Combination of brd7 stabilizer and her2 targeting drug and use thereof

Abstract

This invention discloses a combination formulation of a BRD7 stabilizer and a HER2-targeting drug, and its application, belonging to the field of pharmaceutical molecular biology. This invention is the first to discover that BRD7 can upregulate HER2 protein stability in triple-negative breast cancer, promoting the transformation of triple-negative breast cancer cells to HER2-positive cells, thereby increasing the sensitivity of triple-negative breast cancer cells to HER2-targeting antibody drugs. Further research demonstrates that BRD7 increases HER2 protein stability by inhibiting HER2 protein degradation mediated by the autophagy-lysosomal pathway. Therefore, the combination of a BRD7 stabilizer drug and a HER2-targeting monoclonal antibody drug exhibits significant anti-tumor effects. This invention provides a potential treatment strategy for the clinical treatment of triple-negative breast cancer and has broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical molecular biology technology, specifically relating to the combined formulation of BRD7 stabilizers and HER2-targeting drugs and their applications. Background Technology

[0002] Triple-negative breast cancer (TNBC) is the most malignant type of breast cancer with the worst prognosis. Its molecular characteristics include negativity of the estrogen receptor (ER), progesterone receptor (PR), and the proto-oncogene HER2. Due to the lack of hormone receptors and the HER2 target, patients cannot benefit from endocrine and targeted therapies, and chemotherapy remains the standard treatment. However, chemotherapy is prone to drug resistance and severe toxic side effects.

[0003] HER2 is an important member of the human epidermal growth factor receptor family, playing a crucial role in normal cell proliferation, differentiation, and tumorigenesis. Traditionally, HER2-positive breast cancer patients have significantly benefited from anti-HER2 targeted therapy. However, TNBC is considered to have no or low HER2 expression, making HER2-targeted therapies unsuitable. Therefore, restoring HER2 expression in TNBC and inducing its conversion to HER2-positive status holds promise as a novel treatment option and represents a promising therapeutic strategy.

[0004] In breast cancer, BRD7 has been identified as a tumor suppressor, playing a crucial role in various biological processes. This invention reveals that in TNBC, BRD7 significantly increases HER2 protein stability by inhibiting autophagy-lysosomal pathway-mediated HER2 protein degradation, thereby promoting the conversion of TNBC to HER2-positive status. Summary of the Invention

[0005] The discovery of this invention reveals the crucial role of BRD7 overexpression agents, BRD7-stabilizing peptides, or BRD7-stabilizing small molecule compounds in regulating HER2 expression in TNBC cells. Based on this, this invention proposes novel uses for BRD7 overexpression agents, BRD7-stabilizing peptides, or BRD7-stabilizing small molecule compounds in the preparation of drugs to promote the transformation of triple-negative breast cancer to HER2-positive status and enhance the sensitivity of triple-negative breast cancer to HER2-targeted drugs, as well as corresponding pharmaceutical compositions and therapeutic strategies. This application provides a completely new drug development approach for converting TNBC patients who are traditionally ineligible for HER2-targeted therapy into a potentially benefiting population.

[0006] This invention discovers and confirms that BRD7 can upregulate HER2 protein levels in triple-negative breast cancer, thereby promoting the transformation of triple-negative breast cancer to HER2-positive.

[0007] Specifically, BRD7 can increase the stability of HER2 protein by inhibiting the autophagy-lysosomal pathway-mediated degradation of HER2 protein, thereby ultimately promoting the transformation of triple-negative breast cancer to HER2-positive.

[0008] In this invention, the "promoting the transformation of triple-negative breast cancer to HER2-positive" specifically manifests as increasing the protein expression level of HER2 and / or the immunohistochemical (IHC) score of HER2 in triple-negative breast cancer cells or tissues.

[0009] The first aspect of this invention aims to provide a combination formulation of a BRD7 stabilizer and a HER2-targeting drug, wherein the BRD7 stabilizer comprises at least one of: a BRD7 overexpression reagent, a peptide that increases BRD7 stability, and a small molecule compound that increases BRD7 stability.

[0010] Further, the peptide that increases BRD7 stability comprises at least one of TB16 and TAB12, wherein the sequence of TB16 is KVLETFLAKSRPELLE (see SEQ ID NO.1) and the sequence of TAB12 is IQKHQEHILRFA (see SEQ ID NO.2); the small molecule compound that increases BRD7 stability comprises RN486 and its derivatives, wherein the chemical formula of RN486 is C 35 H 35 FN6O3, the chemical structural formula is shown below:

[0011] ;

[0012] The HER2-targeting drugs mentioned include at least one of HER2 antibody drugs and antibody-drug conjugates.

[0013] Furthermore, the HER2 antibody drug includes at least one of trastuzumab and pertuzumab. HER2 antibody-drug conjugates include at least one of trastuzumab emtansine, trastuzumab botulinum, and vidictetumab.

[0014] The second aspect of this invention aims to provide the application of the combined formulation of the BRD7 stabilizer and the HER2-targeting drug, specifically its application in the preparation of a drug for treating triple-negative breast cancer.

[0015] Furthermore, the combined formulation of the BRD7 stabilizer and the HER2-targeting drug is used in the preparation of a drug that enhances the sensitivity of triple-negative breast cancer to HER2-targeting drugs.

[0016] Furthermore, the combined formulation of the BRD7 stabilizer and the HER2-targeting drug is used in the preparation of drugs for the transformation of triple-negative breast cancer to HER2-positive breast cancer.

[0017] This invention provides a combination drug regimen for treating triple-negative breast cancer, comprising:

[0018] (a) The first active ingredient is a BRD7 stabilizer, including at least one of a BRD7 overexpression reagent, a peptide that increases BRD7 stability, and a small molecule compound that increases BRD7 stability;

[0019] (b) The second active ingredient is an antibody drug or antibody-drug conjugate targeting HER2.

[0020] Preferably, the BRD7 stabilizer is a peptide drug TB16, TAB12, or a small molecule drug RN486, Gracillin, or its derivatives or analogs; the HER2-targeting drug is an anti-HER2 monoclonal antibody or its conjugate, including but not limited to trastuzumab, pertuzumab, trastuzumab emtansine, or combinations thereof.

[0021] Furthermore, the combination therapy involves administering an effective amount of a BRD7 stabilizer and a HER2-targeting drug to subjects in need.

[0022] Furthermore, the combined administration includes simultaneous administration, sequential administration (i.e., administering the BRD7 stabilizer first, followed by the HER2-targeting drug), or other medically acceptable delivery methods.

[0023] Beneficial effects of this invention:

[0024] This study reveals for the first time that the BRD7 protein can stabilize the HER2 protein by inhibiting the autophagy-lysosomal pathway, promoting the transformation of TNBC to HER2-positive, thus providing a new therapeutic target for TNBC. By inducing HER2 expression, TNBC patients who were previously ineligible for HER2-targeted therapy can benefit from this type of drug treatment, expanding the applicable population for targeted drugs. The provided BRD7 stabilizers (such as TB16, TAB12, and RN486, and their stabilizing effects are disclosed in patent documents with publication numbers CN120209082A, CN119751591A, and patent application number CN119080748A, respectively) and the combination therapy strategy of HER2-targeted drugs provide new drug combinations and targeted therapy strategies for the clinical treatment of TNBC, which have significant translational medicine value. Attached Figure Description

[0025] Figure 1 BRD7 increases the protein stability of HER2 through the autophagy-lysosomal pathway;

[0026] in: Figure 1A: Western blot analysis was conducted to investigate the effects of BRD7 overexpression on the protein expression levels of estrogen receptor (ER), progesterone receptor (PR), and HER2 in triple-negative breast cancer cell lines MDA-MB-231, BT549, and 4T1. Figure 1 B: RT-qPCR assay was used to detect the effect of BRD7 overexpression on HER2 mRNA expression levels in MDA-MB-231 and BT549 cells; Figure 1 C: TNBC cell models with BRD7 knockdown expression (MDA-MB-231, BT549, 4T1) were constructed and treated with the proteasome inhibitor MG132 and the autophagy-lysosome inhibitor chloroquine (CQ), respectively, and the protein expression level of HER2 was detected.

[0027] Figure 2 The results of BRD7 overexpression combined with HER2 knockdown synergistically inhibiting the malignant progression of TNBC;

[0028] in: Figure 2 A: Western blot results of protein detection in MDA-MB-231, BT549 and 4T1 cell models of TNBC with BRD7 overexpression and HER2 knockdown; Figure 2 B: CCK8 assay to detect the effect of BRD7 overexpression and HER2 knockdown on the proliferation of TNBC cells (MDA-MB-231, BT549, 4T1); Figure 2 C: The effect of BRD7 overexpression and HER2 knockdown on the colony-forming ability of TNBC cells (MDA-MB-231, BT549, 4T1) was detected by clonogenic assay and the corresponding bar chart.

[0029] Figure 3 The results showed that TB16 and RN486 could increase the protein stability of BRD7 in TNBC cells;

[0030] in: Figure 3 A: Western blot analysis of the effect of TB16 on BRD7 protein expression levels in TNBC cells MDA-MB-231; Figure 3 B: Western blotting to detect the effect of RN486 on BRD7 protein expression levels in TNBC cells MDA-MB-231.

[0031] Figure 4 TB16 and RN486 can increase the protein stability of HER2 in TNBC cells;

[0032] in: Figure 4A: Western blot analysis was conducted to detect the effects of TB16 on the protein expression levels of BRD7 and HER2 in MDA-MB-231, BT549, and 4T1 cells; Figure 4 B: Western blotting experiment to detect the effect of TB16 on the protein stability of HER2 in MDA-MB-231, BT549, and 4T1 cells and the corresponding curve analysis and statistical graph; Figure 4 C: Western blot analysis was used to detect the effect of RN486 on the protein expression levels of BRD7 and HER2 in MDA-MB-231 and BT549 cells.

[0033] Figure 5 Results: TB16 combined with HER2 knockdown can exert a synergistic anti-tumor effect;

[0034] in: Figure 5 A: Western Blot analysis of the effects of TB16 treatment or TB16 combined with HER2 knockdown on the protein expression levels of BRD7 and HER2 in TNBC cells (MDA-MB-231, BT549); Figure 5 B: CCK8 assay to detect the effect of TB16 treatment and HER2 knockdown on the proliferation of TNBC cells (MDA-MB-231, BT549); Figure 5 C: Colony formation assay to detect the effect of TB16 treatment and HER2 knockdown on the colony formation ability of TNBC cells (MDA-MB-231, BT549) and the corresponding bar chart.

[0035] Figure 6 The results showed that BRD7 stabilizers combined with HER2 monoclonal antibodies could exert a synergistic anti-tumor effect.

[0036] in: Figure 6 A: The CCK8 assay was used to detect the effect of TB16 combined with HER2 monoclonal antibody (trastuzumab) on the proliferation of TNBC cells (MDA-MB-231, BT549, 4T1). Figure 6 B: CCK8 assay to detect the effect of RN486 combined with HER2 monoclonal antibody (trastuzumab) on the proliferation of TNBC cells (MDA-MB-231, BT549); Figure 6 C: Clonogenic assay to detect the effect of TB16 combined with HER2 monoclonal antibody (trastuzumab) on the clonogenic ability of TNBC cells (MDA-MB-231, BT549, 4T1) and corresponding bar chart analysis.

[0037] Figure 1The control group was transformed with the blank plasmid pCMV-HA-vector; BRD7 OE was transformed with the BRD7 expression plasmid pCMV-HA-BRD7; and shBRD7 was transformed with the BRD7 knockdown plasmid pSUPER / shBRD7. Figure 1 In the diagram above, cells transfected with plasmid pSUPER / shBRD7 are indicated by a "+", while cells transfected with the blank control plasmid pSUPER / shNC are indicated by a "-".

[0038] Figure 2 The control group consisted of blank plasmids pCMV-HA-vector and pSUPER / shNC, which were transformed into BRD7 and HER2, respectively. BRD7OE was the expression plasmid pCMV-HA-BRD7, which was transformed into BRD7. shHER2 was the HER2 knockdown plasmid pSUPER / shHER2.

[0039] Figure 5 The pSUPER / shNC plasmid was added to both the TAT-TB16 group and the TAT group.

[0040] Figure 6 middle Figure 6 A and Figure 6 The control group for C was the TAT+DMSO treatment group. Figure 6 The control group for B was the DMSO treatment group.

[0041] Figure 4-6 TAT stands for membrane-penetrating peptide, and TAT-TB16 is TB16 linked with the membrane-penetrating peptide.

[0042] Figure 1 , 2 The data in the bar and line charts in sections 5 and 6 are presented as mean ± standard error. ; ; There was no statistically significant difference between the two values ​​(ns). Detailed Implementation

[0043] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but the present invention is not limited to the following technical solutions.

[0044] The MDA-MB-231, BT549, and 4T1 cell lines used in this invention were all purchased from the Biological Cell Laboratory of the Advanced Research Center of Central South University. The MDA-MB-231 cells were cultured in DMEM liquid medium containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin, and grown adherently in a constant temperature incubator at 37°C and 5% CO2. The BT549 and 4T1 cells were cultured in 1640 liquid medium containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin, and grown adherently in a constant temperature incubator at 37°C and 5% CO2.

[0045] Example 1

[0046] BRD7 increases HER2 protein stability through the autophagy-lysosomal pathway, promoting the conversion of TNBC to HER2-positive protein.

[0047] 1.1 Experimental Design:

[0048] (1) Blank plasmid (pCMV-HA-vector, purchased from Prometheus (Beijing) Biotechnology Co., Ltd.) and BRD7 expression plasmid (pCMV-HA-BRD7) were transfected into MDA-MB-231, BT549 and 4T1 cells, respectively. The effect of BRD7 overexpression on the expression levels of ER, PR and HER2 proteins in TNBC cells was detected by Western Blot.

[0049] During the preparation of pCMV-HA-BRD7, the pCMV-HA-vector is inserted with SalⅠ and NotⅠ restriction enzyme sites. The amplification primer sequences for BRD7 are as follows:

[0050] Chain of Justice: TTTGTCGACCGGCAAGAAGCACAAGAAGCAC, see SEQ ID NO.3;

[0051] antisense chain: TTTGCGGCCGCTCAACTTCCACCAGGTCCACACTC, see SEQ ID NO.4.

[0052] (2) The blank plasmid and the BRD7 expression plasmid were transfected into MDA-MB-231 and BT549 cells, respectively. RNA was extracted and the effect of BRD7 overexpression on the expression level of HER2 mRNA in TNBC cells was detected by RT-qPCR.

[0053] (3) pSUPER / shNC (blank plasmid, purchased from Beijing Zhongyuan Heju Biotechnology Co., Ltd.) and the BRD7 knockdown expression plasmid pSUPER / shBRD7 were transfected into MDA-MB-231, BT549, and 4T1 cells, respectively. 24 h after transfection, the cells were treated with the proteasome inhibitor MG132 and the lysosomal inhibitor chloroquine (CQ) for 4 hours, respectively. Cells were collected and proteins were extracted immediately. The protein expression levels of BRD7 and HER2 were detected by Western blotting.

[0054] When preparing the BRD7 knockdown expression plasmid pSUPER / shBRD7, the DNA template sequence of shBRD7 is: 5'-GTGCCAAGATTATCCGTAT-3', see SEQ ID NO.5. The inserted restriction enzyme sites are Bgl II and Hind III. Primer sequences are:

[0055] Chain of Justice: 5'-GATCCCCGTGCCAAGATTATCCGTATTTCAAGAGAATACGGATAATCTTGGCACTTTTTA-3', see SEQ ID NO.6;

[0056] antisense chain: 5'-AGCTTAAAAAGTGCCAAGATTATCCGTATTCTCTTGAAATACGGATAATTCTGGCACGGG-3', see SEQ ID NO. 7.

[0057] 1.2 Experimental Results:

[0058] Experimental results showed that BRD7 overexpression upregulated HER2 protein expression levels in TNBC, while having no significant effect on PR and ER protein expression. This indicates that high BRD7 expression can exert a tumor-suppressive function in TNBC and promote the transformation of TNBC to HER2-positive TNBC. Figure 1 A). Furthermore, RT-qPCR results showed that BRD7 had no significant effect on HER2 mRNA expression levels ( Figure 1B). This indicates that BRD7 may promote HER2 expression in TNBC through post-translational modification rather than transcriptional control. To further identify the pathways involved in BRD7-mediated regulation of HER2 protein stability, a TNBC cell model with BRD7-silenced expression was constructed, and cells were treated with the proteasome inhibitor MG132 and the lysosomal inhibitor chloroquine (CQ), respectively. Western blot results showed that CQ, but not MG132, blocked the decrease in HER2 protein expression caused by BRD7 silencing, suggesting that BRD7 may increase HER2 protein stability through the autophagy-lysosomal pathway. Figure 1 C). The above results indicate that BRD7 participates in the regulation of HER2 protein stability in TNBC through the autophagy-lysosomal pathway, promoting the transformation of TNBC to HER2-positive.

[0059] Example 2

[0060] BRD7 overexpression combined with HER2 knockdown synergistically inhibits the malignant progression of TNBC.

[0061] 2.1 Experimental Procedure:

[0062] (1) PCMV-HA-BRD7 plasmid or co-transfected PCMV-HA-BRD7 and pSUPER / shHER2 plasmids were transfected into MDA-MB-231, BT549, and 4T1 cells. Cells were collected 48 h after transfection and protein extraction was performed immediately. The protein expression levels of BRD7 and HER2 were detected by Western blotting.

[0063] When preparing the pSUPER / shHER2 plasmid, the DNA template sequence of shHER2 is: 5'-GCAGCCAAGTACAACCGTTAA-3', see SEQ ID NO.8, and the restriction sites are: Bgl II and Hind III.

[0064] Primer sequence: positive strand: 5'-GATCCCCGCAGCCAAGTACAACCGTTAATTCAAGAGATTAACGGTTGTACTTGGCTGCTTTTTA -3', see SEQ ID NO.9;

[0065] antisense chain: 5'-AGCTTAAAAAGCAGCCAAGTACAACCGTTAATCTCTTGAATTAACGGTTGTACTTGGCTGCGGG-3', see SEQ ID NO. 10.

[0066] (2) CCK8 experiment:

[0067] ① TNBC cells (MDA-MB-231, BT549, 4T1) transfected with the corresponding plasmids were seeded into 96-well plates at a rate of 1000 cells / well.

[0068] ② Set 6 time points: 0 d, 1 d, 2 d, 3 d, 4 d, and 5 d. Then add 10% CCK8 solution to each well and incubate at 37℃ for 2 h. Measure the absorbance value using a microplate reader (wavelength 450 nm).

[0069] ③ Analyze the data and plot the graphs: Use GraghPad Prism 9.5.0 software to plot the cell proliferation curves.

[0070] (3) Cloning experiment:

[0071] ① Count the TNBC cells (MDA-MB-231, BT549, 4T1) transfected with the corresponding plasmids, seed them in 12-well plates at 500 cells / well, and culture for 10-14 days.

[0072] ② Discard the culture medium, wash with 1×PBS 3 times, 5 minutes each time, then add 4% paraformaldehyde and fix at room temperature for 1 hour.

[0073] ③ Discard the fixative and wash three times with 1×PBS for 5 minutes each time. Then add crystal violet staining solution to each well and stain at room temperature for 30 minutes.

[0074] ④ Discard the crystal violet staining solution, rinse with distilled water until no crystal violet residue remains, air dry, and then use a scanner to scan and record the image.

[0075] ⑤ Count the number of effective cell clones in each group and analyze the data.

[0076] 2.2 Experimental Results:

[0077] To further investigate whether knocking down HER2 expression on top of BRD7 overexpression could further enhance the inhibitory effect of BRD7 overexpression on TNBC cell proliferation and colony formation, a cell model of BRD7 overexpression and HER2 knockdown was constructed using BRD7 overexpressing TNBC cells. Figure 2 A). CCK8 and colony formation assays confirmed that HER2 knockdown further enhanced the inhibitory effect of BRD7 overexpression on TNBC cell proliferation and colony formation. Figure 2 (BC). The above results indicate that BRD7 overexpression combined with HER2 knockdown can synergistically inhibit the malignant progression of TNBC.

[0078] Based on the premise that BRD7 performs its function as a tumor suppressor gene, it was found that BRD7 can upregulate the expression of the oncogene protein HER2. This can be compared to the discovery of the potential side effects of the BRD7 tumor suppressor gene in TNBC. Therefore, further knocking down HER2 can maximize the function of BRD7 as a tumor suppressor gene, eliminate potential side effects, and transform TNBC, a breast cancer lacking a target, into a HER2-positive type.

[0079] Example 3

[0080] TB16 and RN486 can increase the protein stability of BRD7 in TNBC cells.

[0081] 3.1 Experimental Procedure:

[0082] (1) MDA-MB-231 breast cancer cells were treated with different concentrations of TB16 (0 µM, 5 µM, 10 µM, 20 µM, 40 µM). Cells were collected after 24 h of treatment and protein extraction was performed immediately. The protein expression level of BRD7 was detected by Western blotting.

[0083] (2) MDA-MB-231 breast cancer cells were treated with different concentrations of RN486 (0 µM, 4 µM, 8 µM, 12 µM). Cells were collected after 24 h of treatment and protein extraction was performed immediately. The protein expression level of BRD7 was detected by Western blotting.

[0084] 3.2 Experimental Results:

[0085] The results showed that in the TNBC cell line MDA-MB-231, the protein expression level of BRD7 gradually increased with increasing concentrations of TB16 and RN486. Figure 3 (AB). This indicates that both the peptide drug TB16 and the small molecule drug RN486 can function as BRD7 protein stabilizers.

[0086] Example 4

[0087] TB16 and RN486 can increase the protein stability of HER2 in TNBC cells.

[0088] 4.1 Experimental Procedure:

[0089] (1) TNBC cells (MDA-MB-231, BT549, 4T1) were treated with TAT-TB16 (80 µM). Cells were collected after 24 h of treatment and protein extraction was performed immediately. The protein expression levels of BRD7 and HER2 were detected by Western blotting. TB16 alone also had a good effect. TAT is a membrane-penetrating peptide sequence (GRKKRRQRRRPPQ, see SEQ ID NO. 11). When TB16 is linked to it (TAT-TB16), the membrane-penetrating effect and biological effect of TB16 can be further enhanced. Moreover, the membrane-penetrating peptide is not limited to TAT, but also includes other common peptides that have a membrane-penetrating effect.

[0090] (2) TNBC cells (MDA-MB-231, BT549, 4T1) were treated with TAT-TB16 (80 µM) for 24 h, followed by treatment with the protein synthesis inhibitor CHX for 0 h, 4 h, 8 h, and 12 h. Cells were collected and proteins were extracted immediately. The protein expression level of HER2 was detected by Western blotting.

[0091] (3) TNBC cells (MDA-MB-231, BT549) were treated with RN486 (4 µM). After 24 h of treatment, the cells were collected and proteins were extracted immediately. The protein expression levels of BRD7 and HER2 were detected by Western blotting.

[0092] 4.2 Experimental Results:

[0093] Further investigation was conducted to determine whether the BRD7-targeting peptide drug TB16 and the small molecule drug RN486 could promote HER2 protein expression in TNBC, thereby inducing TNBC to HER2-positive transformation. TNBC cells (MDA-MB-231, BT549, and 4T1) were treated with TB16, and Western blot experiments confirmed that TB16 significantly promoted HER2 protein expression levels. Figure 4 A); and compared with the control group, TB16 significantly shortened the protein half-life of HER2 ( Figure 4 B). Furthermore, Western blot experiments also confirmed that RN486 significantly promoted HER2 protein expression levels (B). Figure 4 C). The above results indicate that TB16 and RN486 can promote BRD7 protein expression in TNBC and promote the conversion of TNBC to HER2 positivity.

[0094] Example 5

[0095] TB16 combined with HER2 knockdown can exert a synergistic anti-tumor effect.

[0096] 5.1 Experimental Procedure:

[0097] (1) MDA-MB-231 and BT549 cells were treated with TAT-TB16 (100 µM), or TNBC cells transfected with shHER2 plasmid were further treated with TAT-TB16. Cells were collected and proteins were extracted immediately. The protein expression levels of BRD7 and HER2 were detected by Western blotting.

[0098] (2) CCK8 experiment:

[0099] ① Transfect pSUPER / shNC and pSUPER / shHER2 plasmids into TNBC cells (MDA-MB-231, BT549), respectively, and seed them into 96-well plates at a rate of 1000 cells / well.

[0100] ② After the cells have fully adhered, MDA-MB-231 and BT549 cells were treated with 100 µM TAT-TB16. The control group was the TAT-treated group.

[0101] ③ Set 6 time points: 0 d, 1 d, 2 d, 3 d, 4 d, and 5 d. Then add 10% CCK8 solution to each well and incubate in a 37℃ incubator for 2 h. Measure the absorbance value using an ELISA reader (wavelength 450 nm).

[0102] ④ Analyze the data and plot the graphs: Use GraghPad Prism 9.5.0 software to plot the cell proliferation curves.

[0103] (3) Cloning experiment:

[0104] ① Transfect pSUPER / shNC and pSUPER / shHER2 plasmids into TNBC cells (MDA-MB-231, BT549), respectively. After 24 h of transfection, digest the cells and count them. Seed the cells in 12-well plates at 500 cells / well. After the cells adhered, co-culture the tumor cells with TAT (100 µM) or TAT-TB16 (100 µM) for 10-14 days.

[0105] ② Discard the culture medium, wash with 1×PBS 3 times, 5 minutes each time, then add 4% paraformaldehyde and fix at room temperature for 1 hour.

[0106] ③ Discard the fixative and wash three times with 1×PBS for 5 minutes each time. Then add crystal violet staining solution to each well and stain at room temperature for 30 minutes.

[0107] ④ Discard the crystal violet staining solution, rinse with distilled water until no crystal violet residue remains, air dry, and then use a scanner to scan and record the image.

[0108] ⑤ Count the number of effective cell clones in each group and analyze the data.

[0109] 5.2 Experimental Results:

[0110] HER2 expression was knocked down in TB16-treated TNBC (Figure 5A). CCK8 and colony formation assays showed that HER2 knockdown further enhanced the inhibitory effect of TB16 on TNBC cell proliferation and colony formation (Figures 5B-C). These results indicate that TB16 combined with HER2 knockdown has a synergistic anti-tumor effect in TNBC cells.

[0111] Example 6: The combination of BRD7 stabilizer and HER2 monoclonal antibody (trastuzumab) can exert a synergistic anti-tumor effect.

[0112] 6.1 Experimental Procedure:

[0113] (1) CCK8 experiment:

[0114] ① Seed TNBC cells (MDA-MB-231, BT549, 4T1) in good growth condition into 96-well plates at a rate of 1000 cells / well.

[0115] ② After the cells had completely adhered to the wall, MDA-MB-231, BT549, and 4T1 cells were treated with trastuzumab (purchased from Shanghai Haoyuan Biomedical Technology Co., Ltd.) (2 µM), TAT-TB16 (100 µM), or TAT-TB16 (100 µM) combined with trastuzumab (2 µM), respectively. The control group was the TAT+DMSO treatment group.

[0116] ③ After the cells were fully adhered, MDA-MB-231 and BT549 cells were treated with trastuzumab (2 µM), RN486 (8 µM), or RN486 (8 µM) combined with trastuzumab (2 µM), respectively. The control group was the DMSO treatment group.

[0117] ④ Set 6 time points: 0 d, 1 d, 2 d, 3 d, 4 d, and 5 d. Then add 10% CCK8 solution to each well and incubate at 37℃ for 2 h. Measure the absorbance value using a microplate reader (wavelength 450 nm).

[0118] ⑤ Analyze the data and plot the graphs: Use GraghPad Prism 9.5.0 software to plot the cell proliferation curves.

[0119] (2) Cloning experiment:

[0120] ① Digest and count TNBC cells (MDA-MB-231, BT549, 4T1) in good growth condition, seed them in 12-well plates at 500 cells / well, and after the cells adhere, co-culture the tumor cells with trastuzumab (2 µM), TAT-TB16 (100 µM), or TAT-TB16 (100 µM) combined with trastuzumab (2 µM) for 10-14 days.

[0121] ② Discard the culture medium, wash with 1×PBS 3 times, 5 minutes each time, then add 4% paraformaldehyde and fix at room temperature for 1 hour.

[0122] ③ Discard the fixative and wash three times with 1×PBS for 5 minutes each time. Then add crystal violet staining solution to each well and stain at room temperature for 30 minutes.

[0123] ④ Discard the crystal violet staining solution, rinse with distilled water until no crystal violet residue remains, air dry, and then use a scanner to scan and record the image.

[0124] ⑤ Count the number of effective cell clones in each group and analyze the data.

[0125] 6.2 Experimental Results:

[0126] Figure 6 The results of the CCK8 and colony formation assays showed that TB16 or RN486 combined with trastuzumab had a more significant inhibitory effect on TNBC cell proliferation and colony formation. These results indicate that the BRD7 stabilizer TB16 or RN486, combined with HER2 monoclonal antibody, exerts a synergistic anti-tumor effect in TNBC.

Claims

1. A combination formulation of a BRD7 stabilizer and a HER2-targeting drug, characterized in that, The BRD7 stabilizer includes at least one of the following: a BRD7 overexpression reagent, a peptide that increases BRD7 stability, and a small molecule compound that increases BRD7 stability.

2. The combined formulation according to claim 1, characterized in that, The peptide that increases BRD7 stability comprises at least one of TB16 and TAB12, wherein the sequence of TB16 is KVLETFLAKSRPELLE and the sequence of TAB12 is IQKHQEHILRFA; the small molecule compound that increases BRD7 stability is RN486, with the chemical formula C 35 H 35 FN6O3.

3. The combined formulation according to claim 1, characterized in that, The HER2-targeting drugs include at least one of HER2 antibody drugs and HER2 antibody-drug conjugates.

4. The combined formulation according to claim 3, characterized in that, The HER2 antibody drug includes at least one of trastuzumab and pertuzumab; the HER2 antibody-drug conjugate includes at least one of trastuzumab emtansine, trastuzumab botulinum, and vidictetumab.

5. The application of the combined formulation of the BRD7 stabilizer and the HER2-targeting drug according to any one of claims 1-4, characterized in that, The application of the combined formulation of the BRD7 stabilizer and HER2-targeting drug in the preparation of a drug for treating triple-negative breast cancer.

6. The application of the combined formulation of the BRD7 stabilizer and the HER2-targeting drug according to claim 5, characterized in that, The application of the combined formulation of the BRD7 stabilizer and HER2-targeting drug in the preparation of a drug that enhances the sensitivity of triple-negative breast cancer to HER2-targeting drugs.

7. The application of the combined formulation of the BRD7 stabilizer and HER2-targeting drug according to claim 5, characterized in that, The application of the combined formulation of the BRD7 stabilizer and HER2-targeting drug in the preparation of drugs for the transformation of triple-negative breast cancer to HER2-positive breast cancer.

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