Molecular marker of E3 ubiquitin ligase ASB7 for predicting treatment sensitivity of PARP inhibitor and application of molecular marker

Abstract

The invention discloses an E3 ubiquitin ligase ASB7 as a molecular marker for predicting treatment sensitivity of a PARP inhibitor and application of the molecular marker. According to the application disclosed by the invention, cytological experiments and animal model researches find that tumor cells and tumor tissues with high ASB7 expression show remarkable sensitivity to PARP inhibitors. The specific mechanism is that ASB7 induces homologous recombination to repair (HRR) defects, and the instability of a genome is increased, so that the sensitivity of tumor cells to a PARP inhibitor is enhanced. The ASB7 molecular marker provides a new thought for precise medical treatment, provides an optimized treatment strategy for tumor patients with high ASB7 expression, lays a solid foundation for developing efficient anti-tumor drugs, and has remarkable application prospects and clinical significance.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to E3 ubiquitin ligase ASB7 as a molecular marker for predicting PARP inhibitor treatment sensitivity and application thereof. BACKGROUND

[0002] Poly (ADP-ribose) polymerase (PARP) inhibitors are a class of anti-tumor drugs targeting DNA damage repair mechanisms. By inhibiting the activity of PARP enzyme, PARP inhibitors hinder the repair of single-strand breaks (SSB), and thus lead to the accumulation of double-strand breaks (DSB). In tumor cells with homologous recombination repair (HRR) defects, 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 breast cancer, ovarian cancer and prostate cancer patients carrying BRCA1 / 2 mutations.

[0003] However, not all tumor patients are sensitive to PARP inhibitors. In addition to BRCA1 / 2 mutations, mutations in other homologous recombination repair-related genes (such as ATM, RAD51, PALB2, etc.) are also considered potential markers of PARP inhibitor sensitivity. However, even if some patients carry these gene mutations, the response to PARP inhibitors is still not ideal. Therefore, finding new molecular markers to further accurately screen potential benefit populations has become a hot spot of current research.

[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 is highly amplified in many solid tumors such as sarcoma, lung cancer and esophageal cancer. However, the relationship between ASB7 and PARP inhibitors has not been reported so far. SUMMARY

[0005] The present application aims to overcome the above-mentioned defects and deficiencies in the prior art, and provides E3 ubiquitin ligase ASB7 as a molecular marker for predicting PARP inhibitor treatment sensitivity and application thereof.

[0006] The above-mentioned object of the present application is achieved by the following technical solution:

[0007] The present application proves by experiments that high expression of E3 ubiquitin ligase ASB7 inhibits the homologous recombination repair ability of cells, and further makes the ASB7 high expression tumor cells sensitive to PARP inhibitors. Specifically, the present application research shows that ASB7 high expression inhibits the homologous recombination repair ability of cells: the present application uses HR:DRGFP / NHEJ:EJ5GFP reporter vector to find that ASB7 high expression directly leads to the decrease of homologous recombination repair (HRR) repair ability, and the non-homologous end joining (NHEJ) ability is not affected. Further, the present application research finds that the ASB7 high expression tumor cells are sensitive to PARP inhibitors: the present application uses cytological experiments to prove that the ASB7 high expression cells are sensitive to PARP inhibitors (Olaparib): Olaparib inhibits the proliferation of tumor cells with high expression of ASB7. The animal tumor-bearing model is used to prove that the ASB7 high expression tumor is sensitive to PARP inhibitors (Olaparib): Olaparib inhibits the growth of tumor cells with high expression of ASB7. This reveals that the ASB7 high expression state is a new molecular marker of PARP inhibitor indication. In summary, the present application first discloses that ASB7 high expression can inhibit the homologous recombination repair ability of cells, make the cells sensitive to PARP inhibitors, and enhance the treatment effect of PARP inhibitors. The above results show that ASB7 is a new PARP inhibitor sensitivity marker.

[0008] Therefore, the present application first provides the use of E3 ubiquitin ligase ASB7 as a molecular marker in the preparation of a product for predicting the sensitivity of tumor patients to PARP inhibitor treatment.

[0009] The present application also provides the use of a preparation for detecting the expression level of E3 ubiquitin ligase ASB7 in the preparation of a product for predicting the sensitivity of tumor patients to PARP inhibitor treatment.

[0010] The present application provides the use of a PARP inhibitor in the preparation of a drug for treating ASB7 gene amplification type tumors. The ASB7 gene amplification type refers to an increase in the copy number of the gene, which is a polyploid.

[0011] The present application provides the use of a preparation for promoting the expression of E3 ubiquitin ligase ASB7 in the preparation of a drug for enhancing the sensitivity of tumor patients to PARP inhibitor treatment.

[0012] The present application provides the use of a preparation for promoting the expression of E3 ubiquitin ligase ASB7 in combination with a PARP inhibitor in the preparation of a drug for treating tumors.

[0013] Further, the use of a preparation for promoting the expression of E3 ubiquitin ligase ASB7 in combination with a PARP inhibitor in the preparation of a drug for treating tumors that are not sensitive to PARP inhibitors.

[0014] Further, the preparation for promoting the expression of ASB7 is an overexpression vector containing ASB7 gene.

[0015] Further, the PARP inhibitor is selected from Olaparib, Niraparib, rucaparib / Rubraca or Talazoparib.

[0016] Preferably, the PARP inhibitor is Olaparib.

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

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

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

[0020] The present application firstly provides E3 ubiquitin ligase ASB7 as a molecular marker for predicting PARP inhibitor treatment sensitivity, and the present application finds that tumor cells and tumor tissues with high expression of ASB7 show significant sensitivity to PARP inhibitors through cytological experiments and animal model research. The specific mechanism is that ASB7 induces homologous recombination repair (HRR) defects, increases genomic instability, and thus enhances the sensitivity of tumor cells to PARP inhibitors. The ASB7 molecular marker of the present application provides a new idea for precision medicine, provides an optimized treatment strategy for tumor patients with high expression of ASB7, and lays a solid foundation for the development of efficient anti-tumor drugs, which has a significant application prospect and clinical significance. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The expression of ASB7 gene in the cancer patient sample of TCGA database.

[0022] Figure 2 HR:DRGFP / NHEJ:EJ5GFP report system and the result of evaluating DNA repair efficiency.

[0023] Figure 3 The result of exploring the influence of high expression of ASB7 on the sensitivity to PARP inhibitor treatment; wherein, Figure 3 A is the result of colony formation experiment, B is the tumor image of mice in different treatment groups, C is the statistical result of tumor volume of mice in different treatment groups, and D is the statistical result of tumor weight of mice in different treatment groups. DETAILED DESCRIPTION

[0024] The present application will be further described in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

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

[0026] Example 1 HR:DRGFP / NHEJ:EJ5GFP reporter system verifies that high expression of ASB7 inhibits homologous recombination repair

[0027] TCGA database analysis shows that ASB7 is highly amplified in many solid tumors such as sarcoma, lung cancer, esophageal cancer, etc. Figure 1 ), but the specific mechanism is not clear. The present application first explores the effect of high expression of ASB7 on homologous recombination repair. The specific method is as follows:

[0028] I. Experimental method:

[0029] 1. Construct vectors: Homologous recombination reporter system (HR): DRGFP vector (Addgene-Plasmid#26475) contains two I-SceI enzyme cleavage site interrupted GFP sequences, which need to be repaired by homologous recombination to restore functional GFP. After transfection of I-SceI endonuclease to induce double-strand break (DSB), cells with successful HR repair will express GFP. Non-homologous end joining (NHEJ) reporter system: EJ5GFP vector (Addgene-Plasmid#44026) contains two reverse GFP fragments, which are interrupted by I-SceI site. NHEJ repair of DSB may restore GFP reading frame by random ligation, resulting in fluorescence expression.

[0030] 2. A stable expression of doxycycline (Dox) inducible TetOn-ASB7 HEK293T cell line was used to transfect DRGFP and EJ5GFP, respectively. The experimental group was treated by Dox to induce ASB7 protein expression, and the control group was not given induction treatment, and the transfection or non-transfection I-SceI plasmid group was set in the same group. 48h later, the green fluorescent protein (GFP) expression level was quantitatively detected by flow cytometry (FACS) to evaluate the DNA repair efficiency, and the experiment was repeated independently for 3 times.

[0031] Experimental results:

[0032] As shown in Figure 2 , in the HR reporter system, the fluorescence signal of the ASB7 high expression cell is reduced; the signal of the ASB7 high expression group is unchanged in the NHEJ reporter system. The experimental results show that high expression of ASB7 can inhibit the homologous recombination ability of cells, and does not affect non-homologous end joining.

[0033] Example 2 Clonogenic assay demonstrates that ASB7 high expression makes cells sensitive to PARP inhibitors

[0034] I. Experimental methods:

[0035] 1. The experiment uses 143B cell lines stably overexpressing empty and stably overexpressing ASB7; the PARP inhibitor selected is Olaparib (MCE, HY-10162; dissolved in DMSO).

[0036] 2. Use HEK293T cells to package overexpression ASB7 vector lentivirus, and then infect 143B cell lines according to the system of 1 mL lentivirus + 2 mL medium + 3.5 μL polybrene. After 48 hours, change the screening medium to screen and obtain the 143B cell line stably overexpressing ASB7.

[0037] 2. After trypsinizing the cells, count the two cell lines using a cell counter, and then seed the cells into 6-well cell culture dishes at 500 cells per well. Set up 5 groups when plating, corresponding to Olaparib concentrations: 0 μM, 0.5 μM, 1 μM, 2 μM, and 4 μM.

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

[0039] 4. On the 10th day of plating, collect samples: remove the culture medium, wash twice with PBS, fix with 4% paraformaldehyde at room temperature for 15 minutes, then add 1% methylene blue solution for staining for 1 hour, and slowly rinse with running water until the background is colorless.

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

[0041] II. Experimental results:

[0042] As shown in Fig. A, after Olaparib treatment, the clonogenicity of the ASB7 overexpression group was significantly reduced. The experimental results show that ASB7 high expression makes cells sensitive to PARP inhibitors. Figure 3

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

[0044] I. Experimental methods:

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

[0046] 2. The animals are divided into two groups, 12 in each group, and 1×10 6 empty vector and 143B cells overexpressing ASB7 are injected subcutaneously into the groin of the mice. One week after injection, the two groups of cells are randomly divided into two groups, 6 in each group, and the control group is injected with DMSO (Vehicle), and the drug group is injected with 50 mg / kg Olaparib daily. Then the tumor volume is measured once every three days with a vernier caliper, and the volume calculation formula is: V (volume) = (width 2 × length × π) / 6.

[0047] 3. After 28 days of cell injection, the mice are sacrificed by spinal dislocation, and the tumors are collected and weighed using an electronic balance.

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

[0049] II. Experimental results:

[0050] As Figure 3 shown in Figure B, the mouse tumors are controlled within the scope of animal welfare required by animal ethics. Figure 3 Results of Figures B-D show that in the case of overexpression of empty vector, the cells are not sensitive to Olaparib, and there is no statistically significant difference between DMSO (Vehicle). In the overexpression of ASB7 group, the tumor size is significantly reduced after the use of Olaparib, and the p value is significant, that is, the sensitivity of the mouse tumor overexpressing ASB7 to Olaparib is significantly higher than that of the empty vector group. The experimental results show that ASB7 overexpression tumors are sensitive to PARP inhibitors. ASB7 overexpression can slow down tumor cell proliferation, and the specific mechanism is that ASB7 amplification damages the homologous recombination repair of tumor cells, leading to a decrease in cell proliferation, and also makes the tumor cells sensitive to PARP inhibitors.

Claims

1. Application of E3 ubiquitin ligase ASB7 as a molecular marker in the preparation of products for predicting the sensitivity of cancer patients to PARP inhibitor therapy.

2. Application of formulations that detect the expression level of E3 ubiquitin ligase ASB7 in the preparation of products that predict the sensitivity of cancer patients to PARP inhibitor therapy.

3. Application of PARP inhibitors in the preparation of drugs for treating ASB7 gene amplification tumors.

4. Application of formulations that promote the expression of E3 ubiquitin ligase ASB7 in the preparation of drugs that enhance the sensitivity of cancer patients to PARP inhibitor therapy.

5. Application of agents that promote the expression of E3 ubiquitin ligase ASB7 in combination with PARP inhibitors in the preparation of drugs for treating tumors.

6. The application according to any one of claims 1 to 5, characterized in that, The PARP inhibitor is selected from olaparib, niraparib, rucaparib, or tapolaparib.

7. The application according to claim 6, characterized in that, The PARP inhibitor is olaparib.

8. The application according to any one of claims 1-2 and 4-5, characterized in that, The tumor was selected from ASB7 amplified tumor.

9. The application according to claim 4 or 5, characterized in that, The formulation that promotes ASB7 expression is an overexpression vector containing the ASB7 gene.

10. The application according to claim 8, characterized in that, The tumor is a sarcoma.

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