GRB2 inhibitor combined antitumor drug composition and pharmaceutical application of compound composition

Abstract

The invention discloses an anti-tumor medicine composition combined with a GRB2 inhibitor and pharmaceutical application of a compound composition. The triple negative breast cancer is taken as a research object, and the effects of a GRB2 inhibitor (GRB2i) and a P70S6K inhibitor (PF-4708671) on the triple negative breast cancer are detected through a plate cloning formation experiment, a flow cytometry and construction of a tumor-bearing mouse. The composition of the GRB2 inhibitor (GRB2i) and the P70S6K inhibitor (PF-4708671) is proposed and proved for the first time to be capable of remarkably inhibiting the proliferation of the triple negative breast cancer. The application provides thinking and basis for diagnosis and treatment of triple negative breast cancer, and has certain theoretical and clinical significance. The research finds that the GRB2 inhibitor and the P70S6K inhibitor are combined to achieve a better killing effect on other various cancer cells, and it is indicated that improvement of the effect is broad-spectrum.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to pharmaceutical uses of an anti-tumor drug combination and a compound combination combined with a GRB2 inhibitor. Background Art

[0002] Triple-negative breast cancer (TNBC) is a type of breast cancer that is negative for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). It is characterized by high invasiveness, high metastasis, high recurrence rate, and poor prognosis. Surgery combined with chemoradiotherapy is the mainstay of clinical treatment for TNBC. With advances in research and technology, immunotherapy, endocrine therapy, and targeted therapies have emerged as emerging treatments for TNBC. However, patients exhibit varying degrees of resistance to these treatment options. Therefore, identifying novel targeted drugs to address drug resistance in TNBC is a key focus of clinical research.

[0003] Growth factor receptor-binding protein 2 (GRB2) is an adaptor protein that plays a key role in cell signaling. It is composed of a central Src homology 2 domain (SH2) and two Src homology 3 domains (SH3): the N-terminal SH3 (n-SH3) and the C-terminal SH3 (c-SH3). The SH2 domain recognizes and binds to phosphorylated tyrosine residues (pYxNx motifs), mediating the interaction of GRB2 with various receptor tyrosine kinases (RTKs) such as EGFR, FGFR, and c-Met. The SH3 domain, by recognizing the proline-rich PxxP motif, participates in the binding of GRB2 to downstream effector proteins such as SOS and Gab2. GRB2 exists in cells as both monomers and dimers. The monomeric form is its active state, while the dimer is autoinhibitory. When stimulated by growth factors, GRB2 becomes phosphorylated, causing the dimer to dissociate into monomers, thereby activating the MAPK and PI3K / AKT signaling pathways and regulating cell proliferation, differentiation, migration, and survival. Studies have shown that GRB2 phosphorylation levels increase in malignant tumor tissues, suggesting that increased levels of its monomeric form may be closely related to tumor development and progression. Given its pivotal role in multiple oncogenic signaling pathways, GRB2 has become an important target for anti-tumor therapy.

[0004] p70 ribosomal protein S6 kinase (p70S6K) is a key serine / threonine protein kinase belonging to the AGC kinase family. It plays a central role in the mTOR signaling pathway. It primarily regulates protein synthesis by phosphorylating 40S ribosomal protein S6, thereby promoting cell growth, proliferation, and metabolism. Activation of p70S6K is dependent on the upstream kinase mTORC1. In response to growth factors and nutrients, mTORC1 phosphorylates p70S6K at Thr389, promoting its activation and affecting translation initiation and elongation. p70S6K also participates in regulating physiological activities such as the cell cycle, autophagy, and energy metabolism. Studies have shown that p70S6K is abnormally activated in various tumors, including prostate, gastric, and breast cancers. Its high expression is closely associated with tumor development, progression, and drug resistance, suggesting its potential as an anti-tumor target. Several small molecule inhibitors have been developed to target p70S6K, including PF-4708671, a selective inhibitor that has shown promising anti-tumor activity in vitro and in animal models. Despite this, the efficacy of single-target p70S6K therapies remains limited, necessitating the exploration of combined therapeutic strategies with other targeted agents. Summary of the Invention

[0005] To address the aforementioned technical issues in the prior art, the present invention provides anti-tumor drug combinations and compound combinations combined with GRB2 inhibitors, and their pharmaceutical uses. The present invention demonstrates, through in vitro and in vivo experiments, that a combination of a GRB2 inhibitor (GRB2i) and a P70S6K inhibitor (PF-4708671) significantly inhibits the proliferation of mouse triple-negative breast cancer cells 4T1 and human triple-negative breast cancer cells MDA-MB-231 and MDA-MB-453. Therefore, the combination of a GRB2 inhibitor and a P70S6K inhibitor offers a new potential clinical treatment strategy, providing a novel approach for the treatment of triple-negative breast cancer.

[0006] The present invention first provides pharmaceutical uses of a compound combination, which includes a GRB2 inhibitor and a P70S6K inhibitor. The pharmaceutical use is application in the preparation of anti-tumor drugs.

[0007] Preferably, the structural formula of the GRB2 inhibitor is as shown in Formula 1: Formula 1.

[0008] Preferably, the P70S6K inhibitor is the small molecule compound PF-4708671.

[0009] Preferably, the tumor type is breast cancer, lung cancer, gastric cancer, colon cancer, liver cancer, thyroid cancer, esophageal cancer, or leukemia. More preferably, the breast cancer is triple-negative breast cancer, the lung cancer is lung adenocarcinoma, the gastric cancer is gastric adenocarcinoma, the esophageal cancer is esophageal squamous cell carcinoma, and the leukemia is chronic myeloid leukemia.

[0010] The present invention further provides an anti-tumor drug combination combined with a GRB2 inhibitor, comprising a GRB2 inhibitor and a P70S6K inhibitor.

[0011] Preferably, the structural formula of the GRB2 inhibitor is as shown in Formula 1: Formula 1.

[0012] Preferably, the P70S6K inhibitor is the small molecule compound PF-4708671.

[0013] Compared with the prior art, the present invention has the following beneficial effects.

[0014] This study examined triple-negative breast cancer (TNBC) using plate-based colony formation assays, flow cytometry, and the establishment of tumor-bearing mice to examine the effects of a GRB2 inhibitor (GRB2i) and a P70S6K inhibitor (PF-4708671). This study, for the first time, demonstrates that a combination of a GRB2 inhibitor (GRB2i) and a P70S6K inhibitor (PF-4708671) can significantly inhibit the proliferation of TNBC. This application provides insights and foundations for the diagnosis and treatment of TNBC, possessing both theoretical and clinical significance.

[0015] The present invention found that the combination of GRB2 inhibitors and P70S6K inhibitors also has a better killing effect on a variety of other cancer cells, indicating that the improvement of this effect is broad-spectrum. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The effect of PF-4708671 (denoted as P70S6Ki, the same below) combined with GRB2i on cell activity. Figure 1 AC in the figure are the effects of combined use of PF-4708671 and GRB2i on the activity of HAP1, 4T1, and MDA-MB231 cells detected by checkerboard assay; Figure 1 DE in the figure represent the effects of combined use with 20 μM PF-4708671 on the IC50 values ​​of GRB2 inhibitors in HAP1 and 4T1 cells, respectively; Figure 1 F in the figure represents the effect of 30 μM PF-4708671 combined with 20 μM GRB2i on the activity of MDA-MB-231 cells; Figure 1 Panel G shows the effect of combined treatment with 30 μM PF-4708671 and 75 μM GRB2i on the viability of MDAMB-453 cells after 48 h of treatment. **** indicates p < 0.0001, n = 3.

[0017] Figure 2The effect of PF-4708671 combined with GRB2i on cell proliferation. Figure 2 A in the figure is a colony formation assay to detect the colony formation of HAP1, 4T1 and MDA-MB-231 cells under the combined action of PF-4708671 and GRB2i; Figure 2 Figures B and D are statistical histograms of colony formation in HAP1, 4T1, and MDA-MB-231 cells, respectively. **** represents p < 0.0001, *** represents p < 0.001, ** represents p < 0.01, and * represents p < 0.05. n = 3.

[0018] Figure 3 The effect of PF-4708671 combined with GRB2i on cell apoptosis. Figure 3 A in the figure is the Annexin V / PI double staining method to detect the apoptosis levels of HAP1 and 4T1 under the combined action of PF-4708671 and GRB2i; Figure 3 Figures B and C are histograms showing the percentage of apoptosis in HAP1 and 4T1 cells, respectively, and the Annexin V-positive cell population was counted. **** indicates p < 0.0001, n = 3.

[0019] Figure 4 The effect of combined use of GRB2i and PF-4708671 on autophagic flux. Figure 4 A in the figure is Western Blot analysis of the effect of PF-4708671 on LC3B-II in GRB2 knockout HAP1 cells; Figure 4 B in the figure is Western Blot analysis of the effect of combined use of GRB2i and PF-4708671 on LC3B-II in 4T1 cells.

[0020] Figure 5 The combination of GRB2i and PF-4708671 inhibited the growth of 4T1-Luc tumors. Figure 5 A in the figure is a picture of mouse tumors after combined treatment with GRB2i and PF-4708671; Figure 5 Panel B is a histogram of tumor weight after combined treatment with GRB2i and PF-4708671; Figure 5 C in the figure is the tumor volume growth curve during the combined treatment of GRB2i and PF-4708671; Figure 5 D in the figure is the in vivo imaging of mice on the first and last days, with two mice selected from each group; Figure 5 Figure E shows the statistical graph of luminescence area from in vivo mouse imaging experiments. **** represents p < 0.0001, *** represents p < 0.001, ** represents p < 0.01, * represents p < 0.05, ns represents p > 0.05, n = 5.

[0021] Figure 6 Immunohistochemical staining of tumor tissues after treatment with PF-4708671 and GRB2i. Figure 6 A in the figure shows H&E, Ki-67, TUNEL and merged immunohistochemical staining of tumor tissues. Scale bar: 10 μm. Figure 6 B in the figure is a statistical diagram of Ki-67 positive cells in tumor tissue; Figure 6 Panel C shows the statistical analysis of TUNEL-positive cells in tumor tissue. **** represents p < 0.0001, ** represents p < 0.01, and * represents p < 0.05. n = 5.

[0022] Figure 7 This is an in vivo toxicity experiment after treatment with PF-4708671 and GRB2i. Figure 7 A in the figure is a curve of changes in mouse body weight during GRB2i and PF-4708671 treatment; Figure 7 B is H&E staining of the main organ tissues (heart, liver, spleen, lung and kidney) of each group of mice, scale bar: 10μm.

[0023] Figure 8 The effect of PF-4708671 combined with GRB2i on the activity of tumor cells of different cancers. Figure 8 Figures A to F represent the effects of PF-4708671 combined with GRB2i on the viability of human gastric adenocarcinoma cells AGS, human lung adenocarcinoma cells A549, human colon cancer cells HCT116, human liver cancer cells HepG2, human thyroid cancer cells HTh-7, and human esophageal squamous cell carcinoma cells KYSE150, respectively. **** represents p < 0.0001, *** represents p < 0.001, and ** represents p < 0.01. n = 3. DETAILED DESCRIPTION

[0024] The GRB2 inhibitor used in this application is designated GRB2i, with patent number US2023 / 0366033A1, and its structure is shown in Formula 1: Formula 1.

[0025] The P70S6K inhibitor used in the present invention is PF-4708671, denoted as P70S6Ki, manufactured by MCE, with the product number HY-15773, and the structure is shown in Formula 2: Formula 2.

[0026] Example 1: CCK-8 assay to detect cell activity (1) In vitro experiments were conducted to study the anti-proliferative activity of GRB2i and PF-4708671 combined.

[0027] To explore the cell-killing effect of the combined use of GRB2i and PF-4708671, the drug combination checkerboard assay was first used to detect the killing effects of different concentrations of GRB2i and PF-4708671 alone or in combination on HAP1 cells (human chronic myeloid leukemia cells) after 48 h of treatment, and the cell survival rate was detected using the CCK-8 method.

[0028] The target cells were digested into cell suspension and the cells were counted. 3×10 3 HAP1 cells / well, 2×10 3 4T1 cells / well and 2×10 3 MDA-MB-231 cells were seeded into 96-well plates at a volume of 100 μL / well. After culturing HAP1 and 4T1 cells for 24 hours, PF-4708671 concentrations of 25, 20, 15, 10, 5, and 0 μM were added to rows 2 through 7 of the 96-well plate, and GRB2i concentrations of 25, 12.5, 6.3, 3.1, 1.6, 0.8, and 0 μM were added to columns 2 through 8 of the 96-well plate. After culturing MDA-MB-231 cells for 24 hours, PF-4708671 concentrations of 40, 30, and 0 μM were added to rows 2 through 4 of the 96-well plate, and GRB2i concentrations of 80, 50, 30, and 0 μM were added to columns 2 through 5 of the 96-well plate. A control group consisting of cells without drug and a blank group without drug were also established.

[0029] The results are as follows Figure 1 As shown in A, the cell survival rate of HAP1 after the combination of the two drugs was significantly lower than that under GRB2i and PF-4708671 treatment alone.

[0030] The same results were obtained in triple-negative breast cells. Figure 1 The results of B in Figure 1 show that the combined killing effect of the two drugs on 4T1 cells (mouse breast cancer cells) is stronger than using either drug alone.

[0031] Figure 1 Results from the C checkerboard assay showed that combined treatment with GRB2i and PF4708671 reduced cell survival in MDA-MB-231 cells (a human breast cancer cell line and a leading model for triple-negative breast cancer) compared to treatment with either drug alone. Combined treatment with GRB2i and PF-4708671 exerted a synthetic lethal effect on triple-negative breast cancer cells.

[0032] (2) After confirming the effect of the combination of GRB2i and P70S6Ki through the checkerboard assay, this application further determined the effect of the combined use of PF-4708671 on the IC50 of the GRB2 inhibitor in cells through the CCK-8 assay.

[0033] The target cells were digested into cell suspension and the cells were counted. 3×10 3 HAP1 cells / well, 2×10 3 4T1 cells were seeded into 96-well plates at a volume of 100 μL per well. After 24 hours of culture, the culture medium was discarded. PF-4708671 concentrations of 25, 20, 15, 10, 5, and 0 μM were added to rows 2 through 7 of the 96-well plate. GRB2i concentrations of 25, 12.5, 6.3, 3.1, 1.6, 0.8, and 0 μM were added to columns 2 through 8 of the 96-well plate. A control group consisting of cells without drug and a blank group without drug were also set up, with three replicates for each concentration. The cells were treated in a 37°C, 5% CO2 incubator for 48 hours. The culture medium in the 96-well plate was then discarded, and fresh culture medium containing 10% CCK-8 was added to each well and incubated with the cells for an additional 0.5–4 hours.

[0034] After incubation, measure the absorbance of each well at 450 nm using a microplate reader. Calculate viability using the following equation: "Vitality = (OD experimental group - OD blank group) / (OD control group - OD blank group) × 100%." ​​Use GraphPad Prism 9 software to process experimental data and calculate cell viability, or plot cell survival curves and calculate IC50 values.

[0035] Figure 1 As shown in Figure D, compared with GRB2i treatment alone (IC50=56.50μM), the IC50 value of HAP1 cells to GRB2i was significantly reduced (IC50=37.07μM) under the action of 20μM PF4708671. Figure 1 Figure (E) shows that the IC50 value of 4T1 cells to GRB2i was significantly reduced (IC50 = 16.99 μM) when treated with 20 μM PF-4708671 compared to GRB2i alone (IC50 = 36.34 μM). Combined drug treatment also reduced the IC50 value of cells to GRB2i.

[0036] (3) At the same time, this application conducted drug combination experiments on human triple-negative breast cancer cells MDA-MB-231 and MDA-MB-453 (human breast cancer cells).

[0037] like Figure 1 As shown in Figure F, after 48 h of co-treatment with 30 μM PF-4708671 and 20 μM GRB2i, the survival rate of MDA-MB-231 cells was significantly reduced. The results of MDA-MB-453 cells are shown in Figure F. Figure 1As shown in G, after 48 hours of co-treatment with 30 μM PF-4708671 and 75 μM GRB2i, a complete inhibitory effect was achieved. Through CCK-8 experiments, the present invention found that the combination of GRB2i and PF-4708671 had a better killing effect on tumor cells.

[0038] Example 2: Clone formation assay to detect cell proliferation ability To investigate the effect of combined use of GRB2i and P70S6Ki on cell proliferation, HAP1, 4T1, and MDA-MB-231 cells were evaluated in vitro using a clone formation assay ( Figure 2 A in ).

[0039] Healthy cells in the logarithmic growth phase were seeded into 6-well plates (500 cells per well) and cultured in a 37°C, 5% CO2 incubator. After adherence was observed under a microscope for 2-3 days, HAP1 cells were treated with 5μM GRB2i, 7μM PF-4708671, or their combination; 4T1 cells were treated with 7μM GRB2i, 7μM PF-4708671, or their combination; and MDA-MB-231 cells were treated with 3μM GRB2i, 5μM PF-4708671, or their combination for 14 days. Each group was plated in triplicate. During drug treatment, cell growth and proliferation were carefully monitored. Cultivation was terminated when distinct cell clusters were observed in the 6-well plates. The IMDM medium was discarded, the cells were washed once with PBS, and fixed in a universal tissue fixative (4% paraformaldehyde) for 15 minutes. The cells were washed once more with PBS buffer and stained with 0.1% crystal violet solution (2.5% crystal violet in PBS buffer) for 1 hour. Six-well plates were then stained to obtain colony formation images. The six-well plates were scanned with a scanner to obtain images of colony formation, which were then processed using Image Processing. The number of cell colonies was normalized to that of untreated WT cells.

[0040] The results of clone formation showed that when 5μM GRB2i and 7μM PF-4708671 were used in combination, the number of cell clones was significantly less than that when GRB2i or PF-4708671 were used alone, and there was a significant difference ( Figure 2 B in ).

[0041] The results of the 4T1 cell clone formation experiment showed that the combined effect of 7μM GRB2i and 7μM PF-4708671 reduced the number of cell clones. The inhibition rate of the combined drug on clone formation was greater than that of the single drug, and there was a significant difference ( Figure 2 C in ).

[0042] Under the combined action of 3μM GRB2i and 5μM PF-4708671, the number of MDA-MB-231 cell clones was reduced, and there was a significant difference ( Figure 2 D in the figure).

[0043] Therefore, the combined action of GRB2i and PF-4708671 resulted in a significant decrease in the number of cell clones formed and a significant inhibition of cell proliferation.

[0044] Example 3: In vitro study of the pro-apoptotic activity of GRB2i and PF-4708671 combined The effect of combined use of GRB2i and P70S6Ki on cell apoptosis was investigated by Annexin V / PI double staining flow cytometry experiment.

[0045] HAP1 and 4T1 cells in good condition and in the logarithmic growth phase were seeded into 6-well plates (1×10 cells per well). 5 Cells were cultured in a 37°C, 5% CO2 incubator. After 24 hours, cell attachment was observed. HAP1 cells were treated with 30 μM GRB2i, 20 μM PF-4708671, or their combination. 4T1 cells were treated with 30 μM GRB2i, 10 μM PF-4708671, or their combination for 48 hours. Three replicate wells were set up for each group.

[0046] After drug treatment, the cell culture medium was aspirated into a sterile 15 mL centrifuge tube. The HAP1 cells were washed once with PBS and 1 mL of 0.25% trypsin (without EDTA) was added to each well to digest the cells. When the digestion time was appropriate, the previously collected cell culture medium was added and the cells were gently pipetted off and transferred to a centrifuge tube. The tube was centrifuged at 1100 g for 5 minutes. After centrifugation, the supernatant was discarded and the cells were gently washed once with PBS. The cells were centrifuged at 1100 g for 5 minutes and the supernatant was discarded. The cells were then gently resuspended in PBS and counted. 5 × 10 5 Resuspend the cells and centrifuge at 1000g for 5 minutes. Discard the supernatant and resuspend the cells in 200μL of Annexin V-FITC conjugate solution. Add 5μL of Annexin V-FITC and mix gently. Then add 10μL of propidium iodide staining solution and mix gently. Incubate at room temperature in the dark for 15 minutes, then place on ice and protect from light with aluminum foil.

[0047] Because the emission spectra of each fluorophore overlap, there is a risk that one light type may leak into another channel. Therefore, when using the Attune NxT flow cytometer to measure cell apoptosis, single-stained samples must be set up to compensate for leakage of dual fluorescence.

[0048] Before loading, the samples were filtered through a 300-mesh nylon mesh. Apoptosis was assessed using an Attune NxT flow cytometer. A compensation matrix was calculated using a single-stained sample, followed by sequential loading of samples for apoptosis analysis. Flow cytometric data were analyzed using FlowJo software. The experiment was repeated three times. Apoptosis was assessed by counting the percentage of Annexin V-positive cells.

[0049] Flow cytometry analysis of apoptosis in HAP1 and 4T1 cells under the combined action of GRB2i and P70S6Ki Figure 3 As shown in Figure A, the number of apoptotic cells in HAP1 cells under the combined action of 30 μM GRB2i and 20 μM PF-4708671 for 48 h increased significantly (71.8%), which was higher than the number of apoptotic cells under the treatment of GRB2i (21.3%) and PF-4708671 (13.22%) alone, and there was a statistical difference ( Figure 3 The apoptosis of 4T1 cells also increased significantly (64.73%) when treated with 30μM GRB2i and 10μM PF-4708671 for 48 hours, which was higher than that of GRB2i (11.52%) and PF-4708671 (23.29%) alone, and there was a statistical difference ( Figure 3 C). The combined treatment of GRB2i and PF-4708671 resulted in a significant increase in cell apoptosis.

[0050] Example 4: Combination of GRB2i and PF-4708671 to inhibit cellular autophagy In vitro experiments have shown that the combination of GRB2i and P70S6Ki can inhibit tumor cell proliferation and promote apoptosis, resulting in significant anti-tumor effects. Traditional cancer drug research typically focuses on a single target or signaling pathway. However, single targets often lead to drug resistance, and current cancer treatment approaches often utilize multi-target combination therapies. This application identifies a new inhibitor, PF-4708671, for use in combination with GRB2i for the treatment of TNBC.

[0051] In this regard, we further verified the molecular mechanism by which the combined use of the two drugs achieved a synthetic lethal effect, and verified the effect of the combined use of drugs on the key autophagy protein LC3B-II in HAP1 and KO cells.

[0052] KO cells (Knockout cells) refer to GRB2 knockout cell lines constructed in HAP1 cells using CRISPR-Cas9 technology. These cells lose the expression or function of the target gene and are often used to study the biological role and function of the gene.

[0053] First, the knockout plasmid sgRNA fragment was designed based on the CRISPR-Cas9 system. The corresponding target sequence is as follows: 5′-AGATGGAGCCGGGAAGTACT-3′.

[0054] The target fragment sgRNA was constructed into the CRISPR-Cas9 system to construct a CRISPR-Cas9 plasmid for GRB2 knockout.

[0055] First, 293T cells were cultured at 4×10 5 293T cells were seeded in a 10 cm dish and allowed to adhere the next day, at which point the confluence was approximately 50%. After starving the cells for 2 hours with serum-free DMEM medium, the 293T cells were transfected with the CRISPR-Cas9 plasmid for GRB2 knockout, pMD2.G, and psPAX2 at a ratio of 5 μg:2.5 μg:2.5 μg using jetPRIME (Polyplus, France) according to the manufacturer's protocol. Eight hours later, complete medium was replaced, and the viral supernatant was collected 48 hours later, followed by a second batch of viral supernatant 24 hours later. The collected viral supernatant contained the lentiviral solution for GRB2 gene knockout.

[0056] HAP1 cells were first plated at 3×10 5 Cells were seeded in 6-well plates. The next day, after the cells adhered, the lentiviral solution was mixed with complete culture medium at a 1:1 ratio and added to HAP1 cells for infection for 8 hours. Once the HAP1 cells were in good condition, they were selected with puromycin. All control cells died, and the remaining cells were enzymatically hydrolyzed and seeded at 1 cell per well in 96-well plates. The cell line was then expanded. Protein was extracted from each well and quantified using the BCA assay. Western blotting was used to determine GRB2 knockdown in the HAP1 cell line.

[0057] When HAP1 and KO cells were treated with 50 μM CQ (chloroquine, an autophagy inhibitor) for 3 h, the autophagosomes were blocked before fusion and degradation. The expression level of LC3B-II was downregulated more significantly in cells additionally treated with 50 μM PF-4708671 ( Figure 4 A in Figure 3 indicates that when GRB2 is missing, P70S6Ki treatment leads to a more significant inhibition of cellular autophagy, resulting in the inability of cells to clear damaged proteins and organelles, and inducing increased cell death.

[0058] In addition, this application also verified the changes in the key autophagy protein LC3B-II in 4T1 cells under the combined action of GRB2i and PF-4708671. The results were consistent with those in gene knockout cells. The expression of LC3B-II in 4T1 cells decreased significantly after 24 hours of combined action of 50μM GRB2i and 50μM PF-4708671 ( Figure 4 B in ).

[0059] In this regard, it is reasonable to speculate that the combination of the two drugs leads to the obstruction of the autophagy signaling pathway and may induce the death of 4T1 cells.

[0060] Example 5: In vivo study of the anti-tumor activity of the combination of GRB2i and PF-4708671 The combination of GRB2i and PF-4708671 inhibited tumor growth.

[0061] A mouse xenograft model was established by inoculating a stable mouse 4T1 cell line (4T1-Luc) expressing Luciferase into the subcutaneous tissue of the mouse hind legs. Prepare 4T1-Luc cells in good cell condition and in the logarithmic growth phase, digest and centrifuge them, resuspend and wash them once with PBS buffer (remove serum), resuspend the cells with PBS buffer to make a cell suspension, and store them on ice for use in tumor implantation. In order to ensure that the mice have enough time to recover and adapt, the purchased mice need to adapt for about 1 week before modeling. First, fix the mouse, use a shaver to remove the hair on the left and right hind legs of the mouse, and then use depilatory cream to remove the hair on the left and right hind legs of the mouse to expose clean skin. Gently mix the cell suspension to ensure that a single cell suspension is formed. Spread the mouse skin and use an injection needle to aspirate 100μL (containing 1×10 5 A cell suspension containing 100 cells (100 cells) was implanted subcutaneously on each side of the hind legs of mice. Tumor formation was observed 2-4 days after injection. GRB2i and PF-4708671 were intraperitoneally injected at a dose of 25 mg / kg daily for 13 consecutive days. After treatment, the mice were tested.

[0062] The results showed that the tumor size of the GRB2i and PF-4708671 combined treatment group was significantly smaller than that of the other three groups, such as Figure 5 As shown in A. Compared with the single drug treatment group, the tumor weight of the drug combination group was significantly reduced ( Figure 5 According to the measured tumor growth volume curve, the combined use of GRB2i and PF-4708671 had the most significant inhibitory effect on 4T1 tumor growth ( Figure 5 C in ).

[0063] On the first and last day of drug treatment, mice were imaged intraperitoneally with a luciferin bioluminescent substrate to assess tumor development. Intravital imaging was performed on day 1 and day 13 of drug administration to monitor tumor growth. A 15 mg / ml luciferin solution was prepared in PBS buffer, with 100 μL injected per 10 g of mouse body weight. Intraperitoneal injection was performed: the needle was inserted at a slight angle with the bevel facing upward, and the needle slightly penetrated the abdominal wall. Ten minutes after luciferin injection, isoflurane gas anesthesia was administered, and the mice were positioned in a side-lying position for intravital imaging.

[0064] The results are as follows Figure 5 As shown in D. Compared with single drug, the luminous area of ​​the combined treatment group was significantly reduced after 13 days of treatment, with a significant difference ( Figure 5 E), indicating that the drug combination can significantly inhibit the growth of 4T1-Luc tumors.

[0065] Therefore, the combination of GRB2i and PF-4708671 has significant therapeutic potential for triple-negative breast cancer in mice and warrants further study. This experiment adhered to the basic principles of animal ethics, ensuring the scientific nature of the experiment and the respect and protection of the basic rights of animals through the 3R principle, strict ethical review, and comprehensive consideration of experimental animal welfare.

[0066] GRB2i and PF-4708671 were used together to inhibit tumor proliferation and promote tumor apoptosis. H&E, Ki-67 and TUNEL indicators of tumor tissue were analyzed ( Figure 6 A in ).

[0067] Tumor tissues from the four experimental groups were fixed in 4% formaldehyde and embedded in paraffin. Paraffin tumor tissues were sectioned, dewaxed, and hydrated by immersing the sections in xylene for 15 and 10 minutes, respectively. Dehydration was then performed by immersing the sections in 100% ethanol, 95% ethanol, and 80% ethanol for 1 minute each, followed by two rinses in PBS. The sections were then differentiated with 1% hydrochloric acid in ethanol for 30 seconds, rinsed twice in PBS, and stained with eosin and hematoxylin, respectively, and mounted with neutral resin. Images were captured using a light microscope.

[0068] Tumor tissues were stained for Ki-67. Tissue embedding, sectioning, and dewaxing procedures were performed as for H&E staining. Antigen retrieval was then performed. Tumor tissue sections were placed in a pH 6.0 citric acid antigen retrieval buffer and microwaved on medium heat for 8 minutes until boiling. The microwave was then turned off for 8 minutes, then kept warm and reduced to medium-low heat for 6 minutes. After cooling, sections were washed twice with PBS. Endogenous peroxidase was then blocked with a 3% H₂O₂ solution for 20 minutes and washed three times with PBS. Serum was then blocked with 3% BSA for 30 minutes. After discarding the blocking solution, the primary Ki-67 antibody was added to the tissue and the sections were placed in a humidified chamber at 4°C overnight in the dark. Secondary antibody was incubated for 20 minutes, followed by two PBS rinses. DAB staining was performed for 10 minutes. Finally, sections were dehydrated and mounted. Staining results from five tumors in each group were statistically analyzed.

[0069] Tumor tissues were stained for TUNEL. Tissue embedding, sectioning, and dewaxing procedures were performed as for H&E staining. Antigen retrieval and blocking procedures were the same as for Ki-67 immunohistochemistry. 50 μL of Equilibration Buffer was then added to each tumor sample, covering the entire area to be examined. The sample was incubated at room temperature for 10 minutes. After removing as much of the equilibration buffer as possible, 56 μL of TdT incubation buffer was added to each tissue sample and incubated at 37°C for 1 hour. The tissue samples were then rinsed with PBS four times, each for 5 minutes. In the dark, the slides were immersed in a staining jar containing DAPI solution (freshly prepared and diluted in PBS) for 8 minutes at room temperature. After staining, the tissue samples were washed three times with PBS for 5 minutes each. Excess liquid was gently removed, and the slides were mounted with anti-fading mounting medium. Samples were analyzed immediately under a fluorescence microscope, protecting the slides from light. Staining results from five tumors in each group were statistically analyzed.

[0070] The results showed that compared with the single drug treatment group, the number of Ki-67 positive cells in the tumor tissue of the GRB2i and PF-4708671 drug combination group was significantly reduced ( Figure 6 B in the figure), the tumor proliferation ability decreased. After combined treatment with GRB2i and PF-4708671, the number of TUNEL-positive cells in tumor tissues increased significantly, and the degree of tumor apoptosis was higher than that in the single-drug treatment group ( Figure 6 C). After the combination of drugs, the proliferation of tumor tissue was significantly inhibited and the apoptosis level was significantly increased.

[0071] Example 6: In vivo experimental study of PF-4708671 drug with low toxicity and side effects In addition, the toxic effects of GRB2i and PF-4708671 on the above mice were further investigated. The daily weight data curve of each group of mice showed that there was no significant difference in the weight of the mice in each group ( Figure 7 A in the figure indicates that the mice are in good condition after drug treatment.

[0072] The main organ tissues of the mice (heart, liver, spleen, lung and kidney) were taken for H&E staining. The results are as follows Figure 7 As shown in Figure B. H&E staining results after administration also showed that the cells of various organs were neatly arranged, just like those in the blank group, without inflammatory infiltration or other damage, indicating that GRB2i and PF-4708671, used alone or in combination, had no obvious toxicity to the various organs in mice.

[0073] Example 7: In vitro study of the anti-proliferative activity of GRB2i and PF-4708671 in combination in different cancer types The present application conducted drug combination experiments on human lung adenocarcinoma cells A549, human gastric adenocarcinoma cells AGS, human colon cancer cells HCT116, human liver cancer cells HepG2, human thyroid cancer cells HTh-7, and human esophageal squamous cell carcinoma cells KYSE150.

[0074] The target cells were digested into cell suspension and the cells were counted. 3×10 3 A549, AGS, HCT116, HepG2, HTh-7, and KYSE150 cells were seeded into 96-well plates at a volume of 100 μL per well. After 24 hours of culture, the medium was discarded. PF-4708671 concentrations of 25, 20, 15, 10, 5, and 0 μM were added to rows 2 through 7 of the 96-well plate, and GRB2i concentrations of 25, 12.5, 6.3, 3.1, 1.6, 0.8, and 0 μM were added to columns 2 through 8 of the 96-well plate. A control group containing only cells and a blank group containing no drug were also set up, with three replicates for each concentration. The cells were treated in a 37°C, 5% CO2 incubator for 48 hours. The medium in the 96-well plate was then discarded, and fresh medium containing 10% CCK-8 was added to each well and incubated with the cells for an additional 0.5–4 hours. After incubation, the absorbance of each well was measured at 450 nm using a microplate reader. The viability was calculated using the following equation: Viability = (OD experimental group - OD blank group) / (OD control group - OD blank group) × 100%. GraphPad Prism 9 software was used to process the experimental data and calculate cell viability.

[0075] like Figure 8As shown, after co-treatment with PF-4708671 and GRB2i, the survival rates of different cancer cells were significantly reduced. Through this experiment, the present applicant found that the combination of GRB2i and PF-4708671 has a better killing effect on tumor cells of different cancers.

[0076] In summary, in both cellular and animal studies, this application found that the combination of a GRB2 inhibitor and a P70S6K inhibitor can effectively inhibit tumor proliferation with a good safety profile. This application will provide insights and foundations for the diagnosis and treatment of triple-negative breast cancer, with certain theoretical and clinical significance.

Claims

1. Pharmaceutical use of a compound combination, characterized in that: The compound combination includes a GRB2 inhibitor and a P70S6K inhibitor, and its pharmaceutical use is in the preparation of anti-tumor drugs.

2. The pharmaceutical use of the compound combination according to claim 1, characterized in that The structural formula of the GRB2 inhibitor is shown in Formula 1: Formula 1.

3. The pharmaceutical use of the compound combination according to claim 1, characterized in that: The P70S6K inhibitor is a small molecule compound PF-4708671.

4. The pharmaceutical use of the compound combination according to claim 1, characterized in that: The tumor type was breast cancer, lung cancer, stomach cancer, colon cancer, liver cancer, thyroid cancer, esophageal cancer, or leukemia.

5. The pharmaceutical use of the compound combination according to claim 4, characterized in that: Breast cancer is triple-negative breast cancer, lung cancer is lung adenocarcinoma, gastric cancer is gastric adenocarcinoma, esophageal cancer is esophageal squamous cell carcinoma, and leukemia is chronic myeloid leukemia.

6. An anti-tumor drug combination in combination with a GRB2 inhibitor, characterized in that: Contains a GRB2 inhibitor and a P70S6K inhibitor.

7. The anti-tumor drug combination according to claim 6, characterized in that: The structural formula of the GRB2 inhibitor is shown in Formula 1: Formula 1.

8. The anti-tumor drug combination according to claim 6, characterized in that: The P70S6K inhibitor is a small molecule compound PF-4708671.

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