 50 micrometers. (F) Flow cytometry analysis shows that AURKB knockout leads to an increased proportion of apoptotic cells.

[0084] Figure 6The antitumor activity of AURKB inhibitors in imatinib-sensitive and resistant GIST models is shown. (A) Scatter plot of the results of compound sensitivity screening in GIST430 / 654 cells using the MCE small molecule compound library. (B) Results of CellTiter-Glo growth sensitivity assay after treating GIST430 / 654 cells with different concentrations of ilorasertib or LY3295668 for 72 h; (C) Results of CellTiter-Glo growth sensitivity assay after treating GIST430 / 654 cells with 2 μM ilorasertib, LY3295668 or AZD1152 for 72 h; (D) Results of CellTiter-Glo growth sensitivity assay after treating GIST430 / 654 cells or H1792 cells with different concentrations of ilorasertib for 72 h; (E) Results of CellTiter-Glo growth sensitivity assay after treating imatinib-sensitive or resistant GIST cells and non-GIST cells with different concentrations of ZM447439 or VX680 for 72 h; (F) Half-maximal inhibitory concentrations (IC50) of ZM447439 and VX680 for different tumor cell lines in the GDSC database. This section supplements data on GIST cell lines, as the original GDSC database did not contain data on the half-maximal inhibitory concentrations (IC50) of GIST cell lines against ZM447439 and VX680; (G) Results of CellTiter-Glo growth sensitivity assays after treating imatinib-sensitive or resistant GIST cells and non-GIST cells with different concentrations of AZD1152 for 72 h; (H) Tumor growth curves in GIST-T1 tumor-bearing nude mice treated with different concentrations of AZD1152; (I) Tumor growth curves in imatinib-resistant GIST430 / 654 tumor-bearing nude mice treated with AZD1152; (J) Tumor growth curves in tumor-bearing nude mice inoculated with tissues from fourth-line resistant GIST patients treated with AZD1152.

[0085] Figure 7 The AURKB-specific inhibitor AZD1152 was shown to inhibit GIST proliferation in vivo without significant toxic side effects. (A) Relative body weight changes in GIST-T1 (left) and GIST430 / 654 (right) tumor-bearing nude mice treated with AZD1152; (B) Aspartate aminotransferase (AST) vs. alanine aminotransferase (ALT) activities in GIST-T1 (left) and GIST430 / 654 (right) tumor-bearing mice treated with AZD1152; (C) Complete blood count of GIST-T1 tumor-bearing mice treated with AZD1152; (D) Complete blood count of GIST430 / 654 tumor-bearing mice treated with AZD1152.

[0086] Figure 8The study demonstrated a protein-protein interaction between AURKB and ATAD2. (A) Interaction between exogenous AURKB and endogenous ATAD2 in GIST430 and GIST430 / 654 cells. β-ACTIN was used as an internal control. (B) Interaction between endogenous AURKB and endogenous ATAD2 in GIST430 and GIST430 / 654 cells, an interaction not observed in H1792 and PANC1. (C) Immunoprecipitation assay using AURKB truncated mutant and ATAD2. (D) Co-expression of MYC-labeled ATAD2 and FLAG-labeled wild-type AURKB or FLAG-labeled AURKB kinase domain in HEK 293T cells, followed by immunoprecipitation and immunoblotting. (E) Immunofluorescence assay performed in GIST430 / 654 cells. Scale bar: 20 micrometers; (F) Immunoblotting analysis to analyze the correlation between AURKB and ATAD2 expression in GIST patient samples; (G) Pearson correlation analysis to analyze the correlation of protein expression in (F); (H) Detection of ATAD2 and AURKB mRNA levels in AURKB knockout GIST430 / 654 samples by qRT-PCR. (I) Detection of ATAD2 and AURKB mRNA levels in ATAD2 knockout GIST430 / 654 samples by qRT-PCR.

[0087] Figure 9 This study demonstrates that ATAD2 is an interacting substrate for AURKB. (a) Identification of novel interacting substrates for AURKB in GIST using proteomic profiling following co-IP. (b) Identification of AURKB-related proteins using proteomic profiling. (c) Interaction between exogenous AURKB and endogenous ATAD2 in GIST-T1 and GIST48B cells. (d) Schematic diagram of AURKB truncated mutations; (e) Correlation between AURKB mRNA levels and ATAD2 mRNA levels in GIST patient samples.

[0088] Figure 10This study showed that inhibiting AURKB activity leads to the degradation of ATAD2 via ubiquitination. (A) Immunoblotting analysis of changes in ATAD2 protein levels in AURKB knockout GIST430 and GIST430 / 654 cells; (B) Immunoblotting analysis of changes in ATAD2 protein levels in AURKB knockout and AURKB-replenished GIST430 / 654 cells; (C) Immunoblotting analysis of changes in AURKB protein levels in ATAD2 knockout GIST430 / 654 cells; (D) Changes in ATAD2 protein levels in GIST430 / 654 cells after treatment with different concentrations of AZD1152 for 48 h; (E) Changes in ATAD2 protein levels in GIST430 / 654 cells after treatment with 1 μM and 2 μM AZD1152 for 6 h. The phosphorylation level of histone H3 S10 was used as a reference for AURKB kinase activity; (F) Immunoblotting detection of changes in ATAD2 protein levels in GIST430 / 654 cells treated with AZD1152, PLK1 inhibitor onvansertib, and CDK4 / 6 inhibitor palbociolib; (G) Changes in ATAD2 protein levels in AURKB knockout GIST430 / 654 and GIST430 cells treated with the protein synthesis inhibitor CHX; (H) Changes in ATAD2 protein levels in AURKB knockout GIST430 / 654 and GIST430 cells treated with the proteasome inhibitor MG132 for 6 h; (I) In HEK 293T cells were co-expressed with MYC-labeled ATAD2, HA-labeled ubiquitin (HA-Ub), and FLAG-labeled wild-type AURKB or FLAG-labeled AURKB with missing kinase domain. Immunoprecipitation and immunoblotting were then performed to detect changes in ATAD2 ubiquitination levels. (J) Changes in endogenous ATAD2 ubiquitination levels were detected in GIST48B cells treated with AZD1152.

[0089] Figure 11 This study demonstrates how AURKB regulates ATAD2 protein levels. (a) Immunoblotting analysis of changes in ATAD2 protein levels in AURKB-knockout GIST48 and GIST48B cells; (b) Immunoblotting analysis of changes in ATAD2 protein levels in AURKB-knockout and AURKB-replenished GIST430 cells; (c) Immunohistochemical analysis. Figure 4The protein level of ATAD2 in GIST430 / 654 tumor-bearing mice in F. Scale bar: 30 micrometers; (d) Changes in ATAD2 protein level in GIST48B cells after treatment with different concentrations of AZD1152 for 48 h; (e) Changes in ATAD2 protein level in GIST48B and GIST-T1 cells after treatment with 1 μM and 2 μM AZD1152 for 6 h. The phosphorylation level of histone H3 S10 was used as a reference for AURKB kinase activity; (f) Changes in ATAD2 protein level in AURKB knockout GIST48B and GIST-T1 cells after treatment with the protein synthesis inhibitor CHX.

[0090] Figure 12 This study shows that ATAD2 is an oncogene for GIST. (A) ATAD2 expression levels in GISTs of different risk grades; (B) ATAD2 expression levels in 5 GIST patients and their paired normal adjacent normal tissues; (C) CellTiter-Glo assay revealed that lentivirus-mediated ATAD2 knockout reduced the viability of GIST430 / 654 cells; (D) CellTiter-Glo assay of changes in the viability of GIST430 / 654 cells after ATAD2 knockout and replenishment; (E) CellTiter-Glo assay of changes in the viability of AURKB knockout GIST430 / 654 cells after replenishment with wild-type AURKB, kinase-inactivated AURKB, and ATAD2; (FG) AURKB and ATAD2 replenishment can rescue tumor growth in AURKB knockout GIST430 / 654 tumor-bearing nude mice. (H) The volcano plot shows the significantly upregulated (orange) and downregulated (blue) signaling pathways after GSEA analysis of RNAseq data following ATAD2 knockout. Signaling pathways related to DNA damage repair are marked in red; (I) The heatmap shows the similarity of GSEA signaling pathway changes in AURKB knockout and ATAD2 knockout GIST430 / 654; (J) The GSEA plot shows that signaling pathways related to DNA damage repair are significantly downregulated in AURKB knockout GIST430 / 654; (K) The heatmap shows the changes in the overall chromatin openness after ATAD2 knockout; (L) The chromatin openness of gene promoter regions related to DNA damage repair after ATAD2 knockout.

[0091] Figure 13This study demonstrates the ATAD2-dependent function of GIST cells. (A) CellTiter-Glo assays revealed that lentivirus-mediated ATAD2 knockout reduced the viability of GIST430 cells; (B) CellTiter-Glo assays showed changes in the viability of GIST430 cells after ATAD2 knockout and replenishment; (C) CellTiter-Glo assays showed changes in the viability of AURKB-knockout GIST430 cells after replenishment with wild-type AURKB, kinase-inactivated AURKB, and ATAD2; (D) The DepMap database showed no significant correlation between ATAD2 dependence and AURKB dependence in 1066 tumor cell lines, none of which included GIST cell lines; (E) Density histograms showed the ATAD2 dependence of all tumor cell lines in the DepMap Public 24Q2+Score Chronos database. Red lines represent a gene effect of -1, and gray lines represent a gene effect of 0. (FG) The effect of ATAD2 knockout on the viability of H1792(F) and PANC1(G) cells was detected by CellTiter-Glo; (H) Immunoblotting was used to verify the knockout efficiency of ATAD2 in H1792 and PANC1 cells; (I) Volcano plots showed the gene expression changes caused by ATAD2 knockout in GIST430 / 654 cells; (J) GSEA plots showed that signaling pathways related to DNA damage repair were significantly downregulated in ATAD2 knockout GIST430 / 654 cells; (K) Heatmaps showed the similarity of GSEA signaling pathway changes in GIST430 / 654 cells treated with the AURKB-specific inhibitor AZD1152 after ATAD2 knockout; (L) GSEA plots showed that signaling pathways related to DNA damage repair were significantly downregulated in GIST430 / 654 cells treated with the AURKB-specific inhibitor AZD1152.

[0092] Figure 14This study demonstrates that FOXM1 directly regulates AURKB expression. (A) Correlation between 817 transcription factors and AURKB expression at the mRNA level; (B) Expression levels of the four transcription factors most associated with AURKB expression in GIST patient samples; (C) Expression levels of FOXM1 in GISTs with different risk levels; (D) Pearson correlation analysis showing a significant correlation between AURKB and FOXM1 expression in GIST patients; (E) Immunoblotting detection of AURKB protein expression levels after FOXM1 knockout; (F) Immunoblotting detection of changes in ATAD2 and AURKB expression levels in GIST cells after 6 hours of treatment with the FOXM1 inhibitor Thiostrepton; (G) FOXM1 regulates the AURKB promoter region; (H) FOXM1 regulates AURKB through promoter region 1. Detailed Implementation

[0093] Through extensive and in-depth research, the inventors have for the first time identified AURKB as a key target promoting GIST cell proliferation, and revealed that AURKB promotes GIST cancer development through its interacting substrate ATAD2, and discovered that the transcription factor FOXM1 directly regulates the transcriptional expression of AURKB. Based on the above mechanistic research, the inventors have provided the application of AURKB inhibitors, ATAD2 inhibitors, or combinations thereof in the preparation of drugs for treating GIST, and verified that the knockout or administration of AURKB and / or ATAD2 gene inhibitors produced GIST-inhibiting effects in both cell and animal experiments. Furthermore, since AURKB expression is significantly elevated in high-risk / metastatic GIST patient samples, AURKB can also serve as a target for diagnosing GIST. This invention was completed based on these findings.

[0094] definition

[0095] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0096] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0097] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0098] AURKB gene and protein

[0099] The Aurora kinase family is a group of serine / threonine protein kinases associated with the regulation of centrosome and microtubule function, participating in various events such as centrosome replication and separation, spindle microtubule assembly and stabilization. The mammalian cell Aurora kinase family currently has three members: Aurora-A (AURKA), Aurora-B (AURKB), and Aurora-C (AURKC).

[0100] Aurora kinase B (AURKB) is an important member of the serine / threonine protein kinase family, located on chromosome 17p13.1. The AURKB protein it encodes plays a core regulatory role in mitosis and cytokinesis. AURKB The uniprot sequence number is Q96GD4, and the amino acid sequence is MAQKENSYPWPYGRQTAPSGLSTLPQRVLRKEPVTPSALVLMSRSNVQPTAAPGQKVMENSSGTPDILTRHFTIDDFEIGRPLGKGKFGNVYLAREKKSHFIVALKVLFKSQIEKEGVEHQLRREIEIQAHLHHPNILRLYNYFYDRRRIYLILEYAPRGEL YKELQKSCTFDEQRTATIMEELADALMYCHGKKVIHRDIKPENLLLGLKGELKIADFGWSVHAPSLRRKTMCGTLDYLPPEMIEGRMHNEKVDLWCIGVLCYELLVGNPPFESASHNETYRRIVKVDLKFPASVPMGAQDLISKLLRHNPSERLPLAQVSAHPWVRANSRRVLPPSALQSVA(SEQ ID NO:9). The full-length human AURKB protein contains 344 amino acids, of which positions 78-326 are the kinase domain, the integrity of which is the basis for catalytic activity.

[0101] It should be understood that the term "AURKB" also includes various naturally occurring variants of the AURKB gene. Representative examples include: nucleotide sequences encoding the same AURKB protein as the wild type due to codon degeneracy, and nucleotide sequences of conserved variant polypeptides encoding the wild-type AURKB protein. Furthermore, for mammals other than humans, the term refers to the homologue of the AURKB gene in that animal. For example, in mice, the term refers to mouse AURKB.

[0102] ATAD2 gene and protein

[0103]

[0104] It should be understood that the term "ATAD2" also includes various naturally occurring variants of the ATAD2 gene. Representative examples include: nucleotide sequences encoding the same ATAD2 protein as the wild-type ATAD2 protein due to codon degeneracy, and nucleotide sequences of conserved variant polypeptides encoding the wild-type ATAD2 protein. Furthermore, for mammals other than humans, the term refers to the homologue of the ATAD2 gene in that animal. For example, in mice, the term refers to mouse ATAD2.

[0105] AURKB inhibitors

[0106] Various conventional screening methods can be used to screen for substances that interact with the AURKB gene or protein, especially inhibitors.

[0107] In this invention, the AURKB inhibitor can be used to prepare a drug for treating gastrointestinal stromal tumors.

[0108] The AURKB inhibitors (or antagonists) that can be used in this invention include any substance that can inhibit the expression and / or activity of the AURKB gene or its encoded protein.

[0109] For example, the AURKB inhibitors include antibodies against AURKB, antisense RNA of AURKB nucleic acid, siRNA, shRNA, miRNA, gene editors, or inhibitors of AURKB activity. A preferred AURKB inhibitor refers to an antibody capable of inhibiting AURKB expression.

[0110] In a preferred embodiment, the method and steps for inhibiting AURKB include neutralizing its protein with an antibody against AURKB, silencing the AURKB gene using shRNA or siRNA carried by a virus (such as a lentivirus) or a gene editor.

[0111] The inhibition rate of AURKB is generally at least 50%, preferably 60%, 70%, 80%, 90%, or 95%. The inhibition rate of AURKB can be controlled and detected based on conventional techniques, such as flow cytometry, quantitative real-time PCR, or Western blot.

[0112] ATAD2 inhibitors

[0113] In this invention, the use of ATAD2 inhibitors in the preparation of medicaments for the treatment of gastrointestinal stromal tumors is provided.

[0114] The ATAD2 inhibitors that can be used in this invention include any substance that can inhibit the expression and / or activity of the ATAD2 gene or its encoded protein.

[0115] For example, the ATAD2 inhibitors include antibodies against ATAD2, antisense RNA of ATAD2 nucleic acid, siRNA, shRNA, miRNA, gene editors, or small molecule compounds that inhibit ATAD2 activity. A preferred ATAD2 inhibitor refers to a small molecule compound capable of inhibiting ATAD2 activity.

[0116] In a preferred embodiment, the method and steps for inhibiting ATAD2 include silencing the ATAD2 gene using shRNA or siRNA carried by a virus (such as adeno-associated virus) or a gene editor.

[0117] The inhibition rate of ATAD2 is generally at least 50%, preferably 60%, 70%, 80%, 90%, or 95%. The inhibition rate of ATAD2 can be controlled and detected based on conventional techniques.

[0118] Pharmaceutical Composition

[0119] The AURKB inhibitor, ATAD2 inhibitor, or combinations thereof (including antibodies, antisense nucleic acids, gene editors, and other inhibitors) of the present invention, when administered therapeutically, can inhibit the expression and / or activity of AURKB or ATAD2 proteins, thereby inhibiting gastrointestinal stromal tumors. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH may vary depending on the nature of the formulated substance and the disease to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): local, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, local administration, and reinfusion after autologous cell extraction and culture.

[0120] This invention also provides a pharmaceutical composition containing a safe and effective amount of an inhibitor of the invention (such as an antibody, gene editor, antisense sequence (such as siRNA)) and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of this invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as tablets and capsules can be prepared using conventional methods. Pharmaceutical compositions such as injections, solutions, tablets, and capsules are preferably manufactured under aseptic conditions.

[0121] application

[0122] In this invention, the use of the inhibitors or pharmaceutical compositions of the invention is also provided, administered to subjects in need, for the treatment, prevention and / or relief of gastrointestinal stromal tumors, especially high-risk, metastatic and / or drug-resistant gastrointestinal stromal tumors.

[0123] As used herein, the term "high-risk gastrointestinal stromal tumor" includes high-risk gastrointestinal stromal tumors (GISTs) as defined by the modified National Institutes of Health (NIH) risk grading system for GISTs. The modified NIH risk grading system for GISTs defines each risk level as follows: Low-risk GISTs are defined as tumors originating in any location, with a size of 5 cm or less (tumor size is measured by the longest diameter of the tumor) and a mitotic count of less than 5 per 50x high-power field; intermediate-risk GISTs are defined as tumors originating in the stomach, with a size of less than 5 cm and a mitotic count between 6 and 10 per 50x high-power field, or tumors with a size between 5 and 10 cm and a mitotic count of less than 5 per 50x high-power field. Gastrointestinal stromal tumors (GISTs) are defined as high-risk tumors if they rupture. Tumors originating in any location with a size greater than 5 cm and a mitotic count greater than 5 per 50x high-power field, or tumors larger than 10 cm with a mitotic count greater than 10 per 50x high-power field, are defined as high-risk GISTs. Tumors originating outside the stomach with a size between 2 and 5 cm and a mitotic count greater than 5 per 50x high-power field, or tumors between 5 and 10 cm and a mitotic count less than 5 per 50x high-power field, are also defined as high-risk GISTs.

[0124] As used in this article, the term "treatment" refers to both therapeutic and preventative measures. Those requiring treatment may include individuals who already have gastrointestinal stromal tumors (GISTs) and those who may eventually develop the disease.

[0125] As used herein, the terms “subject” and “object” are used interchangeably and include any human or non-human mammal, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, rats, mice, etc.

[0126] As used herein, the term "effective amount" or "effective dose" refers to an amount that is functional or active in humans and / or animals and is acceptable to them. It refers to the amount of a therapeutic agent that treats, alleviates, or prevents a target disease or condition, or that exhibits a detectable therapeutic or preventative effect. Therapeutic effects also include a reduction in physiological symptoms. The precise effective amount for a given subject depends on the subject's body size and health status, the nature and severity of the condition, and the choice of the therapeutic agent and / or combination of therapeutic agents administered. For a given condition, the effective amount can be determined using routine laboratory methods.

[0127] The effective amount of a pharmaceutical composition can vary depending on the mode of administration and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the drug, such as tissue distribution, bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration.

[0128] The beneficial effects of this invention include:

[0129] (1) This invention reveals for the first time the key role of AURKB in gastrointestinal stromal tumors and provides the application of AURKB in the diagnosis of gastrointestinal stromal tumors, especially high-grade gastrointestinal stromal tumors.

[0130] (2) This invention provides for the first time the application of AURKB inhibitors in the inhibition and treatment of gastrointestinal stromal tumors, especially the application in the treatment of drug-resistant gastrointestinal stromal tumors.

[0131] (3) This invention reveals for the first time that AURKB affects the proliferative activity of gastrointestinal stromal tumor cells through its substrate ATAD2, and provides the application of ATAD2 inhibitors for the inhibition and treatment of gastrointestinal stromal tumors.

[0132] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and fractions are weight percentages and weight fractions.

[0133] Example 1: Screening and Identification of Therapeutic Targets for GIST

[0134] GIST-related genes were integrated and screened using whole transcriptome sequencing, whole-genome CRISPR screening, and small molecule compound library screening. (See schematic diagram.) Figure 1 A. The specific method is as follows:

[0135] (1) Whole transcriptome sequencing: Transcriptome sequencing was performed on 105 GIST patient samples, including 31 low-risk, 18 intermediate-risk, 28 high-risk, and 28 metastatic cases, to analyze differentially expressed genes in GIST patient samples of different risk levels.

[0136] (2) Whole-genome CRISPR screening: The whole-genome CRISPR screen library GeCKOv2 was used to perform whole-genome CRISPR knockout screening on GIST430 (imatinib-sensitive GIST cell line) and GIST430 / 654 (imatinib-resistant GIST cell line).

[0137] (3) Screening of small molecule compound library: GIST430 / 654 cells were screened for drug treatment using the MCE Online Bioactive Compound Library, which contains 4919 small molecule compounds.

[0138] The results are as follows Figure 1 As shown, Figure 1 Images B and C show the differentially expressed genes identified in samples from patients with metastatic GIST and low-risk GIST. Figure 1 D shows the results of the GO analysis. Figure 1 The E-squared values ​​showed a strong correlation between CRISPR score and β-score in GIST430 and GIST430 / 654 cells. In the small molecule compound screening, core small molecule drugs with inhibition rates greater than 0.9 were identified, among which the Aurora kinase inhibitor ilorasertib and the ATAD2 inhibitor BAY-850 were highlighted. Figure 1 The pathway enrichment results of core small molecule drugs with inhibition rates greater than 0.9 (F) showed that they mainly involved signaling pathways such as apoptosis and cell cycle.

[0139] Data from three integrated screening methods were combined to identify targets that are highly expressed in metastatic GISTs, whose knockout inhibits cell proliferation, and whose related inhibitors also have a significant inhibitory effect on GIST cells. Western blot was performed on GIST patients with different risk levels to detect the protein expression level of these targets.

[0140] The results are as follows Figure 2 As shown, integrated screening identified AURKB as a potential target for high-risk and metastatic GIST. Figure 2 Results from C, E, and F showed that both AURKB mRNA and protein expression levels were elevated in high-risk and metastatic patient samples. Therefore, AURKB could serve as a target for diagnosing gastrointestinal stromal tumors.

[0141] Further verification of the critical dependence of AURKB in GIST using CRISPR and RNAi screening yielded the following results: Figure 3As shown, AURKB's CRISPR score was significantly low, and its Z-value was significantly negative in RNAi screening. GIST-T1 cells were highly sensitive to AURKB knockdown, and AURKB was positively correlated with the transcriptional level of the proliferation marker MKI67, indicating that AURKB is a key gene dependent on GIST cell survival.

[0142] Example 2: AURKB promotes GIST cell proliferation and tumor progression

[0143] (I) AURKB and GIST cell proliferation

[0144] To verify the promoting effect of AURKB on GIST proliferation at the cellular level, AURKB was knocked out in GIST cells using CRISPR / Cas9. Cell viability was detected using CellTiter-Glo, cell colony formation ability was detected using crystal violet staining, cell apoptosis and cell cycle changes were detected using flow cytometry, and cell senescence was detected using SA-β-Gal staining.

[0145] The results are as follows Figure 4 As shown in AD. Figure 4 CellTiter-Glo viability assays in A showed that lentivirus-mediated AURKB knockout reduced the viability of GIST-T1 and GIST430 / 654 cells. Figure 4 Flow cytometry results in cell line B showed that AURKB knockout led to cell cycle arrest at the G2 / M phase; similarly, crystal violet staining showed that AURKB knockout reduced the proliferation capacity of GIST-T1 cells. Figure 4 SA-β-Gal staining experiments showed that AURKB knockout increased the proportion of senescent cells (C). Figure 4 (D). The above results indicate that AURKB plays a key role in GIST cell proliferation.

[0146] (II) Tumor Progression of AURKB and GIST

[0147] To verify the association between AURKB and GIST tumor progression in animals, AURKB-knockout GIST cells were inoculated into nude mice, and the effect of AURKB on the in vivo proliferation of GIST cells was examined.

[0148] The sgRNA sequence of the CRISPR / Cas9 system used to knock out AURKB is sgAURKB#1(sg1)forward:CACCGTCTAGAGTATGCCCCCCGCG(SEQ ID NO:1),reverse:AAACCGCGGGGGGCATACTCTAGAC(SEQ ID NO:2); or sgAURKB#2(sg2)forward:CACCGAATCTGCTCTTAGGGCTCAA(SEQ ID NO:3),reverse:AAACTTGAGCCCTAAGAGCAGATTC(SEQ ID NO:4).

[0149] The results are as follows Figure 4 As shown in EK, tumor volume and tumor weight were significantly suppressed in the AURKB knockout group for both GIST430 / 654 (EG) and GIST-T1 (HJ), indicating that AURKB knockout inhibited the tumorigenicity of GIST430 / 654 (EG) and GIST-T1 (HJ) in nude mice.

[0150] The above experimental results were further validated in various GIST cell lines, including GIST430 and GIST-T1 / 820, as shown in the following figures. Figure 5 As shown, consistent results were obtained in experiments on different cell lines: AURKB knockout inhibited cell proliferation and increased the proportion of apoptosis.

[0151] Example 3: Antitumor activity of AURKB inhibitors against GIST

[0152] (I) Sensitivity of GIST cells to AURKB inhibitors

[0153] GIST cells were treated with different AURKB inhibitors, including ilorasertib, ZM447439, VX680, and AZD1152, and the sensitivity of GIST cells to AURKB inhibitors was detected. The AURKB inhibitor LY3295668 was used as a control. Details are as follows:

[0154] GIST430 / 654 cells were treated with different concentrations of ilorasertib or LY3295668 for 72 h, followed by CellTiter-Glo growth sensitivity assays. The results are as follows: Figure 6 As shown in Figure B, the AURKB inhibitor ilorasertib significantly inhibited the activity of GIST430 / 654 cells, while the AURKB inhibitor LY3295668 had no significant effect on GIST cell activity.

[0155] Similarly, GIST430 / 654 cells were treated with 2 μM ilorasertib, LY3295668, or AZD1152 for 72 h, and the results of the CellTiter-Glo growth sensitivity assay were as follows: Figure 6 As shown in Figure C, both AURKB inhibitors ilorasertib and AZD1152 significantly inhibited the activity of GIST430 / 654 cells.

[0156] (II) Tumor-specific sensitivity of GIST cells to AURKB inhibitors

[0157] Non-GIST cells, including lung adenocarcinoma cells H1792, pancreatic cancer cells PANC1, and human skin fibroblasts BJ, were treated with AURKB inhibitors to determine whether the sensitivity of GIST cells to AURKB inhibitors is tumor-specific. The details are as follows:

[0158] GIST430 / 654 cells or lung adenocarcinoma H1792 cells were treated with different concentrations of ilorasertib for 72 hours, followed by CellTiter-Glo growth sensitivity assays. Figure 6 As shown in Figure D, the AURKB inhibitor ilorasertib significantly inhibited the activity of GIST430 / 654 cells, but had no significant effect on the activity of lung adenocarcinoma H1792 cells.

[0159] The effects of different types of AURKB inhibitors were further tested. Imatinib-sensitive or resistant GIST cells and non-GIST cells were treated with different concentrations of ZM447439, VX680, or AZD1152 for 72 hours. The results of the CellTiter-Glo growth sensitivity assay were then performed. Figure 6 As shown in E and G, ZM447439, VX680, or AZD1152 all have inhibitory effects on different types of GIST cells, but no significant effect on non-GIST cells. The effects of different types of AURKB inhibitors on different types of cells show a consistent trend.

[0160] (III) The antitumor effect of AURKB inhibitors in vivo

[0161] Nude mice bearing GIST cells or GIST patient tissue were treated with the AURKB-specific inhibitor AZD1152. The in vivo effects of the AURKB inhibitor were then assessed, as detailed below:

[0162] Tumor growth curves of GIST-T1 tumor-bearing nude mice treated with different concentrations of AZD1152 are shown below. Figure 6As shown in H, the tumor growth curves of AZD1152-treated imatinib-resistant GIST430 / 654 tumor-bearing nude mice are as follows: Figure 6 As shown in Figure I, the tumor growth curves of tumor-bearing nude mice after inoculation with tissues derived from fourth-line drug-resistant GIST patients treated with AZD1152 are as follows: Figure 6 As shown in Figure J, AZD1152 significantly inhibited tumor volume in different GIST tumor model mice.

[0163] Especially Figure 6 The patient-derived PDX model shown in J, who developed resistance to imatinib, sunitinib, regorafenib, and ripetinib, was shown to be inhibited by AZD1152, indicating that AURKB inhibitors can be used to treat drug-resistant GIST.

[0164] Example 4: Safety of AURKB inhibitors in vivo

[0165] To assess the safety of AURKB inhibitors in vivo, changes in body weight of tumor-bearing mice after treatment with AURKB inhibitors were recorded, and blood samples were collected from tumor-bearing mice after treatment with AURKB inhibitors for complete blood count and liver toxicity testing to determine the toxic side effects of the AURKB inhibitor AZD1152.

[0166] The results are as follows Figure 7 As shown. Under AZD1152 treatment, there was no significant change in the relative body weight of GIST-T1 and GIST430 / 654 tumor-bearing nude mice. Figure 7 The ratio of aspartate aminotransferase (AST) to alanine aminotransferase (ALT) did not change significantly in GIST-T1 and GIST430 / 654 tumor-bearing mice after AZD1152 treatment. Figure 7 The results (B) indicate that AZD1152 treatment did not cause significant liver damage in mice. Blood tests showed only a slight decrease in the total white blood cell count and monocyte count in GIST-T1 tumor-bearing mice treated with AZD1152. Figure 7 (C); Blood routine tests showed that blood cells in GIST430 / 654 tumor-bearing mice treated with AZD1152 were not affected. Figure 7 (D).

[0167] In summary, the AURKB-specific inhibitor AZD1152 has no obvious toxic side effects in vivo.

[0168] Example 5: ATAD2 is the substrate of AURKB in GIST

[0169] To understand the substrate of AURKB in GIST, proteomic proteomic analysis was performed. Specifically, GIST cells were infected with AURKB-FLAG overexpressing virus, and cell lysates were subjected to immunoprecipitation using anti-FLAG antibody. The eluted proteins were then identified using SDS-PAGE. Figure 9 a) identified AURKB-related proteins such as Figure 9 As shown in b. ATAD2 (total PEP = 108) was identified as a candidate interacting protein with AURKB.

[0170] To verify this finding, AURKB-FLAG and AURKB-6MYC were overexpressed in GIST-T1 and GIST48B cell lines, respectively. Immunoprecipitation with anti-FLAG or anti-MYC antibodies detected specific binding to endogenous ATAD2. Figure 9 c). Simultaneously, a series of truncated mutants of AURKB were constructed ( Figure 9 (d) to determine the interacting domains. Although experiments confirmed protein-level interactions, mRNA expression analysis of GIST patient samples showed no significant correlation between AURKB and ATAD2 transcriptional levels. Figure 9 The (e) indicates that the interaction between the two may occur at the post-translation level.

[0171] In further validation experiments, an immunoprecipitation assay was used to verify the interaction between exogenous AURKB and endogenous ATAD2. The results showed that an interaction existed between exogenous AURKB and endogenous ATAD2 in GIST430 and GIST430 / 654 cells. Figure 8 (A), and this interaction was not found in H1792 and PANC1. Figure 8 (B).

[0172] To further determine the interacting domains, MYC-ATAD2 and AURKB-FLAG truncated mutants were co-expressed in HEK293T cells. It was found that the kinase domain (KD) of AURKB is the key region for its binding to ATAD2. Figure 8 Immunofluorescence assays showed that in GIST430 / 654 cells, AURKB (red) and ATAD2 (green) exhibited significant colocalization in structures such as the nucleus and mitotic spindle. Figure 8 (E).

[0173] The correlation between AURKB and ATAD2 protein expression levels in GIST patient samples was detected by protein immunoblotting. The results showed a significant positive correlation between AURKB and ATAD2 protein expression levels in GIST patient samples. Figure 8 (FG).

[0174] The correlation between AURKB and ATAD2 mRNA expression levels in GIST cell lines was detected using CRISPR knockout and qRT-PCR techniques. Knockout of AURKB using CRISPR-Cas9 technology (sequences of sgAURKB#1 and sgAURKB#2 as described above) significantly reduced ATAD2 mRNA levels, while knockout of ATAD2 (sgATAD2#4(sg4)forward:CACCGGTACGATTTGATAGTGTTGG(SEQ ID NO:5),reverse:AAACCCAACACTATCAAATCGTACC(SEQ ID NO:6); or SgATAD2#6(sg6)forward:CACCGTTGCCAATGAGTGCAGTCAA(SEQ ID NO:7),reverse:AAACTTGACTGCACTCATTGGCAAC(SEQ ID NO:8)) had no significant effect on AURKB expression. Figure 8 The results suggest that AURKB may affect ATAD2 expression through transcriptional regulation mechanisms.

[0175] The above results confirm the specific interaction between AURKB and ATAD2 in GIST and also reveal its potential regulatory mechanism in the occurrence and development of GIST.

[0176] Example 6: The regulatory mechanism of AURKB on ATAD2

[0177] To understand the regulatory mechanism of AURKB on ATAD2, AURKB was knocked out in the GIST cell line using CRISPR knockout technology, and changes in ATAD2 protein levels were detected. The results of Western blot analysis are shown below. Figure 10 As shown in Figure A, the results indicate that the ATAD2 protein level decreased significantly after AURKB knockout.

[0178] In the GIST cell line, after knocking out AURKB and then reinstating AURKB, changes in ATAD2 protein levels were observed. The results are as follows: Figure 10 As shown in Figure B, ATAD2 protein levels were restored after replacement with wild-type (WT)AURKB; however, replacement with the kinase-inactivating mutant (KDloss) did not fully restore ATAD2 levels.

[0179] In the GIST cell line, ATAD2 was knocked out, and changes in AURKB protein levels were observed. The results are as follows: Figure 10 As shown in Figure C, the AURKB protein level was not affected after ATAD2 knockout.

[0180] GIST cells were treated with the AURKB-specific inhibitor AZD1152, and changes in ATAD2 protein levels were observed. The results are as follows: Figure 10 As shown in D, AZD1152 dose-dependently reduced ATAD2 protein levels.

[0181] After treatment with 1 μM and 2 μM AZD1152 for 6 h, changes in ATAD2 protein levels were detected in GIST430 / 654 samples. Histone H3 S10 phosphorylation levels were used as a reference for AURKB kinase activity. Results are as follows: Figure 10 As shown in E, AZD1152 rapidly reduced ATAD2 protein levels, and histone H3 S10 phosphorylation levels also decreased, confirming that AURKB activity was inhibited.

[0182] GIST cells were treated with other cell cycle-related inhibitors (PLK1 inhibitor onvansertib, CDK4 / 6 inhibitor palbociolib), and changes in ATAD2 protein levels were observed. The results showed that only AZD1152 significantly reduced ATAD2 protein levels, while PLK1 and CDK4 / 6 inhibitors had no effect. This indicates that the change in ATAD2 protein levels after AURKB inhibition was caused by changes in AURKB itself, rather than a secondary effect resulting from cell cycle changes. Figure 10 (F).

[0183] GIST cells after AURKB knockout were treated with the protein synthesis inhibitor CHX and the proteasome inhibitor MG132. The changes in ATAD2 protein levels were observed to determine whether AURKB mediates ATAD2 protein stabilization through the ubiquitin-proteasome pathway.

[0184] The changes in ATAD2 protein levels in AURKB knockout GIST430 / 654 and GIST430 cells after treatment with the protein synthesis inhibitor CHX are as follows: Figure 10 As shown in G. The changes in ATAD2 protein levels in AURKB knockout GIST430 / 654 and GIST430 cells after 6 h of treatment with the proteasome inhibitor MG132 are shown in Figure G. Figure 10 As shown in H.

[0185] In HEK 293T cells, MYC-labeled ATAD2, HA-labeled ubiquitin (HA-Ub), and FLAG-labeled wild-type AURKB or FLAG-labeled AURKB lacking the kinase domain were co-expressed. Immunoprecipitation and immunoblotting were then performed to detect changes in ATAD2 ubiquitination levels. The results are as follows: Figure 10As shown in Figure I. The changes in endogenous ATAD2 ubiquitination levels in GIST48B cells after AZD1152 treatment are shown in Figure I. Figure 10 As shown in J.

[0186] The regulation of ATAD2 protein levels by AURKB in different GIST cell lines and tumor-bearing mice was further validated. Results are shown below. Figure 11 .

[0187] The above results fully verify that AURKB mediates the protein stability of ATAD2 through ubiquitination, and inhibiting AURKB activity can cause ATAD2 to be degraded through ubiquitination.

[0188] Example 7: The role of ATAD2 in the cancer-promoting function of AURKB

[0189] First, the expression level of ATAD2 in GIST patient samples and normal tissues was examined; then, ATAD2 was knocked out in GIST cells, and changes in cell proliferation were examined; finally, ATAD2 was reintroduced into GIST cells with knocked-out AURKB or ATAD2, and changes in cell proliferation were examined.

[0190] The results showed that ATAD2 expression levels were high in high-risk / metastatic GISTs. Figure 12 The expression level of A in the cancer tissues of 5 GIST patients was higher than that in their paired normal adjacent normal tissues. Figure 12 (B); Lentiviral-mediated ATAD2 knockout reduced the viability of GIST430 / 654 cells (B); Figure 12 The C) and the viability of GIST430 / 654 cells was increased after ATAD2 knockout and reinjection ( Figure 12 (D). Changes in cell viability of AURKB knockout GIST430 / 654 cells after rehydration with wild-type AURKB, kinase-inactivated AURKB, and ATAD2 are as follows: Figure 12 As shown in Figure E, ATAD2 and wild-type AURKB supplementation enhances cell viability. This effect was also confirmed in GIST430 / 654 tumor-bearing nude mice, where wild-type AURKB and ATAD2 supplementation rescued tumor growth in AURKB-knockout GIST430 / 654 tumor-bearing nude mice. Figure 12 (F and G).

[0191] GSEA analysis of RNAseq data after ATAD2 knockout identified signaling pathways related to DNA damage repair. Figure 12 The H of AURKB knockout and ATAD2 knockout of GIST430 / 654 show similarities in GSEA signaling pathway changes. Figure 12Furthermore, signaling pathways related to DNA damage repair were significantly downregulated in AURKB knockout GIST430 / 654. Figure 12 J).

[0192] ATAD2 knockout reduces the overall openness of chromatin. Figure 12 In genes involved in DNA damage repair, the degree of chromatin opening in the promoter regions of genes knocked out by ATAD2 was significantly reduced. Figure 12 The L indicates that ATAD2 promotes GIST progression by maintaining chromatin openness.

[0193] In the different GIST cell line GIST430, the effects of ATAD2 knockout and reinstatement, AURKB knockout and reinstatement of wild-type AURKB, kinase-inactivated AURKB, and ATAD2 on cell viability were also verified. Figure 13 AC), results and Figure 12 The results were consistent.

[0194] The DepMap database showed no significant correlation between ATAD2 dependence and AURKB dependence in 1066 tumor cell lines, excluding GIST cell lines. Figure 13 The density histogram shows the dependence of all tumor cell lines on ATAD2 in the DepMap Public 24Q2+Score Chronos database. Figure 13 ATAD2 knockout had no significant effect on the viability of H1792 and PANC1 cells. Figure 13 (F and G). The above results indicate that the effect of ATAD2 on GIST is tumor-specific.

[0195] GIST430 / 654 cells treated with ATAD2 knockout and the AURKB-specific inhibitor AZD1152 showed similar changes in the GSEA signaling pathway. Figure 13 The signaling pathways associated with DNA damage repair were significantly downregulated in GIST430 / 654 cells after treatment with the AURKB-specific inhibitor AZD1152. Figure 13 L).

[0196] Example 8: Transcription factor FOXM1 regulates the transcriptional expression of AURKB

[0197] Upstream regulators of AURKB were identified, and the correlation between all transcription factors and AURKB expression levels in GIST patient transcriptome sequencing data was analyzed. FOXM1, a transcription factor associated with AURKB expression, was identified. Figure 14 (A and B).

[0198] In the validation experiment, the expression level of FOXM1 in GIST patient samples was analyzed, and it was found that the expression level of FOXM1 in high-risk / metastatic patients was higher than that in low / intermediate-risk patients. Figure 14 The expression of FOXM1 was positively correlated with the expression of AURKB (C). Figure 14 (D); Following lentivirus-mediated FOXM1 knockout, AURKB protein levels also decreased. Figure 14 (E); After treatment of GIST cells with the FOXM1 inhibitor Thiostrpton for 6 hours, the expression levels of ATAD2 and AURKB also decreased. Figure 14 The F indicates that FOXM1 is an upstream regulator of AURKB and ATAD2.

[0199] The specific regulatory segment of the AURKB promoter region by FOXM1 was determined by dual fluorescence reporter assay in HEK 293T. Specifically, co-transferring the AURKB-2kb promoter region and FOXM1 in HEK 293T resulted in enhanced fluorescence signal intensity, indicating that FOXM1 has a regulatory effect on the AURKB promoter region. Figure 14 The AURKB promoter region was truncated into three regions and co-transfected with FOXM1 into HEK 293T cells. Fluorescence signal values ​​were then measured to determine which specific region of the AURKB promoter region FOXM1 regulates the cell. The results showed that FOXM1 is regulated through the 0–-666 bp region of the AURKB promoter. Figure 14 H).

[0200] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. Use of an aurora kinase B (AURKB) inhibitor in the preparation of a medicament for the treatment of gastrointestinal stromal tumors.

2. The use as described in claim 1, characterized in that, The AURKB inhibitor is selected from the group consisting of: small molecule compounds, antibodies, microRNA, siRNA, shRNA, antisense oligonucleotides (ASO), nucleic acid aptamers, gene editors, or combinations thereof.

3. The use as described in claim 1, characterized in that, The AURKB inhibitor is a small molecule compound, including: ilorasertib, ZM447439, VX680, AZD1152, AZD2811, or a combination thereof.

4. The use as described in claim 1, characterized in that, The AURKB inhibitor is a gene editor used to knock out or silence the AURKB gene, including: CRISPR / Cas9, TALEN, ZFN, and base editor.

5. Use of an ATAD2 inhibitor in the preparation of a medicament for the treatment of gastrointestinal stromal tumors.

6. The use as described in claim 5, characterized in that, The ATAD2 inhibitor is selected from the group consisting of small molecule compounds, antibodies, microRNA, siRNA, shRNA, antisense oligonucleotides (ASO), nucleic acid aptamers, gene editors, or combinations thereof.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (a) An AURKB inhibitor as the primary active ingredient; (b) An ATAD2 inhibitor as a second active ingredient; and (c) Pharmaceutically acceptable carriers.

8. A medicine box, characterized in that, The medicine box contains: (a) A first pharmaceutical composition comprising (a1) an AURKB inhibitor as a first active ingredient; and (a2) a pharmaceutically acceptable carrier; and (b) a second pharmaceutical composition comprising (b1) an ATAD2 inhibitor as a second active ingredient; and (a2) a pharmaceutically acceptable carrier.

9. The use of an AURKB detection reagent, characterized in that, This is used to prepare a kit for the diagnosis of gastrointestinal stromal tumors.

10. A method for screening candidate compounds for treating gastrointestinal stromal tumors, characterized in that, The method includes the following steps: (a) In the test group, the test compound was added to the cell culture system, and the expression level (E1) and / or activity (A1) of AURKB in the cells of the test group were observed; in the control group, the test compound was not added to the same cell culture system, and the expression level (E0) and / or activity (A0) of AURKB in the cells of the control group were observed. If the expression level (E1) and / or activity (A1) of AURKB in the test group is significantly lower than that in the control group, it indicates that the test compound is a candidate compound for the treatment of gastrointestinal stromal tumors that inhibits the expression and / or activity of AURKB.

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