Application of inhibitors of BEX4-XRCC5 interaction in the preparation of anti-hepatoblastoma drugs

Abstract

This invention discloses the application of an inhibitor of the BEX4-XRCC5 interaction in the preparation of an anti-hepatoblastoma drug, belonging to the field of biomedical technology. The inhibitor, F35-303, specifically disrupts the direct interaction between BEX4 and XRCC5 proteins, inhibiting the DNA non-homologous end joining repair pathway, thereby targeting hepatoblastoma tumor stem cells and overcoming the limitations of existing therapies. It is particularly suitable for treating platinum-resistant or recurrent hepatoblastoma. The inhibitor is a small molecule compound named F35-303. This invention also provides pharmaceutical compositions containing this inhibitor and their combination therapy with platinum-based chemotherapy drugs (such as cisplatin), which can synergistically enhance efficacy and reduce toxic side effects. This invention provides a novel targeted therapy strategy for hepatoblastoma.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of inhibitors of BEX4-XRCC5 interaction in the preparation of anti-hepatoblastoma drugs. Background Technology

[0002] Hepatoblastoma is the most common malignant liver tumor in children. Despite the rapid advancements in liver cancer research, which have greatly enriched our understanding of the pathogenesis of hepatoblastoma and facilitated the development of new drugs, recurrence, metastasis, and drug resistance remain critical issues in the field. Increasing evidence suggests that a small number of cells known as cancer stem cells (CSCs) promote tumor recurrence and metastasis. Like normal stem cells, CSCs possess the capacity for self-renewal, differentiation, and proliferation. This stemness maintenance depends on various molecular targets, such as metabolic reprogramming, cell plasticity, the tumor microenvironment, apoptosis pathways, microRNAs, stem cell differentiation, and drug resistance markers. Therefore, effective treatment strategies rely on targeting CSCs to overcome potential liver cancer recurrence, drug resistance, and treatment resistance. Recently, the successful approval of vemodegary and other new drugs targeting acute myeloid leukemia tumor stem cells demonstrates the broad prospects for clinical translation of cancer stem cell research. However, no drugs specifically targeting hepatoblastoma CSCs have yet been released.

[0003] Currently, research on hepatoblastoma stem cell stem cells (CSCs) mainly focuses on the activation of classic stem cell signaling pathways such as Wnt / β-catenin, Notch, and Hedgehog. These pathways have been shown to promote the self-renewal, differentiation, and invasiveness of CSCs. Small molecule inhibitors targeting these pathways, such as CWP232228 and GANT-6, can also partially eliminate CSCs. However, these existing targets are not specific to CSCs, and inhibitors targeting them often cause significant systemic toxicity, severely limiting their clinical application prospects. The stemness maintenance mechanism of CSCs is complex and not fully elucidated, with unknown key regulatory nodes still existing.

[0004] Based on this, those skilled in the art recognize that the key to developing highly effective and low-toxicity targeted therapies for hepatoblastoma lies in discovering and validating novel key regulatory molecules unique to hepatoblastoma CSCs, and developing highly selective inhibitors based on these molecules. Summary of the Invention

[0005] In view of the lack of effective drugs that can specifically and safely target hepatoblastoma CSCs, this invention aims to provide the application of inhibitors of BEX4-XRCC5 interaction in the preparation of anti-hepatoblastoma drugs.

[0006] To achieve the above objectives, the present invention employs the following technical solution: This invention provides the application of an inhibitor of BEX4-XRCC5 protein interaction in the preparation of a drug for treating hepatoblastoma.

[0007] The inhibitor is used to treat hepatoblastoma that is resistant to or has relapsed from platinum-based chemotherapy.

[0008] The inhibitor suppresses the DNA non-homologous end joining repair pathway by disrupting the direct interaction between the BEX4 and XRCC5 proteins.

[0009] The inhibitor is a small molecule compound, polypeptide, peptide-like substance, antibody or its antigen-binding fragment, or nucleic acid molecule.

[0010] The inhibitor is a small molecule compound of formula (I), or a pharmaceutically acceptable salt, solvate, prodrug, or stereoisomer thereof: .

[0011] The small molecule compound is selected from one of the following specific compounds: a) The compound shown in formula (I); b) An IC having the same parent nucleus structure as the compound shown in formula (I), and which inhibits the BEX4-XRCC5 interaction. 50 Derivatives with values ​​below 10 μM.

[0012] The present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the above-described BEX4-XRCC5 protein interaction inhibitor, and a pharmaceutically acceptable carrier or excipient.

[0013] It also includes one or more chemotherapeutic agents for the treatment of hepatoblastoma, said chemotherapeutic agents being cisplatin, doxorubicin, or a combination of both.

[0014] Preferably, the chemotherapy drug is cisplatin.

[0015] This invention provides a combination drug product comprising: (a) A first formulation comprising the aforementioned BEX4-XRCC5 protein interaction inhibitor; and (b) A second formulation containing platinum-based chemotherapy drugs; The first and second formulations are prepared as a combination drug product for simultaneous, separate or sequential administration to treat hepatoblastoma.

[0016] The present invention provides a kit for screening BEX4-XRCC5 interaction inhibitors, comprising recombinant BEX4 protein, recombinant XRCC5 protein and the compound shown in formula (I) as a positive control.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention provides the application of BEX4-XRCC5 protein interaction inhibitors in the preparation of drugs for treating hepatoblastoma. It is the first time that an inhibitor targeting the BEX4-XRCC5 protein interaction has been proposed for the treatment of hepatoblastoma, offering a novel targeted therapy strategy with a clearly defined mechanism of action, potentially overcoming the limitations of existing therapies. Based on differentially expressed genes from transcriptome data of a mouse model of hepatoblastoma, the key target BEX4 was screened. Analysis of the mechanism of action of BEX4 confirmed that the BEX4-XRCC5 protein interaction complex plays a crucial role in maintaining the stemness of hepatoblastoma.

[0018] Furthermore, by targeting the key DNA repair protein XRCC5, the resistance mechanism of platinum-based drugs (whose action depends on DNA damage) can be reversed or bypassed, providing a new treatment option for patients with refractory hepatoblastoma. By specifically disrupting protein-protein interactions rather than generally inhibiting the function of individual proteins, this approach offers higher selectivity. Inhibiting the non-homologous end joining (NHEJ) pathway weakens the DNA damage repair capacity of tumor cells, leading to genomic instability and cell death, while simultaneously enhancing the tumor's sensitivity to radiotherapy or DNA-damaging chemotherapy. Small molecule drugs typically possess good cell membrane penetration, can be taken orally, and have relatively low production costs, making them one of the most promising candidate forms for drug development, thus laying the material foundation for their rapid entry into preclinical and clinical development.

[0019] The pharmaceutical composition provided by this invention can be formulated into a stable dosage form suitable for different routes of administration (such as oral tablets, injections, etc.) by adding pharmaceutically acceptable carriers or excipients. The combination of drugs has a synergistic effect: the inhibitor disrupts the DNA repair pathway, making tumor cells more sensitive to DNA damage caused by cisplatin, thereby potentially reducing the dosage of chemotherapy drugs, reducing toxic side effects, overcoming or delaying cisplatin resistance, and improving the overall efficacy. Attached Figure Description

[0020] Figure 1BEX4 expression is typically elevated in HB tissue and is an overexpression of cancer stem cell markers in HB. A is a volcano plot indicating differentially expressed genes in c-MYC-induced HB mice; B is the mRNA expression levels of BEX4 and several CSC markers, as shown in the heatmap of mouse HB transcription data; C is a spheroid formation analysis in BEX4 knockout HB cells; DE is the protein expression level of BEX4 in tumors and surrounding tissues of human HB patients, determined by WB (D) and IHC (E); F is the protein expression level of BEX4 in fetal and adult liver specimens assessed by IHC assay; G is an IHC analysis of the correlation between BEX4 expression levels in HB tumor tissue and several CSC markers (SALL4, CD133, EpCAM.BEX1). Scale bar: 50 μm. (H) Correlation between mRNA levels of BEX4 and CSCs markers in the HB dataset (GSE131329), I is the Kaplan-Meier assessment of OS in the cohort, and J is the IF study of EpCAM expression in FACS-purified BEX4+Huh6 and HepG2 cells. Scale bar: 25 μm. (K) IF determination of EpCAM expression in spheroids and non-spheroids of primary BEX4+ human HB tumor cells. Scale bar: 25 μm. Abbreviations: HB, hepatoblastoma; CSCs, cancer stem cells; immunohistochemistry; IF, immunofluorescence; WB, Western blotting; IHC, immunohistochemistry; OS, overall survival; FACS, fluorescence-activated cell sorting system.

[0021] Figure 2BEX4 promotes tumorigenesis and stemness in HB mice; AB represents tumor formation in AAV8-shCtrl and AAV8-shBEX4 mice at different time points after hydrodynamic injection of c-MYC plasmid. Scale bar: 50 μm. C represents the number of tumors, maximum tumor size, AST, ALT, and liver / body weight ratio in the AAV8-shCtrl and AAV8-shBEX4 groups at the sacrifice time point; D represents the comparison of survival curves between the AAV8-shCtrl and AAV8-shBEX4 groups; EF represents the expression analysis of indicator molecules mRNA (E) and protein (F) in liver specimens from AAV8-shCtrl and AAV8-shBEX4 mice; G represents tumor formation experiments performed in Huh6 and HepG2 cells with different BEX4 expression levels treated with pLKO.1-GFP-shBEX4 and pLVX-CMV-Flag-BEX4 viruses. Scale bar: 50 μm. H represents the assessment of HB cell invasiveness by transwell Matrigel assay; I represents the FC assay of EpCAM+ cell proportion using specified treatments; J represents xenograft assay using BEX4-downregulated Huh6 cells via limiting dilution. AST, aspartate aminotransferase; ALT, alanine aminotransferase; FC, flow cytometry. Figure 3BEX4 is essential for DNA damage repair in HB cells. AB represents Western blot analysis of the protein expression level of the DNA damage marker γ-H2AX in HB cells infected with shCtrl or shBEX4 lentivirus (A), or transfected with the empty Flag vector or Flag-BEX4 plasmid (B), after different time points of cisplatin (5 μM) treatment. CD represents immunofluorescence staining of HB cells under corresponding conditions to detect γ-H2AX foci (red). Cell nuclei were counterstained with DAPI (blue). Scale bar: 5 μm; E represents the cell viability of HB cells infected with shCtrl / shBEX4 (top) or transfected with Flag / Flag-BEX4 (bottom) after 48 hours of treatment with different concentrations of cisplatin, determined by the CCK8 assay. FG represents a representative gross liver view (F) and H&E stained sections (G) after AAV8-mediated shCtrl or shBEX4 knockdown combined with cisplatin chemotherapy in a c-MYC-induced HB mouse model. Scale bar: 50 μm. H represents the quantitative analysis of tumor number, maximum tumor size, serum AST / ALT level, and liver / body weight ratio in the above mouse models (mean ± standard deviation, n≥5). *P<0.05, P<0.01. (IK) Huh6 cells stably knocked down by shCtrl or shBEX4 were subcutaneously inoculated into nude mice. After tumor formation, cisplatin was administered. I is a representative photograph of the dissected solid tumor, J is the tumor growth curve, and K is the final tumor weight. All data are from at least three independent experiments. γ-H2AX, phosphorylated Ser139H2A histone family member X.

[0022] Figure 4BEX4 promotes NHEJ repair by stabilizing XRCC5 in HB cells. AB represents IP-MS (A) and co-IP (C) analyses to identify XRCC5 as a protein interacting with BEX4 in HB cells. C represents changes in the expression of BEX4, XRCC5, and XRCC6 detected by IP in Huh6 cells with XRCC5 knockout. DE represents the NHEJ repair activity of the I-Scel NHEJ reporter system and the HR repair activity of the DR-GFP reporter system in Huh6 cells expressing shCtrl or shBEX4, assessed by flow cytometry. F represents changes in the expression levels of BEX4, XRCC5, and RAD51 (a key regulator of HR repair) in Huh6 cells downregulated by BEX4. Scale bar: 5 μm. G represents the changes in expression levels of different markers associated with double-strand break reactions in Huh6 cells downregulated by BEX4. H represents the CHX tracking analysis of XRCC5 protein degradation in HB cells, where BEX4 downregulation is normalized to β-actin. I represents the WB assessment of protein expression in HB cells downregulated by BEX4 after 12 hours of treatment with 10 μM MG132. J represents the IP analysis of ubiquitin expression in BEX4-dysregulated HB cells treated with MG132. K represents the effect of restoring XRCC5 expression downregulated by BEX4 on γ-H2AX levels in HB cells. L represents the relative levels of γ-H2AX detected in HB cells transduced with shBEX4 and Flag-XRCC5, which were treated with cisplatin (5 μM) at each time point. M represents the determination of NHEJ activity in Huh6 cells downregulated by BEX4 and in XRCC5 rescue. N represents the IHC staining of BEX4 and XRCC5 in human HB specimens. Scale bar: 50 μm, O represents stratified survival analysis of HB patients grouped by combination based on BEX4 and XRCC5 expression levels. Abbreviations: IP-MS, Immunoprecipitation Mass Spectrometry; Co-IP, Co-immunoprecipitation; CHX, Cycloheximide; NHEJ, Non-homologous end joining; XRCC5, X-ray retrieval cross-complement 5; RAD51, Radiation-sensitive 51 recombinase.

[0023] Figure 5 F35-303 significantly inhibited cisplatin resistance in HB cells; A is a schematic diagram of the virtual screening process, B is the molecular docking result of F35-303 targeting the BEX4-XRCC5 interaction interface, and C is the IC50 value evaluated in Huh6 cells. 50Values, D represents the interaction between BEX4 and XRCC5 in Huh6 cells treated with F35-303 for 12 hours, analyzed by IP; E represents the result of surface plasmon resonance analysis of the binding affinity between F35-303 and BEX4; FG represents the relative γ-H2AX level of Huh6 cells pre-incubated with F35-303 treated with cisplatin (5 μM) as assessed by WB (F) and IF (G) assays at each specified time point. γ-H2AX staining is red, and fusion with DAPI results in pink; H represents NHEJ activity measured in F35-303-treated Huh6 cells; I represents cell viability assessed by the CCK8 assay in HB cells pre-incubated with F35-303, followed by treatment with different concentrations of cisplatin for 48 hours; JL represents xenograft experiments using Huh6 cells treated with F35-303 and cisplatin, expressed as total tumor (I), tumor volume (J), and tumor weight (K); M represents tumor formation in mice treated with cisplatin plus F35-303 for approximately 3 weeks at a time point 3 weeks after c-MYC plasmid injection. Scale bar: 50 μm; N represents the number of tumors, maximum tumor size, AST, ALT, and liver / body weight ratio in harvested mice treated with cisplatin plus F35-303 at the sacrifice time point.

[0024] Figure 6 The identification and toxicity assays of F35-303 in a xenograft HB model are as follows: A represents candidate small molecule compounds screened from the HY-L0067V immuno-oncology library (MCE) using the third round of XP mode in Schrödinger Maestro software; B represents the top 10 potential compounds selected by cell viability assay; and CD represent the toxicity assessment of F35-303 by histological H&E examination (C) and serum biochemical indicators (ALT, AST, BUN, and Cre) (D).

[0025] Figure 7 For the identification and toxicity assay of F35-303 in the oncogene-induced HB model, the scale bar is 100 μm. Detailed Implementation

[0026] To enable those skilled in the art to understand the features and effects of the present invention, the following descriptions and definitions are only general descriptions of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in the event of any conflict, the definitions in this specification shall prevail.

[0027] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications in the art, unless otherwise stated.

[0028] BEX4: Brain Expressed X-linked 4 is a member of the human BEX gene family. It is a regulatory gene located on the X chromosome and mainly expressed in the brain. It is also often simply referred to as BEX family member 4. XRCC5: X-ray Repair Cross Complementing 5 is the name of a human gene that encodes a protein more commonly known as Ku80 (also called Ku86). It is a key initiation protein in the core DNA damage repair pathway of non-homologous end joining.

[0029] The Huh6 (hepatoblastoma cell line) used in this invention (purchased from Wuhan Pronosai Biotechnology Co., Ltd.) was cultured under the following conditions (DMEM high glucose medium containing 10% fetal bovine serum, 37°C, 5% CO2).

[0030] Animal Model: c-MYC Oncogene-Driven Mouse Model: A hepatoblastoma mouse model was established using a hydrodynamic tail vein injection method. The specific method was as follows: 6-8 week old C57BL / 6 mice (purchased from the Experimental Animal Center of Air Force Medical University) were rapidly injected via tail vein (within 5-8 seconds) with a saline solution containing the pT3-EF5α-c-MYC plasmid (a gift from Professor Chen Xin of the University of California, San Francisco, obtained from the Addgene plasmid sharing platform). The dosage was 28 μg of plasmid per mouse body weight, and the total volume was calculated at 1 mL of saline solution per 10 g mouse body weight.

[0031] Clinical samples: Human HB tissue microarray or fresh / frozen tissue were obtained from Xi'an Children's Hospital and approved by the Ethics Committee of Xi'an Children's Hospital (Approval No.: 2025-060-01), with informed consent from the patients.

[0032] Antibodies: The specific Western blot (WB), IHC, and IF antibodies used in this invention are shown in the table below.

[0033]

[0034] Example 1: Identification of BEX4 as a key molecule for maintaining stemness in hepatoblastoma (1) Screening and validation of BEX4 as a differentially upregulated gene in a c-MYC-driven mouse hepatoblastoma model. A c-MYC oncogene-driven mouse model was established via tail vein injection using hydrodynamics. Six- to eight-week-old male C57BL / 6 mice (obtained from the Experimental Animal Center of Air Force Medical University) were randomly divided into a model group and a control group. Mice in the model group were injected via tail vein over 5-7 seconds with a saline solution containing the pT3-EF1α-c-MYC expression plasmid (plasmid concentration of 10 μg / mL), at a dose of 0.01 mL per gram of body weight. Mice in the control group received an equal volume of empty plasmid solution. Eight weeks after injection, the mice were euthanized by cervical dislocation, and liver tumor tissue (model group) and normal liver tissue (control group) were dissected and separated. The tissues were then flash-frozen in liquid nitrogen and stored at -80°C for later use.

[0035] Total RNA was extracted from the frozen tissues using TRIzol reagent (Invitrogen, USA). After passing agarose gel electrophoresis and NanoDrop ND-1000 spectrophotometer testing, mRNA libraries were constructed and paired-end 150 bp sequencing was performed using the Illumina NovaSeq 6000 platform by Beijing Qingke Biotechnology Co., Ltd. Fastp software was used for quality control filtering of the raw data. Clean sequences were aligned to the mouse reference genome (GRCm39), and gene counting was performed using featureCounts. Differential expression analysis was performed using the DESeq2 package (version 1.34.0) in R, with a significance threshold of |log2FoldChange| > 1 and a p-value (Padj) adjusted for false discovery rate (FDR) < 0.05. Transcriptomic analysis comparing the tumor group and the control group showed that the expression of BEX4, a member of the X-linked protein family, was significantly upregulated in c-MYC-induced liver tumors. Figure 1 (BEX4 was found to be positive for AB), suggesting that BEX4 may be involved in the development and progression of hepatoblastoma.

[0036] (2) Effect of BEX4 gene knockout on stem function of hepatoblastoma cells To test its functional relevance, shBEX4 plasmid was transfected into human hepatoblastoma Huh6 cells, and stably knocked-down Huh6 cells were obtained through resistance drug selection. Both cell lines were then seeded in low-adhesion 24-well plates, supplemented with B27 (1:50; Invitrogen), bFGF (20 ng / mL), and EGF (20 ng / mL). The shBEX4-1 group showed a significantly decreased spheroidization ability compared to the shCtrl control group, indicating that BEX4 is a key factor in maintaining the stemness of hepatoblastoma cells. Figure 1 (C)

[0037] (3) Expression of BEX4 in human hepatoblastoma and its clinical relevance This validated the clinical significance of the finding. Compared to adjacent non-tumor tissues of hepatoblastoma samples obtained from Xi'an Children's Hospital, BEX4 protein levels were consistently elevated in human HB tissues, which was associated with elevated levels of the stemness marker EpCAM. Figure 1 To further investigate the pathophysiological relevance of BEX4 in the liver, we evaluated its expression in human fetal and adult livers. Significantly higher levels of BEX4 were observed in fetal livers, suggesting it may be a carcinoembryonic molecule (DE). Figure 1 (F). The above results indicate its potential role as an oncogenetic molecule. Crucially, in the applicant's established clinical sample cohort, immunohistochemical staining revealed a strong positive correlation between BEX4 and key cancer stem cell (CSC) markers (including SALL4, CD133, EpCAM, and BEX1) expression at the protein level, supporting the potential function of BEX4 in maintaining cancer stem cells. Simultaneously, using the public data GSE131329, the applicant also observed this correlation at the transcriptional level, where BEX4 mRNA expression was positively correlated with BEX1, BEX2, and EpCAM. Figure 1 (H). Clinically, high expression of BEX4 is associated with lower overall survival in patients with hepatitis B (HB). Figure 1 Middle I).

[0038] (4) Enrichment and identification of tumor stem cell subpopulations based on BEX4 promoter activity Functional characterization of BEX4 was performed using a lentiviral promoter reporter system to isolate tumor cells in which GFP expression was driven by the human BEX4 gene promoter. Immunofluorescence confirmed high EpCAM expression in BEX4 cells, indicating their stem cell-like characteristics. Figure 1 (J). In addition, EpCAM is rich in BEX4. + Comparison of tumor spherical and non-spherical adherent cultures ( Figure 1 These results collectively indicate that BEX4 is a novel CSC-related marker in HB.

[0039] Example 2: Key functions of BEX4 in the development and progression of hepatoblastoma (1) Effect of BEX4 knockout in vivo on c-MYC-induced hepatoblastoma in mice To further elucidate the role of BEX4 in hepatoblastoma (HB) malignancy, a series of in vivo and in vitro functional assays were performed. To assess the contribution of BEX4 to tumorigenesis, mice were co-administered via hydrodynamic tail vein injection with an adeno-associated virus serotype 8 (AAV8) vector encoding a short hairpin RNA targeting mouse BEX4 and a c-MYC plasmid. Figure 2 (A) For example Figure 2 As shown in the BC diagram, BEX4 knockout significantly inhibited tumor formation; simultaneously, compared with the control group, the percentage of Ki67-positive cells detected by immunohistochemical staining, the number and size of tumors observed grossly, and serum alanine aminotransferase (ALT) and aspartate aminotransferase levels were also decreased in BEX4 knockdown mice. Furthermore, compared with AAV8-shCtrl-treated mice, AAV8-shBEX4-treated mice had prolonged survival time. Figure 2 (D). Using qRT-PCR and Western blotting, it was found that the mRNA and protein expression levels of BEX4, EpCAM, and CD133 in the liver tissue of BEX4 knockout mice were also lower than those in the control group. Figure 2 Medium EF).

[0040] (2) In vitro regulation of stemness and invasiveness of hepatoblastoma cells by BEX4 In vitro functional assays further demonstrated that knocking down BEX4 using a human short hairpin RNA interference vector, followed by cell spheroidization and transpore invasion assays, revealed that knocking down BEX4 weakened the stemness maintenance and invasive ability of Huh6 cells, while forced expression of BEX4 in HepG2 cells enhanced tumor spheroid formation and transpore invasion activity. Figure 2 (Zhong GH).

[0041] (3) Effects of BEX4 on the expression of tumor stem cell markers and tumor initiation ability Flow cytometry analysis showed that BEX4 overexpression increased the percentage of EpCAM-positive cells in HepG2 cells, while BEX4 knockout decreased the number of EpCAM-positive cells in Huh6 cells. Figure 2 In addition, limiting dilution assays showed that BEX4 downregulation reduced the tumor initiation capacity of HB cells in nude mice ( ). Figure 2 (J). In summary, these findings suggest that BEX4 promotes tumorigenesis and cancer stemness maintenance in HB.

[0042] Example 3: Mechanism by which BEX4 mediates chemotherapy resistance in hepatoblastoma by promoting DNA damage repair (1) BEX4 regulates the response of hepatoblastoma cells to DNA damage. To investigate the mechanism by which BEX4 promotes hepatoblastoma (HB) malignancy, we further evaluated the role of BEX4 in DNA damage repair using 5 μM cisplatin. Immunoblot analysis showed that cisplatin-induced shCtrl-Huh6 cells exhibited decreased γH2AX levels after 4 hours, but these levels remained elevated in shBEX4-induced Huh6 cells. Figure 3 (A). Conversely, HepG2 cells overexpressing BEX4 showed reduced γH2AX accumulation after cisplatin exposure ( Figure 3 Consistent with these results, immunofluorescence (IF) staining showed that cisplatin treatment increased γH2AX lesions in shBEX4 Huh6 cells and decreased lesions in BEX4-overexpressing HepG2 cells. Figure 3 (C and D) support the role of BEX4 in promoting DNA damage repair in HB cells. To determine whether BEX4 contributes to cisplatin resistance in HB cells, cell viability was assessed using a CCK-8 assay. BEX4 knockout sensitized Huh6 cells to cisplatin, while BEX4 overexpression enhanced cisplatin resistance in HepG2 cells. Figure 3 (E).

[0043] (2) Targeting BEX4 enhances the in vivo sensitivity of c-MYC-driven mouse hepatoblastoma to cisplatin. To assess whether targeting BEX4 enhances in vivo chemosensitivity, AAV8-shCtrl and AAV8-shBEX4 were injected hydrodynamically, followed by cisplatin therapy. Figure 3 (F). BEX4 knockout mice treated with cisplatin showed the strongest inhibitory effect on tumor progression, with the highest γH2AX levels and the lowest Ki67 levels. + Cell percentage, tumor number, tumor size, ALT / AST level, and liver to body weight ratio ( Figure 3 (G and H).

[0044] (3) Targeting BEX4 enhances the sensitivity of xenograft tumor models to cisplatin. Model establishment and drug treatment: Huh6-shNT and Huh6-shBEX4 cells (5×10⁻⁶) were used. 6 Each of the transplanted tumor cells was mixed with an equal volume of Matrigel and subcutaneously injected into the right axilla of 4-6 week old female BALB / c nude mice (n=6 per group). Administration was administered after the tumor volume had grown to approximately 100 mm. 3 Tumor-bearing mice were randomly divided into four groups: shNT+Vehicle group: intraperitoneal injection of solvent (5% DMSO + 5% Cremophor EL + 90% saline); shNT+cisplatin group: intraperitoneal injection of cisplatin (5 mg / kg); shBEX4+Vehicle group; and shBEX4+cisplatin group. Dosage regimen: cisplatin was administered once weekly for 3 weeks. The Vehicle group received an equal volume of solvent.

[0045] Observation and Assessment: Tumor Growth Monitoring: The long diameter (L) and short diameter (W) of the tumor were measured every 3 days using calipers, and the result was calculated using the formula V = 0.5 × L × W. 2 Calculate tumor volume and plot growth curve ( Figure 3 (I). Endpoint analysis: Mice were sacrificed 3 days after the last administration, and tumor weight was measured (I). Figure 3 (J). A portion of tumor tissue was taken for: TUNEL assay: Tumor cell apoptosis was detected using an in situ cell death assay kit (TUNEL method, purchased from Roche, Germany), and the apoptosis index (percentage of positive cells) was calculated. γ-H2AX IHC: The method was the same as above, to assess the level of DNA damage in the tumor tissue (…). Figure 3 (Middle K). Results are attached. Figure 3 As shown in IK, cisplatin treatment significantly enhanced the tumor-suppressive effect in Huh6-shBEX4 cell-derived xenografts. Compared with the shNT+cisplatin group, the shBEX4+cisplatin group had a lower tumor volume growth rate, lighter final tumor weight (P<0.01), a higher TUNEL apoptosis index in tumor tissue (P<0.05), and a higher γ-H2AX positivity rate (P<0.05). This further confirmed in an immunodeficient mouse model that targeting BEX4 can effectively reverse cisplatin resistance in hepatoblastoma. The above series of experiments demonstrates that BEX4 mediates cisplatin chemoresistance in hepatoblastoma by promoting DNA damage repair (specifically, by accelerating the clearance of cisplatin-induced DNA double-strand break marker γ-H2AX). Inhibiting BEX4 can effectively block this repair process, increase DNA damage accumulation, and thus significantly enhance the sensitivity of tumor cells to cisplatin both in vivo and in vitro.

[0046] Example 4: The specific mechanism by which BEX4 promotes the repair of non-homologous end connections by binding to and stabilizing XRCC5. (1) Identify the interaction between BEX4 and XRCC5 and verify its specificity. To elucidate the specific mechanism by which BEX4 promotes DNA damage repair in hepatoblastoma (HB) cells, we identified X-ray repair cross-complement 5 (XRCC5), a known regulator of DNA damage repair, as a candidate BEX4 interacting protein by immunoprecipitation combined with mass spectrometry. This identified XRCC5 as the first protein among those associated with DNA damage repair. Figure 4 (A). Subsequent co-IP experiments in HB cells also demonstrated strong BEX4-XRCC5 interaction ( Figure 4 (B1B). Although BEX4 was found to be associated with XRCC6, it does not bind to other core non-homologous end joining (NHEJ) factors, such as DNA-dependent protein kinase catalytic subunits (DNA-PKcs), DNA ligase IV (LIG4), or XRCC4. Given that XRCC5 and XRCC6 form a functional complex essential for NHEJ, does XRCC5 mediate the interaction between BEX4 and XRCC6? Knocking out XRCC5 in HB cells did indeed attenuate the binding of BEX4-XRC6 (BEX4-XRCC6). Figure 4 The presence of C indicates that XRCC5 promotes this interaction.

[0047] (2) BEX4 specifically promotes the NHEJ repair pathway by stabilizing the XRCC5 protein. Previous studies have shown that NHEJ and HR are two major DNA double-strand break (DSB) repair pathways in mammalian cells. Since BEX4 expression was previously found to be negatively correlated with homologous recombination (HR) activity but interacts with the core NHEJ factor XRCC5, we sought to elucidate its role in DNA double-strand break (DSB) repair. Using GFP-based reporter analysis (EJ5-GFP for NHEJ; DR-GFP for HR), we found that BEX4 knockout significantly reduced the efficiency of NHEJ (…). Figure 4 (Middle E). Consistent with this, IF staining showed that BEX4 downregulation reduced XRCC5 protein levels, but did not affect the key HR protein RAD51 ( Figure 4 (F). Immunoblot analysis further confirmed that BEX4 silencing reduced the expression of XRCC5 and XRCC6, while the protein levels of DNA-PKcs, LIG4, XRCC4, and RAD51 remained unchanged. Figure 4 These results indicate that BEX4 preferentially supports the NHEJ pathway. To explore how BEX4 affects XRCC5, we performed a cycloheximide (CHX) tracking analysis and observed that BEX4 knockout shortened the half-life of XRCC5 (G). Figure 4The presence of H indicates that BEX4 stabilizes XRCC5. Inhibition of the proteasome using MG132 restores XRCC5 levels in BEX4-deficient cells. Figure 4 In the middle I), ubiquitination detection showed that multiubiquitination of XRCC5 increased after BEX4 knockout ( Figure 4 These data indicate that BEX4 attenuates ubiquitin-mediated proteasome degradation of XRCC5. We then investigated whether restoring XRCC5 could reverse the DNA repair defects induced by BEX4 loss. Ectopic expression of XRCC5 in BEX4 knockout Huh6 cells reduced basal and cisplatin-induced γH2AX levels (…). Figure 4 K and L), and rescued NHEJ activity ( Figure 4 (M).

[0048] (3) Clinical relevance of BEX4 and XRCC5 in patients with hepatoblastoma Clinically, a strong positive correlation has been observed between BEX4 and XRCC5 expression in a cohort of HB patients. Figure 4 Patients with higher levels of BEX4 and XRCC5 (in the middle N group) had the worst overall survival. Figure 4 In summary, these results indicate that BEX4 binds to and stabilizes XRCC5 by inhibiting its ubiquitin-dependent degradation, thereby promoting error-prone NHEJ repair and leading to HB resistance to cisplatin.

[0049] Example 5: Screening and identification of small molecule compounds as inhibitors of BEX4-XRCC5 interaction (1) Inhibitors of BEX4-XRCC5 interaction Based on the above findings, it is indicated that the BEX4-XRCC5 interaction promotes NHEJ repair and induces cisplatin resistance in hepatoblastoma (HB), and therapeutic targeting of this interaction could reverse the resistance phenotype. To identify potential small molecule inhibitors, the predicted structure (e.g., predicted by AlphaFold-Multimer) or co-crystallized structure of the complex formed by the human BEX4 protein (UniProt ID: Q9HBW9) and the XRCC5 protein (UniProt ID: P13010) was used as the docking receptor. Protein structures were hydrogenated, side-chain optimized, and energy minimized using molecular modeling software (e.g., Maestro, Schrödinger). A protein-protein interaction (PPI) target compound library (catalog number: HY-L0067V) from MCE, containing 52,935 small molecules, was used. The compound library was converted to three-dimensional structures and pretreated with protonated states, stereoisomer enumeration, and energy minimization. Using Glide's ultra-precise (XP) docking mode, the compound library was docked to the predicted interface region of the BEX4-XRCC5 interaction. Based on the GlideScore and interaction modes with key residues (such as GLU121, HIS120, and ASP116 on XRCC5), all compounds were ranked. Based on the docking score and binding mode analysis, the top 10 candidate compounds were selected for experimental validation. Figure 5 China A and Figure 6 (A). Human hepatoblastoma Huh6 cells were seeded in 96-well plates (5 × 10⁶ cells per well). 3 Cells were cultured for 24 hours, and then the above 10 candidate compounds were added. Five concentration gradients (0.1, 1, 10, 20, 50 μM) were set for each compound, with three replicates for each concentration. A DMSO solvent control group was also included. After 72 hours of treatment, cell viability was assessed using the CCK-8 assay, as shown in the attached figure. Figure 5 China B and Figure 6 As shown in Figure B, compound Z2418580759 (hereinafter referred to as F35-303) exhibited significant inhibition of Huh6 cell growth at low concentrations, and its inhibition curve on cell viability was consistent with a typical dose-dependent effect, making it the most promising candidate for targeting the BEX4-XRCC5 interaction.

[0050] The chemical structure of compound Z2418580759 (hereinafter referred to as F35-303) is shown in the following general formula (I): , IUPAC name (free base): (S)-2-amino-N-(3-(5-(4-fluorophenyl)oxazol-2-yl)propyl)-4,5-dihydrothiazole-4-carboxamide; chemical formula: C 16 H 18 FN3O2; molecular weight 303.33; CAS number (free base): 1585408-68-9.

[0051] (2) Binding mode, affinity and destructive effect of F35-303 on target complex Molecular docking analysis was performed using high-precision molecular docking (using the Induced Fit Docking module in Schrödinger Suite 202X-Y software) to connect the chemical structure of F35-303 to the BEX4-XRCC5 complex. F35-303 formed two hydrogen bonds with GLU121 and HIS120 of XRCC5, two π-π stacking interactions with XRCC5-HIS120, and two salt bridges with XRCC5 residues GLU121 and ASP116. Furthermore, F35-303 exhibited hydrophobic contacts with multiple residues, including VAL113, LEU83, VAL117, VAL79, PRO67, and ILE123 on XRCC5, and ILE90, MET88, and MET87 on BEX4. F35-303 produced profound functional consequences consistent with its targeting mechanism. In Huh6 cells, the IC50 of F35-303 for Huh6 cells was calculated using dose-response curve fitting. 50 Value 2.39 μM ( Figure 5 (C). Co-immunoprecipitation assay confirmed that F35-303 treatment disrupted the interaction between BEX4 and XRCC5 in HB cells (C). Figure 5 Surface plasmon resonance (SPR) assays determined the binding affinity between F35-303 and XRCC5, yielding a KD value of 0.751 μM (D). Figure 5 E), indicating its high affinity. Consistent with this, Western blot analysis showed that F35-303 downregulated BEX4 and XRCC5 protein levels and increased γH2AX expression (E). Figure 5 F35-303 further enhanced cisplatin-induced γH2AX lesion formation (F35-303). Immunofluorescence staining further showed that F35-303 enhanced cisplatin-induced γH2AX lesion formation (F35-303). Figure 5 The presence of G indicates that disruption of the BEX4-XRCC5 complex makes HB cells sensitive to DNA damage. Furthermore, F35-303 treatment inhibited NHEJ repair activity and reduced cell viability. Figure 5(HI). Considering that F35-303 binds to BEX4 through hydrophobic interactions, the binding affinity between F35-303 and XRCC5 was tested.

[0052] (3) Evaluation of the in vivo antitumor efficacy and safety of F35-303 To evaluate the in vivo efficacy of F35-303, a hepatoblastoma (HB) xenograft model was established in Balb / c nude mice. Establishment and grouping of the xenograft model: Human hepatoblastoma Huh6 cells in logarithmic growth phase were digested with trypsin, centrifuged, and resuspended in serum-free DMEM medium for counting. 5 × 10⁶ cells were then... 6 One cell was mixed with an equal volume of Matrigel (catalog number: 356234, Corning) and subcutaneously injected into the right axilla of 4-6 week old female BALB / c nude mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., SPF-grade environment). The cells were allowed to grow to approximately 100 mm in size. 3 Tumor-bearing mice were randomly divided into four groups (n=8 per group): Solvent control group: intraperitoneal injection of the solvent (5% DMSO + 10% Solutol HS-15 + 85% saline). F35-303 monotherapy group: intraperitoneal injection of F35-303 (10 mg / kg, Zhejiang Qixin Biotechnology). Cisplatin monotherapy group: intraperitoneal injection of cisplatin (5 mg / kg). Combination therapy group: intraperitoneal injection of F35-303 (10 mg / kg) and cisplatin (5 mg / kg). Drug preparation and administration regimen: F35-303 preparation: Accurately weigh F35-303 powder, dissolve it in 10% dimethyl sulfoxide (DMSO), vortex until completely dissolved, and then serially dilute with saline solution containing 10% Solutol HS-15 (catalog number: 42966, Sigma) to prepare a working concentration (2 mg / mL). The final DMSO concentration in the preparation is 5%. The preparation should be stored at 4℃ protected from light and used immediately after preparation. Cisplatin preparation: Use clinical cisplatin injection (catalog number: H20040813, Qilu Pharmaceutical), diluted with physiological saline to the required concentration (1 mg / mL). Dosage regimen: In the F35-303 monotherapy and combination therapy groups, F35-303 was administered intraperitoneally at a dose of 10 mg / kg; in the cisplatin monotherapy and combination therapy groups, cisplatin was administered intraperitoneally at a dose of 5 mg / kg. In the combination therapy group, the two drugs were injected 1 hour apart. All administration volumes were 10 mL / kg. The treatment regimen was once every 3 days for 4 weeks (a total of 10 administrations). The solvent control group received an equal volume of solvent. Efficacy observation indicators and methods: From the first administration date, the long diameter (L) and short diameter (W) of the tumor were measured every 3 days using an electronic digital caliper, calculated using the formula V = 0.5 × L × W. 2Tumor volume was calculated, and tumor growth curves were plotted. Mouse body weight was measured and recorded concurrently. 72 hours after the last administration, all mice were euthanized via cervical dislocation after anesthesia with an inhalation of an overdose of isoflurane. Tumor tissue was dissected, weighed using a precision electronic balance, and the final tumor weight was recorded. Whole blood and tissues from major organs such as the heart, liver, spleen, lungs, kidneys, and small intestine were collected simultaneously. A portion of tumor tissue was fixed in 4% paraformaldehyde for 24 hours, routinely embedded in paraffin, and sectioned (4 μm thick). Sections were stained with hematoxylin and eosin to observe tumor cell morphology and necrotic areas; DNA damage marker γ-H2AX (primary antibody: rabbit, catalog number 83307-2-RR, Proteintech, 1:500 dilution) and cell proliferation marker Ki-67 (primary antibody: rabbit, catalog number 27309-1-AP, Proteintech, 1:8000 dilution) were detected. Color development was performed using a DAB chromogenic kit (catalog number: ZLI-9018, Zhongshan Jinqiao), followed by hematoxylin counterstaining. Five non-overlapping fields were randomly selected from each slide under 200x magnification, and the percentage of positive cells was counted using Image-ProPlus 6.0 software. Collected whole blood was allowed to stand for 30 minutes, then centrifuged at 3000 rpm for 15 minutes to separate serum. The concentrations of alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, and creatinine in serum were measured using an automated biochemical analyzer (model: Hitachi 7180). Tissues from the heart, liver, spleen, lung, kidney, and small intestine were collected, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with H&E. Two pathologists, whose groups were unknown, evaluated the tissues under an optical microscope in a double-blind manner for lesions such as inflammation, necrosis, vacuolar degeneration, and fibrosis. pT3-EF5α-c-MYC plasmid was injected into 6-8 week old male C57BL / 6 mice via tail vein injection (method as in Example 1). On day 21 post-injection, after confirming liver tumor formation using an IVIS Spectrum (PerkinElmer) system, the mice were randomly divided into a solvent control group, a cisplatin monotherapy group, an F35-303 monotherapy group, and a combination therapy group (n=6 per group). The drug administration regimen was the same as above. Three weeks after treatment, the mice were sacrificed, and the livers were completely removed for the following: Gross observation: The number of visible tumor nodules on the liver surface was counted, and the major and minor axes of the largest tumor nodule were measured using calipers. Liver index: The weight of the intact liver was weighed and compared with the mouse's body weight to calculate the liver / body weight ratio. Histological analysis: After fixation, embedding, and sectioning, the liver tissue was stained with H&E and immunohistochemically stained with γ-H2AX and Ki-67. Compared with the solvent control group, F35-303 monotherapy significantly inhibited tumor growth (tumor inhibition rate 40%), and the combination therapy of F35-303 and cisplatin showed a significant synergistic effect, with tumor growth almost completely halted (tumor inhibition rate 85%), and the final tumor weight was significantly lower than that of each monotherapy group (P < 0.01). Figure 5According to organ histology and blood biochemical markers, no obvious toxicity was observed. Figure 6 (middle CD). Furthermore, due to the synergistic effect of F35-303 and cisplatin, its efficacy in c-MYC-induced HB mice was tested. Results showed that, compared with the carrier mice, the combination of F35-303 and cisplatin significantly reduced tumor formation ( Figure 5 (MN). Furthermore, H&E staining was used to observe the histopathological changes in the major organs (heart, liver, spleen, lung, kidney, intestine, muscle, and brain) of mice after treatment with F35-303 (DM / SO group) or the solvent control (Control group). No obvious side effects were observed in the histology of the major organs. Figure 7 These results demonstrate that the small molecule inhibitor F35-303 effectively disrupts the BEX4-XRCC5 interaction, inhibits NHEJ activity, and restores cisplatin sensitivity in the HB model. F35-303, at effective doses, significantly inhibits hepatoblastoma growth both alone and in combination with cisplatin, and exhibits good tolerability and safety in experimental animal models, providing solid preclinical evidence for its potential as a novel targeted therapy strategy to overcome cisplatin resistance in hepatoblastoma.

[0053] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. The use of an inhibitor of BEX4-XRCC5 protein interaction in the preparation of a drug for the treatment of hepatoblastoma.

2. The application according to claim 1, characterized in that, The inhibitor is used to treat hepatoblastoma that is resistant to or has relapsed from platinum-based chemotherapy.

3. The application according to claim 1, characterized in that, The inhibitor suppresses the DNA non-homologous end joining repair pathway by disrupting the direct interaction between the BEX4 and XRCC5 proteins.

4. The application according to any one of claims 1-3, characterized in that, The inhibitor is a small molecule compound, polypeptide, peptide-like substance, antibody, or its antigen-binding fragment or nucleic acid molecule.

5. The application according to claim 4, characterized in that, The inhibitor is a small molecule compound of formula (I), or a pharmaceutically acceptable salt, solvate, prodrug, or stereoisomer thereof: 。 6. The application according to claim 5, characterized in that, The small molecule compound is selected from one of the following specific compounds: a) The compound shown in formula (I); b) An IC having the same parent nucleus structure as the compound shown in formula (I), and which inhibits the BEX4-XRCC5 interaction. 50 Derivatives with values ​​below 10 μM.

7. A pharmaceutical composition, characterized in that, It comprises a therapeutically effective amount of the BEX4-XRCC5 protein interaction inhibitor as described in any one of claims 1-6, and a pharmaceutically acceptable carrier or excipient.

8. The pharmaceutical composition according to claim 7, characterized in that, It also includes one or more chemotherapeutic agents for the treatment of hepatoblastoma, said chemotherapeutic agents being cisplatin, doxorubicin, or a combination of both.

9. A combination drug product, characterized in that, Include: (a) A first formulation comprising a BEX4-XRCC5 protein interaction inhibitor as defined in any one of claims 1-6; and (b) A second formulation containing platinum-based chemotherapy drugs; The first and second formulations are prepared as a combination drug product for simultaneous, separate or sequential administration to treat hepatoblastoma.

10. A kit for screening inhibitors of BEX4-XRCC5 interaction, characterized in that, It contains recombinant BEX4 protein, recombinant XRCC5 protein, and the compound shown in formula (I) as a positive control.

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