E3 ubiquitin ligase ASB7 as a molecular marker for predicting PARP inhibitor therapy sensitivity and its application

Abstract

This invention discloses the E3 ubiquitin ligase ASB7 as a molecular marker for predicting PARP inhibitor therapy sensitivity and its application. Through cell biology experiments and animal model studies, this invention found that tumor cells and tissues with high ASB7 expression exhibit significant sensitivity to PARP inhibitors. Specifically, ASB7 induces homologous recombination repair (HRR) deficiency, increasing genomic instability and thus enhancing the sensitivity of tumor cells to PARP inhibitors. This ASB7 molecular marker provides new insights for precision medicine, offers an optimized treatment strategy for tumor patients with high ASB7 expression, and lays a solid foundation for the development of highly effective anti-tumor drugs, demonstrating significant application prospects and clinical significance.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to E3 ubiquitin ligase ASB7 as a molecular marker for predicting PARP inhibitor treatment sensitivity and its application. Background Technology

[0002] Poly(ADP-ribose) polymerase (PARP) inhibitors are a class of anti-tumor drugs that target DNA damage repair mechanisms. By inhibiting PARP enzyme activity, they hinder the repair of single-strand breaks (SSBs), leading to the accumulation of double-strand breaks (DSBs). In tumor cells deficient in homologous recombination repair (HRR), PARP inhibitors can induce a "synthetic lethality" effect, thereby selectively killing tumor cells. PARP inhibitors have shown significant clinical efficacy in various tumor types, especially in patients with breast cancer, ovarian cancer, and prostate cancer carrying BRCA1 / 2 mutations.

[0003] However, not all cancer patients are sensitive to PARP inhibitors. Besides BRCA1 / 2 mutations, mutations in other homologous recombination repair-related genes (such as ATM, RAD51, and PALB2) are also considered potential biomarkers for PARP inhibitor sensitivity. Nevertheless, some patients, even those carrying these gene mutations, still do not respond ideally to PARP inhibitors. Therefore, finding new molecular markers to more precisely screen potential beneficiaries has become a current research focus.

[0004] ASB7 (Ankyrin Repeat and SOCS Box Containing 7) is a member of the E3 ubiquitin ligase family and is involved in protein degradation. TCGA database analysis shows that ASB7 exhibits high-frequency amplification in various solid tumors, including sarcoma, lung cancer, and esophageal cancer. However, the relationship between ASB7 and PARP inhibitors has not yet been reported. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide E3 ubiquitin ligase ASB7 as a molecular marker for predicting PARP inhibitor treatment sensitivity and its application.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution:

[0007] This invention experimentally demonstrates that high expression of the E3 ubiquitin ligase ASB7 inhibits cellular homologous recombination repair (HRR), thereby sensitizing ASB7-overexpressing tumor cells to PARP inhibitors. Specifically, this invention shows that high ASB7 expression inhibits cellular HRR: using the HR:DRGFP / NHEJ:EJ5GFP reporter vector, it was found that high ASB7 expression directly leads to a decrease in HRR, while non-homologous end joining (NHEJ) remains unaffected. Furthermore, this invention found that ASB7-overexpressing tumor cells are sensitive to PARP inhibitors: Cellular experiments demonstrated that ASB7-overexpressing cells are sensitive to the PARP inhibitor (Olaparib): Olaparib inhibits the proliferation of tumor cells overexpressing ASB7. Animal tumor models further demonstrated that ASB7-overexpressing tumors are sensitive to the PARP inhibitor (Olaparib): Olaparib inhibits the growth of tumors overexpressing ASB7. This reveals that high ASB7 expression is a novel molecular marker for the indication of PARP inhibitors. In summary, this invention reveals for the first time that high expression of ASB7 inhibits the homologous recombination repair capacity of cells, making cells sensitive to PARP inhibitors and enhancing the therapeutic effect of PARP inhibitors. These results indicate that ASB7 is a novel biomarker for PARP inhibitor sensitivity.

[0008] Therefore, this invention first provides the application of E3 ubiquitin ligase ASB7 as a molecular marker in the preparation of products that predict the sensitivity of cancer patients to PARP inhibitor therapy.

[0009] This invention also provides the use of formulations for detecting the expression level of E3 ubiquitin ligase ASB7 in the preparation of products that predict the sensitivity of cancer patients to PARP inhibitor therapy.

[0010] This invention provides the application of PARP inhibitors in the preparation of drugs for treating ASB7 gene amplification tumors. The ASB7 gene amplification refers to an increased copy number of the gene, indicating polyploidy.

[0011] This invention provides the use of a formulation that promotes the expression of E3 ubiquitin ligase ASB7 in the preparation of a medicament that enhances the sensitivity of cancer patients to PARP inhibitor therapy.

[0012] This invention provides the application of a formulation that promotes the expression of E3 ubiquitin ligase ASB7 in combination with a PARP inhibitor in the preparation of a drug for treating tumors.

[0013] Furthermore, the application of formulations that promote the expression of E3 ubiquitin ligase ASB7 in combination with PARP inhibitors in the preparation of drugs for treating tumors insensitive to PARP inhibitors.

[0014] Furthermore, the formulation that promotes ASB7 expression is an overexpression vector containing the ASB7 gene.

[0015] Furthermore, the PARP inhibitor is selected from olaparib, niraparib, rucaparib / rubraca, or talazoparib.

[0016] Preferably, the PARP inhibitor is olaparib.

[0017] Furthermore, the tumor is selected from ASB7 amplified tumors.

[0018] Preferably, the tumor is selected from sarcoma; more preferably, osteosarcoma.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] This invention first provides the E3 ubiquitin ligase ASB7 as a molecular marker for predicting sensitivity to PARP inhibitor therapy. Through cell experiments and animal model studies, this invention has found that tumor cells and tissues with high ASB7 expression exhibit significant sensitivity to PARP inhibitors. Specifically, ASB7 induces homologous recombination repair (HRR) deficiency, increasing genomic instability and thus enhancing the sensitivity of tumor cells to PARP inhibitors. This invention's ASB7 molecular marker provides new insights for precision medicine, offers an optimized treatment strategy for tumor patients with high ASB7 expression, and lays a solid foundation for the development of highly effective anti-tumor drugs, demonstrating significant application prospects and clinical significance. Attached Figure Description

[0021] Figure 1 This shows the expression of the ASB7 gene in cancer patient samples from the TCGA database.

[0022] Figure 2 This is the result of the HR:DRGFP / NHEJ:EJ5GFP reporter system and the assessment of DNA repair efficiency.

[0023] Figure 3 To investigate the effect of ASB7 overexpression on sensitivity to PARP inhibitor therapy; among which, Figure 3 In the figure, A represents the results of the clonogenic assay, B represents the tumor images of mice in different treatment groups, C represents the statistical results of the tumor volume of mice in different treatment groups, and D represents the statistical results of the tumor weight of mice in different treatment groups. Detailed Implementation

[0024] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0025] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0026] Example 1: HR:DRGFP / NHEJ:EJ5GFP reporter system validation that ASB7 overexpression inhibits homologous recombination repair

[0027] TCGA database analysis showed that ASB7 exhibited high-frequency amplification in various solid tumors, including sarcoma, lung cancer, and esophageal cancer. Figure 1 However, the specific mechanism is still unclear. This invention first investigates the effect of ASB7 high expression on homologous recombination repair. The specific method is as follows:

[0028] I. Experimental Methods:

[0029] 1. Vector Construction: Homologous Recombination Reporter System (HR): The DRGFP vector (Addgene-Plasmid#26475) contains two GFP sequences separated by an I-SceI restriction site, requiring homologous recombination to restore functional GFP. After transfection with I-SceI endonuclease-induced double-strand breaks (DSBs), cells that successfully repair HR will express GFP. Non-Homologous End Ligation (NHEJ) Reporter System: The EJ5GFP vector (Addgene-Plasmid#44026) contains two inverted GFP fragments separated by an I-SceI site. During NHEJ repair of DSBs, the GFP reading frame may be restored through random ligation, leading to fluorescent expression.

[0030] 2. HEK293T cell lines stably expressing doxycycline (Dox)-inducible TetOn-ASB7 were transfected with DRGFP and EJ5GFP, respectively. The experimental groups received Dox treatment to induce ASB7 protein expression, while the control group received no induction treatment. The groups were also divided into those transfected with or without I-SceI plasmid. After 48 hours, the expression level of green fluorescent protein (GFP) was quantitatively detected by flow cytometry (FACS) to assess DNA repair efficiency. The experiment was independently repeated three times.

[0031] Experimental results:

[0032] like Figure 2 As shown, in the HR reporter system, the fluorescence signal of cells with high ASB7 expression decreased; in the NHEJ reporter system, the signal remained unchanged in the ASB7-high expression group. These results indicate that high ASB7 expression can inhibit homologous recombination in cells without affecting non-homologous end joining.

[0033] Example 2, a colony formation experiment, demonstrated that high expression of ASB7 sensitizes cells to PARP inhibitors.

[0034] I. Experimental Methods:

[0035] 1. The experiment used 143B cell lines that stably overexpressed the empty vector and stably overexpressed ASB7; the PARP inhibitor used was Olaparib (MCE, HY-10162; dissolved in DMSO).

[0036] 2. Lentiviral virus overexpressing ASB7 was packaged in HEK293T cells, and then 143B cell lines were infected with the following mixture: 1 mL lentivirus + 2 mL culture medium + 3.5 μL polybrene. After 48 hours, the selection medium was changed to obtain a stable 143B cell line overexpressing ASB7.

[0037] 2. After trypsin digestion of the cells, the two cell lines were counted using a cell counter. The cells were then seeded into 6-well cell culture dishes, with 500 cells per well. Five groups were set up for seeding, corresponding to Olaparib concentrations of 0 μM, 0.5 μM, 1 μM, 2 μM, and 4 μM.

[0038] 3. After 24 hours of plating, add Olaparib at the set concentration for treatment.

[0039] 4. On day 10 after plating, collect the sample: remove the culture medium, wash twice with PBS, fix with 4% paraformaldehyde at room temperature for 15 minutes, then stain with 1% gentian violet solution for 1 hour, and slowly rinse with running water until the background is colorless.

[0040] 5. Take images of the entire hole, use ImageJ to automatically identify and count the number and area of ​​clones, and analyze the data.

[0041] II. Experimental Results:

[0042] like Figure 3 As shown in Figure A, the colony formation rate was significantly reduced in the ASB7 overexpression group after Olaparib treatment. These results indicate that high ASB7 expression sensitizes cells to PARP inhibitors.

[0043] Example 3: Animal tumor-bearing model demonstrates that tumors with high ASB7 expression are sensitive to PARP inhibitors.

[0044] I. Experimental Methods:

[0045] 1. The experiment used 143B cell lines stably overexpressing the empty vector and stably overexpressing ASB7; BALB / c nude mice, obtained from Zhuhai Best Biotechnology Co., Ltd. (Zhuhai, China), weighing 18-20 grams, 4 weeks old, male; tumor bearing method was subcutaneous tumor formation. This animal experiment has been approved by the Animal Research Committee of Sun Yat-sen University Cancer Center (approval number L0255202107022).

[0046] 2. Divide the animals into two groups of 12 each, and place 1×10⁻⁶ animals in each group. 6 143B cells, containing both empty and ASB7-overexpressing vectors, were subcutaneously injected into the groin area of ​​mice. One week after injection, the cells were randomly reassigned into two groups: a control group and a treatment group, with six mice in each group. The control group received DMSO injection (Vehicle), while the treatment group received 50 mg / kg Olaparib via intraperitoneal injection daily. Tumor volume was then measured every three days using calipers. The formula for calculating volume was: V (volume) = (width) / (width). 2 (×length×π) / 6.

[0047] 3. Twenty-eight days after cell injection, mice were euthanized by spinal dislocation, tumors were collected, and tumor weight was measured using an electronic balance.

[0048] 4. Data analysis of tumor volume and weight in mice.

[0049] II. Experimental Results:

[0050] like Figure 3 As shown in Figure B, the tumors in the mice were all controlled within the animal welfare range required by animal ethics. Figure 3 Results B-D showed that, under the empty vector overexpression condition, cells were not sensitive to Olaparib, and the difference between Olaparib and DMSO (Vehicle) was statistically insignificant. However, in the ASB7 overexpression group, tumor size shrank significantly after Olaparib treatment (p-value significant), indicating that mouse tumors overexpressing ASB7 were significantly more sensitive to Olaparib than the empty vector group. These results demonstrate that tumors with high ASB7 expression are sensitive to PARP inhibitors. ASB7 overexpression leads to slower tumor cell proliferation; specifically, ASB7 amplification impairs homologous recombination repair in tumor cells, resulting in slower cell proliferation, and also increases sensitivity to PARP inhibitors.

Claims

1. The application of a formulation for detecting the expression level of the E3 ubiquitin ligase ASB7 in the preparation of products for predicting the sensitivity of cancer patients to PARP inhibitor therapy, characterized in that, The PARP inhibitor is olaparib, and the tumor is osteosarcoma.

2. The application of a vector overexpressing E3 ubiquitin ligase ASB7 in combination with a PARP inhibitor in the preparation of drugs for treating tumors, characterized in that, The PARP inhibitor is olaparib, and the tumor is osteosarcoma.

Images