Application of small-molecule inhibitor combined medicine in preparation of medicine for treating multiple myeloma

Abstract

The invention discloses an application of a small-molecule inhibitor combined medicine in preparation of a medicine for treating multiple myeloma. The application comprises the step of combining a proteasome inhibitor or an immunomodulator with an STAT3 small-molecule inhibitor. According to the present invention, the killing effect of the STAT3 targeting small molecule inhibitor BBI608 (Napabucasin) on MM cells and MM stem cells is provided, and the STAT3 targeting small molecule inhibitor BBI608 (Napabucasin) is combined with bortezomib (BTZ) or lenalidomide (Len) to improve the synergistic killing effect on multiple myeloma (MM) cells;

Description

Technical Field

[0001] The present invention belongs to the technical field of gene therapy biomedicine, and specifically relates to the application of a small molecule inhibitor combined with a drug in the preparation of a drug for treating multiple myeloma. Background Art

[0002] Multiple myeloma (MM) is a common malignant tumor of the blood system, characterized by the accumulation of clonal malignant plasma cells in the myeloma, leading to clinical manifestations such as hypercalcemia, anemia, renal impairment, and bone destruction. In recent years, the emergence of proteasome inhibitors, CAR-T cell therapy and other methods have greatly improved the survival of patients, but multiple myeloma is still incurable.

[0003] STAT3 is a gene located on chromosome 17. The protein encoded by this gene is a member of the STAT protein family.

[0004] It is very necessary and of great practical significance to further explore the pathogenesis of multiple myeloma (MM) and find methods to overcome MM. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of embodiments of the present invention and to briefly introduce some preferred embodiments.

[0006] As one aspect of the present invention, the present invention provides an application of a small molecule inhibitor combined with a drug in the preparation of a drug for treating multiple myeloma, wherein a proteasome inhibitor or an immunomodulator is used in combination with a small molecule inhibitor targeting STAT3.

[0007] As a preferred embodiment of the use of the present invention: the proteasome inhibitor includes bortezomib.

[0008] As a preferred embodiment of the application described in the present invention: the immunomodulator includes lenalidomide.

[0009] As a preferred embodiment of the application of the present invention: the small molecule inhibitor targeting STAT3 includes the STAT3 small molecule inhibitor BBI608.

[0010] As a preferred embodiment of the application of the present invention: the molar ratio of lenalidomide to the small molecule inhibitor targeting STAT3 is (10-20): (50-100).

[0011] As a preferred embodiment of the application of the present invention: the molar ratio of bortezomib to the small molecule inhibitor targeting STAT3 is (0.125-0.25): (50-100).

[0012] The small molecule inhibitor combined drug is also used to prepare a drug for inhibiting the proliferation of multiple myeloma cells. The small molecule inhibitor combined drug is also used to prepare a drug for promoting the apoptosis of multiple myeloma cells.

[0013] Beneficial effects of the present invention: The small molecule inhibitor BBI608 (Napabucasin) targeting STAT3 of the present invention has a killing effect on MM cells and MM stem cells, and it is combined with bortezomib (BTZ) or lenalidomide (Len) to enhance the synergistic killing effect on multiple myeloma (MM) cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the following briefly introduces the drawings required for describing the embodiment, wherein:

[0015] Figure 1 MM cells were treated with different concentrations of BBI608.

[0016] Figure 2 The effect of different concentrations of BBI608 on cell cycle distribution in MM cells.

[0017] Figure 3 The effect of BBI608 on the osteoclast formation ability induced in vitro.

[0018] Figure 4 Flow cytometry was used to detect the effect of BBI608 on apoptosis of MM cell lines.

[0019] Figure 5 Western blot was used to detect the changes in apoptosis-related proteins in MM cells after treatment with BBI608.

[0020] Figure 6 Flow cytometry was used to analyze the effect of BB1608 on MM stem cells.

[0021] Figure 7 This is the effect of BBI608 on primary MM stem cells and normal umbilical cord blood stem cells.

[0022] Figure 8 Analyze cell death rate by flow cytometry. DETAILED DESCRIPTION

[0023] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0024] Experimental Materials and Methods

[0025] 1. CCK8 method cell viability assay: MM cells in the logarithmic growth phase were taken at 2×10 4 Each well was inoculated with BBI608 at different drug concentrations and cultured in an incubator for 24 h, 48 h, and 72 h. 10 ul CCK-8 solution was added to each well 3 h in advance. After incubation for another 3 h, the OD value at 450 nm was detected with an enzyme reader.

[0026] 2. Cell cycle detection: MM cells in the logarithmic growth phase were inoculated in a 6-well plate at a density of 1×100,000 wells, BBI608 was added at final concentrations of 0, 400, 800, and 1600 nM, EdU was used for incubation for 2 h, the cells were collected, the staining was eluted according to the EdU reagent method, and the flow cytometer was used for detection and analysis of cell cycle distribution.

[0027] 3. Osteoclast formation ability test: 20ml of peripheral blood from normal volunteers was used to separate mononuclear cells with Ficoll and cultured with a-MEM medium containing 10% fetal bovine serum. After 72h, the supernatant was discarded, the cells were collected and inoculated into 24-well plates with trypsin digestion, M-CSF (25ng / ml) and RANKL (50ng / ml) were added, and the cells were cultured in an incubator. After 48h, control DMSO or different concentrations of BBI608 were added to different groups, and then the medium was completely replaced every other day and cytokines and drugs were added. After 14 days, tartrate-resistant acid phosphatase staining was performed, and the cells were observed and photographed under a microscope.

[0028] 4. Cell apoptosis detection: 5×10 MM cells in logarithmic growth phase were taken 5 The cells were inoculated at a density of 100 wells in a 12-well plate, and BBI608 was added at a final concentration of 0, 400, 800, and 1600 nM. The cells were cultured in an incubator for 24 h. Annexin V FITC staining was used, and PI staining was added 5 min before loading. The proportion of apoptotic cells was detected and analyzed by flow cytometry.

[0029] 5. Analysis of the activity of side population cells and myeloma stem cells: (1) Side population cells: MM cells in the logarithmic growth phase were taken and 1×10 6100,000 cells were seeded in a 6-well plate, and different concentrations of BBI608 were added for incubation for 24 hours. Hochest33342 (5 ng / ml) was used for staining for 90 minutes, and PBS was used for washing. Flow cytometry was used for detection. (2) Myeloma stem cells: Bone marrow blood samples of MM patients were collected, and mononuclear cells were separated by Ficoll. 1×100,000 cells were seeded in a 6-well plate per well. Different concentrations of BBI608 were added and incubated in a cell culture incubator for 16 hours. Cells were collected and labeled with flow cytometry antibodies CD138, CD19, CD20, and CD27. Annexin V FITC was used for staining, and PI was added for staining 5 minutes before loading. Flow cytometry was used to detect and analyze the proportion of apoptotic cells in myeloma stem cells.

[0030] 6. Umbilical cord blood stem cell activity test: 7 fetal umbilical cord blood samples were collected and mononuclear cells were separated by Ficoll. 6 Cells were seeded in 6-well plates, and different concentrations of BBI608 were added. The cells were placed in a cell culture incubator for 16 hours, and the cells were collected and labeled with flow cytometry antibody CD34. Annexin V FITC staining was then used, and PI staining was added 5 minutes before the machine. Flow cytometry was used to detect and analyze the proportion of apoptotic cells in umbilical cord blood stem cells.

[0031] 7. T cell co-culture killing experiment: Peripheral blood mononuclear cells were isolated from normal human peripheral blood and inoculated into 6-well plates for 3 hours. The suspended cells were removed and adherent cells were cultured in RPMI16 medium containing 10% fetal bovine serum, and 100ng / ml IL-4 and 100ng / ml GM-CSF were added. After 5 days of culture, DCs were collected for subsequent experiments. At the same time, T cells were isolated from PBMC using a T cell isolation kit. 2×10 5 RPMI8226 cells or IM9 cells pre-stained with CFSE were seeded in 6-well plates and divided into the following 5 groups: BBI608 pre-treated group; 5 , the same below) co-culture group; and T cells (1×10 6 , the same below) co-culture group; co-culture group with DC and T cells and pre-treated cells co-cultured with DC and T cells. RPMI8226 or IM9 cells were used alone as the control group. After 48 hours of co-culture, the cells were washed twice with PBS and then resuspended in a flow centrifuge tube with 100ulPBS. 1ul live&dead reagent (Invitrogen, L34994) was added to each tube and stained at room temperature in the dark for 30 minutes, and finally washed twice with PBS, and the mortality rate of RPMI8226 cells or IM9 cells was detected on the machine.

[0032] Embodiment 1:

[0033] Effects of different concentrations of BBI608 (Napabucasin) on the viability of multiple myeloma (MM) cells:

[0034] IM9 (human peripheral blood B lymphocytes), U266 (human multiple myeloma cells) and OPM2 (human myeloma cells) were selected, which were in good condition and in the logarithmic proliferation phase, and 1×10 4 Cells were seeded in 96-well plates, and the final concentration of BBI608 was 200, 400, 800 or 600, 1200, 2400 nM. The cell status was observed after 24, 48 and 72 hours, and CCK-8 reagent was added. The absorbance was detected by microplate reader. The results showed that with the increase of concentration and drug treatment time, cell viability decreased significantly, and it was statistically significant compared with the control group. Figure 1 MM cells were treated with different concentrations of BBI608 and cell viability was detected. (Compared with the control group, *p<0.05)

[0035] Embodiment 2:

[0036] Effects of BBI608 on MM cell cycle distribution:

[0037] To investigate the effect of STAT3 inhibitor BBI608 on MM cell cycle, we seeded U266 and IM9 cells at 2×10 5 The number of cells was seeded in a 24-well plate, and BBI608 was added to a final concentration of 400 and 800 nM, respectively. After 24 hours of treatment, EDU staining was used to analyze the changes in cell cycle distribution. The results showed that compared with the control group, the proportion of cells in the G1 phase increased and the S phase decreased after drug treatment. Figure 2 The effect of different concentrations of BBI608 on cell cycle distribution in MM cells.

[0038] Embodiment 3:

[0039] Effects of BBI608 on osteoclast formation:

[0040] After 14 days of induction of human peripheral blood mononuclear cells in vitro and the addition of BBI608 (200, 400 nM), the results showed that the number of osteoclasts and the number of cell nuclei decreased with the increase of drug concentration, indicating that BBI608 inhibited the formation of osteoclasts. Figure 3 The effect of BBI608 on the osteoclast formation ability induced in vitro.

[0041] Embodiment 4:

[0042] Effects of BBI608 on apoptosis of MM cells:

[0043] In 5×10 5Different concentrations of BBI608 (400, 800, 1600 nM) were added to the cell suspension of MM cells, and the apoptosis state of cells was detected by flow cytometry after 24 hours of action. The results showed that BBI608 promoted the apoptosis of MM cells. Figure 4 Flow cytometry was used to detect the effect of BBI608 on apoptosis of MM cell lines.

[0044] Embodiment 5:

[0045] Detection of changes in apoptosis-related genes:

[0046] 1×10 6 MM cells were cultured in medium containing different concentrations of BBI608 for 12 hours, and the cells were harvested to measure the expression levels of apoptosis-related proteins. The results showed that with tubulin as the internal reference, the bcl-2 / bax ratio decreased, PARP hydrolysis increased, and caspase-3 cleavage activation increased. This indicates that BBI608 activated the endogenous apoptosis pathway. Figure 5 Western blot was used to detect the changes in apoptosis-related proteins in MM cells after treatment with BBI608.

[0047] Embodiment 6:

[0048] Detection of the effect of BBI608 on myeloma stem cells and primary myeloma stem cells:

[0049] Side population cells (SP cells) promote differentiation and promote tumor formation and growth, which may be the root cause of tumor occurrence, metastasis and recurrence, and play an important role in metastasis and recurrence. In a group of cells, the DNA-binding dye Hoechst 33342 fluorescent dye can be quickly pumped out of the cells, and these unstained cells are called swarm cells. This group of side population cells in multiple myeloma is considered to be myeloma stem cells. After Hochest 33342 staining the sample, the double low-stained cells were SP population cells. The flow cytometry results showed that compared with the control group without BBI608, the proportion of SP cells decreased after adding BBI608. Figure 6 Flow cytometry was used to analyze the effect of BBI608 on MM stem cells.

[0050] In addition, we also tested the effect of BBI608 on primary MM stem cells. The results showed that BBI608 can effectively kill primary MM stem cells, while the same concentration of BBI608 has almost no effect on normal umbilical cord blood stem cells. Figure 7 This is the effect of BBI608 on primary MM stem cells and normal umbilical cord blood stem cells.

[0051] Embodiment 7:

[0052] Detect the effect of combined use of BBI608, BTZ and Len on proliferation and apoptosis of MM cells:

[0053] Drug resistance of MM is one of the main reasons affecting the prognosis of MM. In order to improve the prognosis of patients, the present invention adopts a combined drug therapy to study the effect of combined drug therapy on killing tumors. The present invention studies the effect of BBI608 combined with Len or BTZ in treating MM and its effect on MM cell apoptosis. MM cells in the logarithmic phase were cultured at 1×10 4 The number of cells were inoculated in a 96-well plate and treated with 50, 100 nM BBI608 alone, 0.125, 0.25 nM BTZ or 10, 20 nM Len alone or in combination for 48 hours. The combination index (CI) of the two drugs was calculated. The results showed that low concentrations of BBI608 and Len or BTZ had a synergistic inhibitory effect on the killing of MM.

[0054] Table 1 shows the synergistic index of BBI608 combined with Len, and Table 2 shows the synergistic index of BBI608 combined with BTZ.

[0055] Table 1

[0056]

[0057] Table 2

[0058]

[0059] Embodiment 8:

[0060] Detection of the effect of BBI608 on T cell killing of MM cells:

[0061] The patient's own T cells also play a key role in killing tumor cells in the anti-tumor process. We further explored the effect of BBI608 combined with T cells to kill myeloma. DCs, as antigen-presenting cells, cross-present tumor-associated antigens to cytotoxic CD8 T lymphocytes. To study whether BBI608 enhances DC-mediated anti-tumor responses, BBI608 pre-treated / untreated MM cells were co-cultured with DCs and T cells in six-well plates for 48 hours, and MM cell mortality was detected by flow cytometry. The experimental groups were: MM, MM / DC, MM / T cells, MM / DC / T cells, MM pretreated with BBI608, and MM / DCs / T cells pretreated with BBI608. The cell death rates of RMPI8226 were 17.3%, 40.0%, 19.2%, 38.5%, 42.8%, and 77.4%, respectively; the cell death rates of IM9 were 1.11%, 31.6%, 1.77%, 2.46%, 3.15%, and 43.2%, respectively. Therefore, pretreatment with BBI608 enhanced the cytotoxicity of DC-mediated T cells in vitro. Figure 8 The cell death rates of the following groups were analyzed by flow cytometry: RPMI8226, RPMI8226 pretreated with 1000 nM BBI608, RPMI8226 / DCs, RPMI8226 / T cells, RPMI8226 / DCs / T cells, RPMI8226 pretreated with 1000 nM BBI608 / DCs / T cells, and IM9 cells were treated with RPMI8226.

[0062] In summary, the present invention explores the effect of BBI608 on MM cell proliferation and apoptosis through in vitro experiments; BBI608 can also effectively kill MM stem cells; at the same time, it is found that BBI608 can not only be combined with BTZ or Len to improve the effect of killing MM cells; but BBI608 can also enhance the effect of T cells killing MM cells.

[0063] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. Use of a small molecule inhibitor combined drug in the preparation of a drug for treating multiple myeloma, characterized in that: The combination of proteasome inhibitors or immunomodulators and small molecule inhibitors targeting the STAT3 gene synergistically improves the therapeutic effect of multiple myeloma.

2. The use according to claim 1, characterized in that: The proteasome inhibitor is bortezomib.

3. The use according to claim 1, characterized in that: The immunomodulator is lenalidomide.

4. The use according to any one of claims 1 to 3, characterized in that: The small molecule inhibitor targeting the STAT3 gene is the small molecule inhibitor BBI608 targeting the STAT3 gene.

5. The use according to any one of claims 1 to 3, characterized in that: The molar ratio of lenalidomide to the small molecule inhibitor targeting the STAT3 gene is (10-20): (50-100).

6. The use according to any one of claims 1 to 3, characterized in that: The molar ratio of bortezomib to the small molecule inhibitor targeting the STAT3 gene is (0.125-0.25): (50-100).

7. The use according to claim 6, characterized in that: The concentration ratio of bortezomib to the STAT3 small molecule inhibitor is (0.125nM-0.25nM): (50nM-100nM).

8. The use according to claim 5, characterized in that: The concentration ratio of lenalidomide to the STAT3 small molecule inhibitor is (10nM-20nM): (50nM-100nM).

9. Use of the small molecule inhibitor combination drug according to claim 1 in the preparation of a drug for inhibiting the proliferation of multiple myeloma cells.

10. Use of the small molecule inhibitor combination drug according to claim 1 in the preparation of a drug for promoting apoptosis of multiple myeloma cells.

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