Pharmaceutical use of a combination of an antitumor drug and a grb2 inhibitor

Abstract

The application discloses a pharmaceutical use of an anti-tumor drug combination and a compound combination combined with a GRB2 inhibitor. The application takes triple-negative breast cancer as a research object, and detects the effect of a GRB2 inhibitor (GRB2i) and a P70S6K inhibitor (PF-4708671) on triple-negative breast cancer through a plate colony formation experiment, flow cytometry and construction of tumor-bearing mice. It is firstly proposed and proved that the combination of the GRB2 inhibitor (GRB2i) and the P70S6K inhibitor (PF-4708671) can significantly inhibit the proliferation of triple-negative breast cancer. The application provides a thought and a basis for diagnosis and treatment of triple-negative breast cancer, and has certain theoretical and clinical significance. The application finds that the combination of the GRB2 inhibitor and the P70S6K inhibitor has a better killing effect on other various cancer cells, which indicates that the promotion of the effect is broad-spectrum.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to the pharmaceutical use of an anti-tumor drug combination and a compound combination combined with a GRB2 inhibitor. BACKGROUND

[0002] Triple-negative breast cancer (TNBC) is a breast cancer with negative expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), which shows four characteristics of high invasiveness, high metastasis, easy recurrence and poor prognosis. Surgery combined with radiotherapy and chemotherapy is the main means for the treatment of triple-negative breast cancer in clinic. With the development of research technology, immunotherapy, endocrine therapy and targeted therapy have become new means for the treatment of TNBC. However, patients show different degrees of drug resistance to clinical treatment programs. Therefore, finding new targeted drugs to solve the problem of drug resistance in the treatment of TNBC is the focus of clinical research.

[0003] Growth factor receptor binding protein 2 (GRB2) is a linker protein that plays a key role in cell signal transduction. It is composed of a central Src homology domain 2 (SH2) and two Src homology domains 3 (SH3), namely N-terminal SH3 (n-SH3) and C-terminal SH3 (c-SH3). The SH2 domain can recognize and bind to phosphorylated tyrosine residues (pYxNx motif), mediating the interaction of GRB2 with various receptor tyrosine kinases (RTKs) such as EGFR, FGFR, c-Met, etc.; while the SH3 domain participates in the binding of GRB2 to downstream effector proteins such as SOS, Gab2, etc. by recognizing the proline-rich PxxP motif. GRB2 exists in two forms in cells, monomers and dimers. The monomer form is the active state for its function, while the dimer is the self-inhibitory state. When stimulated by growth factors, GRB2 is phosphorylated, prompting the dissociation of dimers into monomers, thereby activating the MAPK and PI3K / AKT signaling pathways, regulating cell proliferation, differentiation, migration and survival, etc. Studies have shown that the increase in the level of GRB2 phosphorylation in malignant tumor tissues suggests that the increase in its monomer form may be closely related to the occurrence and development of tumors. Given the pivotal role of GRB2 in multiple oncogenic signaling pathways, it 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, which plays a core role in the mTOR signaling pathway. It mainly regulates protein synthesis by phosphorylating 40S ribosomal protein S6, and then promotes cell growth, proliferation and metabolism. The activation of p70S6K depends on the upstream kinase mTORC1. Under the stimulation of growth factors, nutrients and other factors, mTORC1 phosphorylates the Thr389 site of p70S6K, which promotes its activation, thereby affecting the process of translation initiation and elongation. In addition, p70S6K is also involved in the regulation of cell cycle, autophagy and energy metabolism and other physiological activities. Studies have shown that p70S6K is abnormally activated in many tumors, such as prostate cancer, gastric cancer and breast cancer. Its high expression is closely related to the occurrence, progression and drug resistance of tumors, suggesting that it can be used as a potential anti-tumor target. At present, a number of small molecule inhibitors have been developed for targeting p70S6K. Among them, PF-4708671, as a selective inhibitor, has shown good anti-tumor activity in vitro and animal models. However, the efficacy of single targeting p70S6K is still limited, and it is urgent to explore the combination therapy strategy with other target drugs. SUMMARY

[0005] In view of the above technical problems existing in the prior art, the present application provides the pharmaceutical use of the anti-tumor drug combination and the compound combination combined with the GRB2 inhibitor. The present application proves by in vivo and in vitro experiments that the drug combination of the GRB2 inhibitor (GRB2i) and the P70S6K inhibitor (PF-4708671) can significantly inhibit 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 the GRB2 inhibitor and the P70S6K inhibitor serves as a new potential clinical treatment strategy, and provides a new idea for the treatment of triple-negative breast cancer.

[0006] The present application first provides the pharmaceutical use of the compound combination, which comprises the GRB2 inhibitor and the P70S6K inhibitor, and the pharmaceutical use is the use in the preparation of the anti-tumor drug.

[0007] Preferably, the structure of the GRB2 inhibitor is shown as formula 1:

[0008] Formula 1.

[0009] Preferably, the P70S6K inhibitor is the small molecule compound PF-4708671.

[0010] 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 myelogenous leukemia.

[0011] The present application also provides an anti-tumor drug combination combined with a GRB2 inhibitor, comprising a GRB2 inhibitor and a P70S6K inhibitor.

[0012] Preferably, the structure of the GRB2 inhibitor is shown in Formula 1:

[0013] Formula 1.

[0014] Preferably, the P70S6K inhibitor is a small molecule compound PF-4708671.

[0015] Compared with the prior art, the present application has the following beneficial effects.

[0016] The present application takes triple negative breast cancer as the research object, and detects the effect of a GRB2 inhibitor (GRB2i) and a P70S6K inhibitor (PF-4708671) on triple negative breast cancer through a plate colony formation experiment, flow cytometry and construction of tumor-bearing mice. It is first proposed and confirmed that the combination of a GRB2 inhibitor (GRB2i) and a P70S6K inhibitor (PF-4708671) can significantly inhibit the proliferation of triple negative breast cancer. The present application provides ideas and basis for the diagnosis and treatment of triple negative breast cancer, and has certain theoretical and clinical significance.

[0017] The present application researches and finds that the combination of a GRB2 inhibitor and a P70S6K inhibitor has better killing effect on other cancer cells, indicating that the improvement of the effect is broad-spectrum. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 PF-4708671 (denoted as P70S6Ki, the same below) and GRB2i are used in combination to affect cell activity. Among them, Figure 1 A-C in FIG. 1 are respectively the effects of PF-4708671 and GRB2i used in combination on HAP1, 4T1 and MDA-MB231 cell activity detected by the checkerboard method; Figure 1 D-E in FIG. 2 are respectively the effects of 20 μM PF-4708671 on the IC50 value of the GRB2 inhibitor in HAP1 and 4T1 cells; Figure 1 F in FIG. 3 is the effect of 30 μM PF-4708671 and 20 μM GRB2i used in combination on MDA-MB-231 cell activity; Figure 1G is the effect of combined use of PF-4708671 and GRB2i on the activity of MDA MB-453 cells after 48h co-treatment with 30μM PF-4708671 and 75μM GRB2i. **** represents p<0.0001, n=3.

[0019] Figure 2 A is the effect of combined use of PF-4708671 and GRB2i on cell proliferation. Wherein Figure 2 A is the effect of combined use of PF-4708671 and GRB2i on cell proliferation. Wherein Figure 2 B-D are the statistical columnar charts of HAP1, 4T1 and MDA-MB-231 cell colony formation, respectively. **** represents p<0.0001, *** represents p<0.001, ** represents p<0.01, * represents p<0.05, n=3.

[0020] Figure 3 A is the effect of combined use of PF-4708671 and GRB2i on cell proliferation. Wherein Figure 3 A is the effect of combined use of PF-4708671 and GRB2i on cell proliferation. Wherein Figure 3 B, C are the statistical columnar charts of HAP1 and 4T1 cell apoptosis percentage, respectively, and the Annexin V positive cell population is counted. **** represents p<0.0001, n=3.

[0021] Figure 4 A is the effect of combined use of PF-4708671 and GRB2i on cell proliferation. Wherein Figure 4 A is the effect of combined use of PF-4708671 and GRB2i on cell proliferation. Wherein Figure 4 A is the effect of combined use of PF-4708671 and GRB2i on cell proliferation. Wherein

[0022] Figure 5 A is the effect of combined use of PF-4708671 and GRB2i on cell proliferation. Wherein Figure 5 A is the effect of combined use of PF-4708671 and GRB2i on cell proliferation. Wherein Figure 5 A is the effect of combined use of PF-4708671 and GRB2i on cell proliferation. Wherein Figure 5 A is the effect of combined use of PF-4708671 and GRB2i on cell proliferation. Wherein Figure 5D in FIG. 6 is the first and last day of mice live imaging, two mice were selected from each group; Figure 5 E in FIG. 6 is the graph of the area of light emission of mice live imaging experiment. **** represents p<0.0001, *** represents p<0.001, ** represents p<0.01, * represents p<0.05, ns represents p>0.05, n=5.

[0023] Figure 6 FIG. 7 is the immunohistochemical staining of tumor tissues after PF-4708671 and GRB2i treatment. Among them, Figure 6 A in FIG. 7 is the immunohistochemical staining graph of H&E, Ki-67, TUNEL and Merge of tumor tissues, scale bar: 10 µm; Figure 6 B in FIG. 7 is the statistical graph of Ki-67 positive cells in tumor tissues; Figure 6 C in FIG. 7 is the statistical graph of TUNEL positive cells in tumor tissues. **** represents p<0.0001, ** represents p<0.01, * represents p<0.05, n=5.

[0024] Figure 7 FIG. 8 is the in vivo toxicity experiment after PF-4708671 and GRB2i treatment. Among them, Figure 7 A in FIG. 8 is the graph of the change of body weight of mice during GRB2i and PF-4708671 treatment; Figure 7 B in FIG. 8 is the H&E staining of the main organ tissues (heart, liver, spleen, lung and kidney) of each group of mice, scale bar: 10 µm.

[0025] Figure 8 FIG. 9 is the effect of the combination of PF-4708671 and GRB2i on the activity of tumor cells of different cancers. Among them, Figure 8 A-F in FIG. 9 are the effects of the combination of PF-4708671 and GRB2i on the activity of human gastric adenocarcinoma cells AGS, human lung adenocarcinoma cells A549, human colon cancer cells HCT116, human hepatocarcinoma cells HepG2, human thyroid carcinoma cells HTh-7, and human esophageal squamous cell carcinoma cells KYSE150, respectively. **** represents p<0.0001, *** represents p<0.001, ** represents p<0.01, n=3. DETAILED DESCRIPTION

[0026] The GRB2 inhibitor used in this application is denoted as GRB2i, with the patent number: US2023 / 0366033A1, and the structure is shown as formula 1:

[0027] Formula 1.

[0028] The P70S6K inhibitor used in the application is PF-4708671, recorded as P70S6Ki, produced by MCE, with the article number HY-15773, and the structure is shown as formula 2:

[0029] Formula 2.

[0030] Example 1: CCK-8 experiment for detecting cell activity

[0031] (1) In vitro experiment of GRB2i and PF-4708671 combination for anti-proliferative activity.

[0032] In order to explore the killing effect of GRB2i and PF-4708671 combination on cells, first, the killing effect of different concentrations of GRB2i and PF-4708671 alone or in combination on HAP1 cells (human chronic myeloid leukemia cells) after 48h treatment was detected by drug combination chessboard method, and the cell survival rate was detected by CCK-8 method.

[0033] After the target cells were digested into cell suspension, the cells were counted, and 3×10 3 4T1 cells and 2×10 3 4T1 cells and 2×10 3 MDA-MB-231 cells were inoculated into 96-well plates at a volume of 100 μL / well. After HAP1 and 4T1 cells were cultured for 24h, PF-4708671 concentrations of 25, 20, 15, 10, 5 and 0 μM were added to rows 2-7 of the 96-well plate, respectively, and GRB2i concentrations of 25, 12.5, 6.3, 3.1, 1.6, 0.8 and 0 μM were added to columns 2-8 of the 96-well plate, respectively. After MDA-MB-231 cells were cultured for 24h, PF-4708671 concentrations of 40, 30 and 0 μM were added to rows 2-4 of the 96-well plate, respectively, and GRB2i concentrations of 80, 50, 30 and 0 μM were added to columns 2-5 of the 96-well plate, respectively. At the same time, a control group without drug and a blank group without cells were set.

[0034] The results are shown in A of Figure 1 The cell survival rate of HAP1 after the combination of the two drugs was significantly lower than that of GRB2i and PF-4708671 alone.

[0035] The same results were obtained in triple-negative breast cells, Figure 1 The results in B show that the killing effect of the combination of the two drugs on 4T1 cells (mouse breast cancer cells) is stronger than that of using one of them alone.

[0036] Figure 1The results of the C-checkerboard assay showed that the cell survival rate of MDA-MB-231 cells (a human breast cancer cell line, a typical research model of triple-negative breast cancer) treated with GRB2i and PF4708671 was lower than that treated with either drug alone. The combined treatment with GRB2i and PF-4708671 had a synthetic lethal effect on triple-negative breast cancer cells.

[0037] (2) After confirming the effect of the combination of GRB2i and P70S6Ki by the checkerboard method experiment, this application further determined the effect of the combined action of PF-4708671 on the GRB2 inhibitor IC50 by the CCK-8 experiment.

[0038] After digesting the target cells into a cell suspension, cell counting was performed, with 3×10⁶ cells being counted. 3 HAP1 cells per well, 2 × 10 3 Four T1 cells per well were seeded at a volume of 100 μL per well into 96-well plates. After 24 h of culture, the culture medium was discarded. PF-4708671 concentrations of 25, 20, 15, 10, 5, and 0 μM were added sequentially to rows 2–7 of the 96-well plates, and GRB2i concentrations of 25, 12.5, 6.3, 3.1, 1.6, 0.8, and 0 μM were added sequentially to columns 2–8 of the 96-well plates. A control group (no drugs, only cells) and a blank group (no drugs, no cells) were also set up, with three replicates for each concentration. The plates were incubated at 37°C with 5% CO2 for 48 h. The culture medium in the 96-well plates was then discarded, and fresh medium containing 10% CCK-8 was added to each well, followed by incubation with the cells for another 0.5–4 h.

[0039] After incubation, the absorbance of each well at 450 nm was measured using a microplate reader. Cell 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, or cell survival curves were plotted and IC50 values ​​were calculated.

[0040] Figure 1 As shown in Figure D, compared to GRB2i alone (IC50 = 56.50 μM), the IC50 value of HAP1 cells treated with 20 μM PF4708671 was significantly reduced to 37.07 μM for GRB2i (IC50 = 37.07 μM). For 4T1 cells (… Figure 1Compared to GRB2i alone (IC50 = 36.34 μM), the IC50 of 4T1 cells treated with 20 μM PF-4708671 was also significantly reduced (IC50 = 16.99 μM). Combined drug treatment resulted in a decrease in the IC50 of cells against GRB2i.

[0041] (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).

[0042] like Figure 1 As shown in Figure F, after co-treatment with 30 μM PF-4708671 and 20 μM GRB2i for 48 h, the survival rate of MDA-MB-231 cells was significantly reduced. The results for MDA-MB-453 cells are shown in Figure F. Figure 1 As shown in G, after 48 hours of co-treatment with 30 μM PF-4708671 and 75 μM GRB2i, near-complete inhibition was achieved. Through CCK-8 assays, this application found that the combined use of GRB2i and PF-4708671 resulted in better tumor cell killing effects.

[0043] Example 2: Colony formation assay to detect cell proliferation capacity

[0044] 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 colony formation assay. Figure 2 (A in the middle).

[0045] Cells in good condition and in logarithmic growth phase were inoculated into 6-well plates (500 cells per well), and cultured in a 37°C, 5% CO2 incubator. After 2-3 days of observation under a microscope, HAP1 cells were treated with 5 μM GRB2i, 7 μM PF-4708671, and a combination of the two, 4T1 cells were treated with 7 μM GRB2i, 7 μM PF-4708671, and a combination of the two, and MDA-MB-231 cells were treated with 3 μM GRB2i, 5 μM PF-4708671, and a combination of the two, for 14 days, with 3 replicates in each group. During drug treatment, the growth state and proliferation of the cells were observed, and the culture was stopped when obvious cell clusters were observed in the 6-well plates. The IMDM medium was discarded, the cells were washed once with PBS buffer, and general tissue fixative (4% paraformaldehyde) was added for 15 min. The cells were washed once again with PBS buffer, then stained with 0.1% crystal violet solution (2.5% crystal violet + PBS buffer) for 1 h to obtain the stained 6-well plate of colony formation. The 6-well plate was scanned with a scanner to obtain the colony formation picture, which was processed with Image. The number of cell colonies was normalized relative to the WT cell group without drug treatment.

[0046] The results of colony formation showed that when 5 μM GRB2i and 7 μM PF-4708671 were used in combination, the number of cell colonies was significantly less than that when GRB2i and PF-4708671 were used alone, and there was a significant difference (P < 0.05) between the two groups (A in FIG. 6). Figure 2

[0047] The results of 4T1 cell colony formation showed that when 7 μM GRB2i and 7 μM PF-4708671 were used in combination, the number of cell colonies decreased. The inhibition rate of colony formation by drug combination was greater than that by single drug, and there was a significant difference (P < 0.05) between the two groups (C in FIG. 6). Figure 2

[0048] Under the combined action of 3 μM GRB2i and 5 μM PF-4708671, the number of MDA-MB-231 cell colonies decreased, and there was a significant difference (P < 0.05) between the two groups (D in FIG. 6). Figure 2

[0049] Therefore, the combined use of GRB2i and PF-4708671 resulted in a significant decrease in the number of cell colonies and a significant inhibition of cell proliferation.

[0050] Example 3: In vitro experimental study of the pro-apoptotic activity of GRB2i and PF-4708671 in combination

[0051] ​​​The effect of GRB2i combined with P70S6Ki on cell apoptosis was explored by Annexin V / PI double staining method flow experiment.

[0052] The HAP1 and 4T1 cells in good condition and in logarithmic growth phase were inoculated into 6-well plates (1x10 5 cells per well), and were placed in a 37°C, 5% CO2 incubator for culture. After 24h, cell adhesion was observed, and 30μM GRB2i, 20μM PF-4708671 and their combination were used to treat HAP1 cells, and 30μM GRB2i, 10μM PF-4708671 and their combination were used to treat 4T1 cells for 48h, with 3 replicates in each group.

[0053] After drug treatment, the cell culture solution was aspirated into a sterile 15mL centrifuge tube, and the HAP1 cells were washed once with PBS. 1mL 0.25% trypsin (without EDTA) was added to each well to digest the cells. After digestion for an appropriate time, the previously collected cell culture medium was added, and the cells were gently blown down and transferred to a centrifuge tube. Centrifugation was performed at 1100g for 5min. After centrifugation, the supernatant was discarded, and the cells were gently washed once with PBS. Centrifugation was performed at 1100g for 5min, and the supernatant was discarded. Then the cells were gently resuspended with PBS and counted, and 5x10 5 The resuspended cells were centrifuged at 1000g for 5min, and the supernatant was discarded. 200μL Annexin V-FITC binding solution was added to resuspend the cells. 5μL Annexin V-FITC was added and gently mixed. 10μL propidium iodide staining solution was added and gently mixed. After incubation at room temperature for 15min in the dark, the cells were then placed on ice and protected from light using aluminum foil.

[0054] Since the emission spectra of each fluorophore overlap with each other, there is a risk that one light will leak into another channel. Therefore, when using the Attune NxT flow cytometer to measure cell apoptosis, a single-stained sample needs to be set up to adjust the compensation leakage of double fluorescence.

[0055] Before loading, filter with a 300-mesh nylon net. Use the Attune NxT flow cytometer to detect cell apoptosis. First, calculate the compensation matrix using a single-stained sample, and then sequentially load and detect cell apoptosis. After sample detection, use FlowJo software to analyze flow cytometry data. The experimental results were repeated 3 times. The percentage of Annexin V-positive cell population was calculated for cell apoptosis.

[0056] The flow cytometry graphs of HAP1 and 4T1 cell apoptosis under the combined action of GRB2i and P70S6Ki are shown in Figures 1 and 2, respectively. Figure 3A). The number of apoptosis of HAP1 cells under the combined action of 30 μM GRB2i and 20 μM PF-4708671 for 48 h was significantly increased (71.8%), higher than that under the action of GRB2i (21.3%) and PF-4708671 (13.22%) alone, and there was a statistical difference (P < 0.05) Figure 3 B). The apoptosis of 4T1 cells under the combined action of 30 μM GRB2i and 10 μM PF-4708671 for 48 h was also significantly increased (64.73%), higher than that under the action of GRB2i (11.52%) and PF-4708671 (23.29%) alone, and there was a statistical difference (P < 0.05) Figure 3 C). The combined action of GRB2i and PF-4708671 led to a significant increase in the level of apoptosis.

[0057] Example 4: GRB2i and PF-4708671 combination inhibit autophagy

[0058] In vitro experiments found that the combination of GRB2i and P70S6Ki can inhibit the proliferation of tumor cells, promote their apoptosis, and has a significant anti-tumor effect. In the research of traditional tumor treatment drugs, only one target or signal pathway is usually focused on, while in fact, single targeting often leads to drug resistance. The current cancer tumor method often uses a multi-target combination therapy. The present application finds a new inhibitor PF-4708671 for treating TNBC in combination with GRB2i.

[0059] To this end, the molecular mechanism of the combination of the two to achieve synthetic lethality was further verified, and the effect of drug combination on autophagy key protein LC3B-II was verified in HAP1 and KO cells.

[0060] KO cells (Knockout cell) refers to GRB2 knockout cell lines constructed in HAP1 cells by using CRISPR-Cas9 technology. Such cells lose the expression or function of the target gene and are often used to study the biological role and function of the gene.

[0061] First, the sgRNA fragment of the knockout plasmid was designed according to the CRISPR-Cas9 system, and the corresponding target was as follows:

[0062] 5'-AGATGGAGCCGGGAAGTACT-3'.

[0063] The target fragment sgRNA was constructed into the CRISPR-Cas9 system, thereby constructing the CRISPR-Cas9 plasmid for GRB2 knockout.

[0064] First, 293T cells were transfected with 4 x 10 5Cells were seeded in 10 cm dishes at a density of 1 cell per cm2. The next day, after the cells adhered, the confluence was about 50%. After 2 hours of starvation in serum-free DMEM medium, the CRISPR-Cas9 plasmid for GRB2 knockout, pMD2.G and psPAX2 were used to transfect 293T cells at a ratio of 5 μg:2.5 μg:2.5 μg using jetPRIME (Polyplus, French) according to the manufacturer's protocol. After 8 hours, the complete medium was replaced, and 48 hours later, the virus supernatant was collected. Another 24 hours later, the second batch of virus supernatant was collected. The collected virus supernatant was the lentivirus containing the GRB2 gene knockout.

[0065] First, HAP1 cells were seeded in 6-well plates at a density of 3 x 10 5 cells per well. The next day, after the cells adhered, the lentivirus and complete medium were mixed at a ratio of 1:1 and added to the HAP1 cells for 8 hours of infection. After the HAP1 cells were in good condition, they were selected with puromycin. Only the control cells died completely, and the remaining cells were seeded in 96-well plates at a density of 1 cell per well after enzymatic digestion and expanded into cell lines. The protein in each well of the cell line was extracted and quantified by the BCA method, and the WB was used to detect the knockout of GRB2 in HAP1 cell lines.

[0066] When HAP1 and KO cells were treated with 50 μM CQ (chloroquine, an autophagy inhibitor) for 3 h, the autolysosome was blocked before fusion and degradation. The expression level of LC3B-II in cells treated with 50 μM PF-4708671 was down-regulated more obviously (Fig. 1A), indicating that when GRB2 is absent, P70S6Ki treatment can more significantly inhibit autophagy in cells, leading to the inability of cells to remove damaged proteins and organelles, and inducing an increase in cell death. Figure 4

[0067] In addition, the present application also verified the changes in the autophagy key protein LC3B-II in 4T1 cells under the combined action of GRB2i and PF-4708671, which was consistent with the results in the gene knockout cells. The expression of LC3B-II in 4T1 cells decreased obviously under the combined action of 50 μM GRB2i and 50 μM PF-4708671 for 24 h (Fig. 1B). Figure 4

[0068] Therefore, it is reasonably speculated that the combination of the two drugs can block the autophagy signaling pathway, which can induce 4T1 cell death.

[0069] Example 5: In vivo experimental study of the anti-tumor activity of the combination of GRB2i and PF-4708671

[0070] The combination of GRB2i and PF-4708671 inhibits tumor growth. The combination of GRB2i and PF-4708671 inhibits tumor growth.​

[0071] A mouse xenograft model was established by subcutaneously inoculating Luciferase-expressing mouse 4T1 cells (4T1-Luc) into the hind legs of mice. The 4T1-Luc cells in good condition and in the logarithmic growth phase were digested and centrifuged, resuspended and washed once with PBS buffer (to remove serum), resuspended in PBS buffer to prepare a cell suspension, and stored on ice for use in tumor implantation. To ensure that the mice have enough time to adapt, the purchased mice need to adapt for about 1 week before modeling. First, the mice were fixed, and the hair on the left and right hind legs of the mice was removed using a hair clipper, and then the hair on the left and right hind legs of the mice was removed with depilatory cream to expose clean skin. The cell suspension was gently mixed to ensure the formation of a single cell suspension. The mouse skin was spread, and 100 μL (containing 1 x 10 5 cells) of the cell suspension was sucked into the injection needle and subcutaneously implanted into the left and right hind legs of the mice. The mice were observed for tumor formation 2-4 days after injection. GRB2i and PF-4708671 were continuously injected intraperitoneally at a dose of 25 mg / kg per day for 13 days, and after drug treatment, detection was performed.

[0072] It was found that the tumor size of the combination treatment group of GRB2i and PF-4708671 was significantly smaller than that of the other three groups, as shown in A of Figure 5 Compared with the single-drug treatment group, the tumor weight of the drug combination group was significantly reduced (B of Figure 5 ). According to the measured tumor growth volume curve, the combination of GRB2i and PF-4708671 had the most obvious inhibitory effect on 4T1 tumor growth (C of Figure 5 ).

[0073] In vivo imaging was performed on the mice by intraperitoneal injection of luciferin bioluminescent substrate on the first and last days of drug treatment to evaluate tumor development. In vivo imaging was performed on the first and thirteenth days of drug administration to observe tumor growth. A 15 mg / ml luciferin solution was prepared with PBS buffer, and the injection dose was 100 μL of luciferin solution per 10 g of mouse body weight. The injection was performed by intraperitoneal injection: the needle should be slightly inclined at an angle when it is inserted into the abdominal cavity, and the needle should be slightly inclined upward. The needle was slightly inserted into the abdominal wall. After 10 min of luciferin injection, isoflurane was used for gas anesthesia, and the mouse posture was adjusted to lie on its side for in vivo imaging.

[0074] The results are shown in D of Figure 5 Compared with single-drug treatment, the luminescence area of the combination treatment group was significantly reduced after 13 days of treatment, and there was a significant difference (E of Figure 5 , indicating that drug combination can significantly inhibit the growth of 4T1-Luc tumors.

[0075] Therefore, GRB2i and PF-4708671 combination has great therapeutic potential for mouse triple-negative breast cancer tumors, and is worthy of further study. This experiment follows the basic principles of animal ethics, through the 3R principle, strict ethical review and comprehensive consideration of the welfare of experimental animals, to ensure the scientificity of the experiment and the basic rights of animals are respected and protected.

[0076] GRB2i and PF-4708671 combination inhibited tumor proliferation and promoted tumor apoptosis. The H&E, Ki-67 and TUNEL indicators of tumor tissues were analyzed (Fig. 4A). Figure 6

[0077] The tumor tissues of the four groups were fixed in 4% formaldehyde and embedded in paraffin. Paraffin tumor tissue sections were dehydrated and deparaffinized, and the sections were soaked in xylene for 15 min and 10 min, respectively. Then dehydrated in 100% ethanol, 95% ethanol and 80% ethanol for 1 min each, and rinsed with PBS twice. Differentiated with 1% hydrochloric acid ethanol solution for 30 s, and then rinsed with PBS twice, and finally stained with eosin and hematoxylin, and sealed with neutral resin. Optical microscope was used to capture images.

[0078] Ki-67 staining of tumor tissue, tissue embedding, sectioning and deparaffinization steps are the same as H&E staining experiment operation. Then antigen repair work was carried out, the tumor tissue sections were placed in citric acid antigen repair buffer with pH 6.0, in the microwave oven for 8 min to boiling, stop fire 8 min, and then reheat and turn to low fire for 6 min. After natural cooling, washed with PBS for 2 times. Then endogenous peroxidase blocking was carried out, using 3% H2O2 solution for 20 min, and PBS for 3 times. Then serum blocking was carried out, using 3% BSA for 30 min. After removing the blocking solution, add the primary antibody Ki-67 to cover the tissue, then put it in the wet box at 4℃ overnight in the dark. After incubation with secondary antibody for 20 min, PBS was washed for 2 times. DAB staining for 10 min. Finally, dehydrate and seal. The staining results of 5 tumors in each group were selected for statistical analysis.

[0079] ​Tumor tissues were stained with TUNEL. The embedding, sectioning, and dewaxing procedures were the same as for H&E staining, while the antigen retrieval and blocking procedures were the same as for Ki-67 immunohistochemistry. Afterward, 50 μL of Equilibration Buffer was added to each tumor sample to completely cover the sample area, and incubated at room temperature for 10 min. After removing as much equilibration buffer as possible, 56 μL of TdT incubation buffer was added to each tissue sample, and incubated at 37°C for 1 h. The tissue samples were then rinsed with PBS four times, 5 min each time. In the dark, slides were immersed in a staining jar containing DAPI solution (freshly prepared and diluted with PBS) and incubated at room temperature for 8 min. After staining, the tissue samples were washed three times with PBS for 5 min each time, then excess liquid was gently removed, and anti-fluorescence quenching mounting medium was added for mounting. The samples were analyzed immediately under a fluorescence microscope after the experiment, with the slides protected from light. The staining results of five tumors from each group were statistically analyzed.

[0080] The results showed that, compared with the single-drug treatment group, the number of Ki-67 positive cells in the tumor tissue of the combination group of GRB2i and PF-4708671 was significantly reduced. Figure 6 In the B group, tumor proliferation ability decreased. Combined treatment with GRB2i and PF-4708671 significantly increased the number of TUNEL-positive cells in tumor tissue, and the degree of tumor apoptosis was higher than in the single-drug treatment group. Figure 6 (C) The combination of drugs significantly inhibited tumor proliferation and significantly increased apoptosis levels.

[0081] Example 6: In vivo experimental study of the low toxicity and side effects of PF-4708671 drug

[0082] Furthermore, the toxic effects of GRB2i and PF-4708671 on the aforementioned mice were further investigated. The daily body weight data curves for each group of mice showed no significant differences in body weight among the groups. Figure 7 The A in the figure indicates that the mice were in good condition after receiving drug treatment.

[0083] H&E staining was performed on the major organ tissues (heart, liver, spleen, lung, and kidney) of mice. The results are as follows. Figure 7 As shown in B in the figure. The H&E staining results after drug administration also showed that the cells of each organ were arranged neatly, just like the blank group, without inflammatory infiltration or other damage, indicating that GRB2i and PF-4708671, whether used alone or in combination, had no obvious toxicity to the organs of mice.

[0084] Example 7: In vitro experimental study on the anti-proliferative activity of GRB2i and PF-4708671 in combination in different cancer types

[0085] The application carried out drug combination experiments on human lung adenocarcinoma cells A549, human gastric adenocarcinoma cells AGS, human colon cancer cells HCT116, human hepatocarcinoma cells HepG2, human thyroid carcinoma cells HTh-7, and human esophageal squamous cell carcinoma cells KYSE150.

[0086] After the target cells were digested into a cell suspension, the cells were counted, and 3x10 3 A549, AGS, HCT116, HepG2, HTh-7, and KYSE150 cells were seeded into a 96-well plate at a volume of 100 μL per well. After 24 h of culture, the culture medium was discarded. PF-4708671 was configured at concentrations of 25, 20, 15, 10, 5, and 0 μM and added to rows 2-7 of the 96-well plate, respectively. GRB2i was configured at concentrations of 25, 12.5, 6.3, 3.1, 1.6, 0.8, and 0 μM and added to columns 2-8 of the 96-well plate, respectively. At the same time, a control group without drug addition and a blank group without cell addition were set up, and each concentration had three replicate wells. The cells were treated in a 37°C, 5% CO2 incubator for 48 h. Then the culture medium in the 96-well plate was discarded, fresh culture medium containing 10% CCK-8 was added to each well, and the cells were incubated for another 0.5-4 h. After incubation, the absorbance of each well at 450 nm was measured using a microplate reader. The survival rate was calculated using the following equation: survival rate = (OD experimental group-OD blank group) / (OD control group-OD blank group) x 100%. The experimental data were processed and the cell survival rate was calculated using GraphPad Prism 9 software.

[0087] As shown in Figure 8 After co-treatment with PF-4708671 and GRB2i, the survival rates of different cancer cells were significantly reduced. Through this experiment, the application found that the combination of GRB2i and PF-4708671 had better killing effect on tumor cells of different cancers.

[0088] In summary, at the cellular level and in animal experiments, the application found that the combination of GRB2 inhibitor and P70S6K inhibitor can effectively inhibit tumor proliferation and has good safety. The application will provide ideas and basis for the diagnosis and treatment of triple-negative breast cancer, and has certain theoretical and clinical significance.

Claims

1. Pharmaceutical use of a combination of compounds, characterized in that, The compound combination comprises a GRB2 inhibitor and a P70S6K inhibitor, and the pharmaceutical use is for preparing an anti-tumor drug; The structural formula of the GRB2 inhibitor is shown in Formula 1: Formula 1; The P70S6K inhibitor is a small molecule compound PF-4708671; The tumor type is breast cancer; The concentration of the GRB2 inhibitor and the P70S6K inhibitor is 25 mg / kg of body weight.

2. The use of the combination of the compound according to claim 1, characterized in that, The breast cancer is triple negative breast cancer.

3. An antitumor pharmaceutical composition in combination with a GRB2 inhibitor, characterized by, The compound combination comprises a GRB2 inhibitor and a P70S6K inhibitor; The structural formula of the GRB2 inhibitor is shown in Formula 1: Formula 1; The P70S6K inhibitor is a small molecule compound PF-4708671; The tumor type is breast cancer; The concentration of the GRB2 inhibitor and the P70S6K inhibitor is 25 mg / kg of body weight.

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