Use of rineterkib in combination with a dna damaging agent for the preparation of a slfn11 deficient tumor treatment

Abstract

The application discloses application of Rineterkib combined with a DNA damaging agent in preparation of a SLFN11-deficient tumor treatment drug. Rineterkib can significantly restore the sensitivity of SLFN11-deficient tumor cells to DNA damaging agents (such as 2-fluoroadenine, cisplatin, bleomycin and etoposide). Further research shows that Rineterkib can achieve a synergistic killing effect on SLFN11-deficient tumor cells by blocking DNA damage repair, inducing G2 / M phase arrest and promoting cell apoptosis.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to the application of Rineterkib in combination with DNA damaging agents in the preparation of therapeutic drugs for SLFN11-deficient tumors. Background Technology

[0002] DNA damaging agents are commonly used chemotherapeutic drugs for treating advanced malignant tumors. They induce DNA single-strand breaks (SSBs) and double-strand breaks (DSBs) in tumor cells, hindering genome replication and transcription, and ultimately leading to cell death. However, the development of chemotherapeutic resistance severely limits the clinical efficacy of these drugs, and epigenetic silencing or loss of function of the SLFN11 gene is one of the key resistance mechanisms.

[0003] SLFN11 (Schlafen11) is a key protein determining the sensitivity of tumor cells to DNA-damaging chemotherapeutic agents. It mediates the sensitivity of tumor cells to various chemotherapeutic drugs through corresponding replication stress (RS) and regulation of DNA damage response (DDR). Studies have shown that approximately 50% of human malignant tumors exhibit SLFN11 expression deficiency. These tumor cells can compensate for DNA damage by activating the ATR-CHK1 signaling pathway, leading to resistance to drugs such as cisplatin and etoposide, thus limiting clinical efficacy. Therefore, developing drugs that can restore the sensitivity of SLFN11-deficient tumor cells to DNA-damaging agents is one of the important directions in current anti-tumor research. Summary of the Invention

[0004] The purpose of this invention is to provide the application of Rineterkib in combination with a DNA damaging agent in the preparation of a therapeutic drug for SLFN11-deficient tumors.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] Application of Rineterkib in combination with DNA damaging agents in the preparation of SLFN11-deficient tumor therapeutic drugs;

[0007] Furthermore, Rineterkib acts as an inhibitor of the ATR-CHK1 signaling pathway;

[0008] Furthermore, the DNA damaging agents include, but are not limited to, 2-fluoroadenine, cisplatin, bleomycin, and etoposide;

[0009] Furthermore, the tumors include, but are not limited to, cervical cancer, colon cancer, and breast cancer.

[0010] A pharmaceutical composition for treating SLFN11-deficient tumors, the pharmaceutical composition comprising Rineterkib and a DNA damaging agent;

[0011] Furthermore, the DNA damaging agents include, but are not limited to, 2-fluoroadenine, cisplatin, bleomycin, and etoposide;

[0012] Furthermore, the tumors include, but are not limited to, cervical cancer, colon cancer, and breast cancer.

[0013] The beneficial effects of this invention are as follows:

[0014] This invention is the first to discover that Rineterkib can effectively inhibit the activation of the ATR-CHK1 signaling pathway induced by DNA damaging agents in SLFN11-deficient tumor cells, and based on this mechanism, a synergistic anti-tumor strategy of combining it with DNA damaging agents was established. Studies show that ERK1 / 2 kinase inhibitors cannot reproduce this synergistic effect, further confirming the unique value and innovation of the combined application strategy of this invention in the treatment of SLFN11-deficient tumors. Attached Figure Description

[0015] Figure 1 The expression of SLFN11 in HeLa, HCT116, MDA-MB-231 cells and SLFN11-overexpressing HeLa cells was determined by Western blot in Example 1.

[0016] Figure 2 The image shows the cell viability of HeLa, HCT116, and MDA-MB-231 cells after treatment with Rineterkib in combination with 2-fluoroadenine, cisplatin, bleomycin, and etoposide in Example 2.

[0017] Figure 3 This is a cell viability graph of HeLa cells after treatment with the control compound SCH772984 (ERK1 / 2 inhibitor) and 2-FA in Example 3.

[0018] Figure 4 This is a diagram showing the colony formation results of HeLa cells after combined treatment with Rineterkib and a DNA damaging agent in Example 4. (**P<0.01; ****P<0.0001).

[0019] Figure 5 The image shows the invasion results of HeLa cells after combined treatment with Rineterkib and a DNA damaging agent in Example 5. (**P<0.01; ****P<0.0001).

[0020] Figure 6 The effect of Rineterkib combined with a DNA damaging agent on DNA damage repair in HeLa cells was determined by immunofluorescence assay in Example 6 (*P<0.05).

[0021] Figure 7 The effect of Rineterkib combined with a DNA damaging agent on DNA damage repair in HeLa cells was determined by Western blot in Example 6 (*P<0.05).

[0022] Figure 8 The effect of Rineterkib on the ATR-CHK1 pathway was determined by Western blot in Example 7 (*P<0.05; **P<0.01).

[0023] Figure 9 In Example 8, the effect of the control compound SCH772984 (ERK1 / 2 inhibitor) on the ATR-CHK1 pathway was determined by Western blot.

[0024] Figure 10 This is a diagram showing the cell cycle distribution of HeLa cells after combined treatment with Rineterkib and a DNA damaging agent in Example 9.

[0025] Figure 11 This is a diagram showing the cell cycle distribution of HeLa cells after treatment with the control compound SCH772984 (an ERK1 / 2 inhibitor) and a DNA damage agent in Example 10.

[0026] Figure 12 This is a diagram showing the distribution of apoptosis in HeLa cells after combined treatment with Rineterkib and a DNA damaging agent in Example 11.

[0027] Figure 13 This is a diagram showing the distribution of apoptosis in HeLa cells after combined treatment with the control compound SCH772984 (an ERK1 / 2 inhibitor) and a DNA damage agent in Example 12. Detailed Implementation

[0028] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0029] To verify the synergistic antitumor effect of Rineterkib and DNA damaging agents discovered in this invention, and to rule out that this effect is merely a non-specific background effect caused by pan-kinase inhibitory activity, this invention introduced the commercially available selective ERK1 / 2 kinase inhibitor SCH772984 as a control compound in several key mechanism verification experiments. ERK1 / 2 is a core node in another classic kinase signaling pathway closely related to cell proliferation and survival. Using its highly selective inhibitors for parallel comparison helps determine whether the synergistic antitumor effect is a generalized and universal effect resulting from broad-spectrum kinase inhibition.

[0030] The CAS Registry Numbers of Rineterkib, 2-fluoroadenine (2-FA), ...

[0031] Example 1: Construction and identification of SLFN11 overexpression stable cell lines

[0032] A stable SLFN11 overexpression cell line was constructed using a lentiviral system: HEK293T cells were seeded in 10cm culture dishes and transfected when confluence reached 70%-80% to package lentivirus carrying the SLFN11 gene (GenBank ID: 1908122797). The specific transfection steps were as follows: the expression plasmid pGenLenti-SLFN11 was mixed with three backbone plasmids (PRRE, VSVG, REV) at a mass ratio of 5:1:1:1 in Opti-MEM medium, followed by the addition of linear polyethyleneimine (PEI) transfection reagent and incubation at room temperature for 15-20 min to prepare the transfection complex. This complex was then added to HEK293T cells for transfection. 72 h after transfection, the supernatant containing viral particles was collected, filtered through a 0.45μm filter membrane, and used to infect human cervical cancer HeLa cells. 48 hours after infection, a stable HeLa cell pool overexpressing SLFN11 was obtained by screening with 2 μg / ml puromycin.

[0033] Human cervical cancer HeLa cells, human colon cancer HCT116 cells, human breast cancer MDA-MB-231 cells, and the aforementioned SLFN11-overexpressing HeLa cells were collected, and total protein was extracted. The expression level of SLFN11 protein was analyzed by Western blot using anti-SLFN11 antibody.

[0034] The results are as follows Figure 1As shown, SLFN11 protein expression was almost undetectable in human cervical cancer HeLa cells, human colon cancer HCT116 cells, and human breast cancer MDA-MB-231 cells; in contrast, a significant SLFN11 protein band was observed in SLFN11-overexpressing HeLa cells. This confirms that human cervical cancer HeLa cells, human colon cancer HCT116 cells, and human breast cancer MDA-MB-231 cells are endogenously negative tumor cells, i.e., SLFN11-deficient tumor cells.

[0035] Example 2: Effect of combined use of Rineterkib and DNA damaging agents on the survival rate of SLFN11-deficient tumor cells

[0036] Human cervical cancer HeLa cells, human colon cancer HCT116 cells, or human breast cancer MDA-MB-231 cells in logarithmic growth phase were seeded at 100 μL per well in 96-well plates, with three replicates per group. Cells were cultured at 37°C in a 5% CO2 incubator until adherence, after which the original culture medium was discarded. The experiment was divided into two groups: the combined treatment group received DMEM medium containing different concentrations of DNA damaging agents and 10 μM Mineterkib, while the single-drug treatment group received DMEM medium containing only the corresponding concentration of DNA damaging agent, with 100 μL per well for each group. After culturing for 72 h, 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37°C in the dark for 2 h. The absorbance was measured at 450 nm using a microplate reader.

[0037] The results are as follows Figure 2 As shown, compared with the use of DNA damaging agents alone (2-fluoroadenine, cisplatin, bleomycin, or etoposide), the combined use of Rineterkib with DNA damaging agents significantly enhanced the inhibitory effect on the viability of various SLFN11-deficient tumor cells.

[0038] Example 3: Cell viability graph of HeLa cells after treatment with control compound SCH772984 (ERK1 / 2 inhibitor) and 2-FA.

[0039] The effect of the commonly used ERK inhibitor SCH772984 on the chemosensitivity of human cervical cancer HeLa cells was detected using the CCK-8 assay. Logarithmically growing human cervical cancer HeLa cells were seeded at 100 μL per well in 96-well plates, with three replicates per group. Cells were cultured at 37°C in a 5% CO2 incubator until adherence, after which the original culture medium was discarded. The experiment was divided into two groups: a combined treatment group received DMEM medium containing 5 μM 2-fluoroadenine and different concentrations of SCH772984, and a single-drug treatment group received DMEM medium containing only the corresponding concentration of SCH772984, with 100 μL per well for each group. After 72 h of further culture, 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37°C in the dark for 2 h. The absorbance was measured at 450 nm using a microplate reader.

[0040] The results are as follows Figure 3 As shown in the figure, within the measured concentration range, the combination of SCH772984 and 2-fluoroadenine did not have a significant synergistic inhibitory effect on the viability of human cervical cancer HeLa cells compared to SCH772984 alone. This result contrasts sharply with the effect of Rineterkib, indicating that not all combinations of kinase inhibitors and DNA damaging agents can produce chemosensitizing effects.

[0041] Example 4: Effect of Rineterkib in combination with DNA damaging agents on the proliferation of SLFN11-deficient tumor cells

[0042] The effect of Rineterkib on the proliferation of SLFN11-deficient tumor cells was detected using the colony formation assay. Human cervical cancer HeLa cells in logarithmic growth phase were seeded at 2 mL per well in 6-well plates. Cells were cultured at 37°C with 5% CO2 until adherence, after which the original culture medium was discarded. Experimental groups were as follows: ① Blank control group: DMEM medium without the drug was added; ② DNA damaging agent group: DMEM medium containing 5 μM 2-fluoroadenine was added; ③ Rineterkib group: a) DMEM medium containing 5 μM Rineterkib was added; b) DMEM medium containing 10 μM Rineterkib was added; ④ Combined drug group: a) DMEM medium containing 5 μM 2-fluoroadenine and 5 μM Rineterkib was added; b) DMEM medium containing 5 μM 2-fluoroadenine and 10 μM Rineterkib was added. Each group had 3 replicates, with 2 mL of medium added to each well. After culturing for another 72 hours, the cells were replaced with fresh, drug-free DMEM medium and cultured for another 7-10 days. Subsequently, the cells were fixed with paraformaldehyde, stained with crystal violet, and images were acquired and counted using an imaging system.

[0043] The results are as follows Figure 4As shown in the figure. Compared with the blank control, the use of 2-fluoroadenine or Rineterkib alone had no significant effect on the colony formation of human cervical cancer HeLa cells, while the combined use of Rineterkib and 2-fluoroadenine significantly inhibited the proliferation of human cervical cancer HeLa cells in a concentration-dependent manner.

[0044] Example 5: Effect of Rineterkib in combination with a DNA damaging agent on the invasion of SLFN11-deficient tumor cells

[0045] The Transwell invasion assay was used to detect the effect of Rineterkib combined with a DNA damaging agent on the invasive ability of SLFN11-deficient tumor cells. Human cervical cancer HeLa cells were cultured in fresh DMEM medium for 24 h, then digested with trypsin and collected. Cells were resuspended in DMEM medium as described below and cultured at 5 × 10⁶ cells / well. 4 Cells were seeded at a density of 1000 mcg / mL in the upper chamber of a Transwell containing matrix gel: ① Blank control group: DMEM medium without drug; ② DNA damaging agent alone group: DMEM medium containing 5 μM 2-fluoroadenine; ③ Rineterkib alone group: a) DMEM medium containing 5 μM Rineterkib; b) DMEM medium containing 10 μM Rineterkib; ④ Combined drug group: a) DMEM medium containing 5 μM 2-fluoroadenine and 5 μM Rineterkib; b) DMEM medium containing 5 μM 2-fluoroadenine and 10 μM Rineterkib. Complete DMEM medium containing 20% ​​fetal bovine serum was added to the lower chamber. Cells were incubated at 37°C and 5% CO2 for 72 h, then fixed, stained, and images were acquired and analyzed using an imaging system.

[0046] The results are as follows Figure 5 As shown, the combined use of 5µM Rineterkib and 5µM 2-fluoroadenine reduced the invasive ability of human cervical cancer HeLa cells by approximately 90%, while the two drugs, when used alone, had almost no significant effect on cell invasiveness.

[0047] Example 6: Effect of combined use of Rineterkib and DNA damaging agents on DNA damage repair in SLFN11-deficient tumor cells

[0048] (1) Immunofluorescence assay: Human cervical cancer HeLa cells were seeded in 96-well clear-bottom black cell culture plates and cultured at 37°C in a 5% CO2 incubator until adherence. The original culture medium was discarded, and the cells were treated with fresh DMEM medium containing different concentrations of Rineterkib and / or different concentrations of 2-fluoroadenine. After incubation for 72 h, the cells were fixed and incubated with anti-phosphoH2A.X (Ser139) primary antibody for 2 h; after washing with PBS, the cells were incubated with anti-Mouse IgG AF594 secondary antibody for 1 h. Finally, high-content images were acquired and data were analyzed.

[0049] Depend on Figure 6 The results showed that the combined treatment of 2-fluoroadenine and Rineterkib significantly enhanced the γH2AX signal intensity in human cervical cancer HeLa cells compared with the single-drug treatment group, indicating that the combined treatment can significantly impair the DNA damage repair capacity of SLFN11-deficient tumor cells.

[0050] (2) Western blot method: Human cervical cancer HeLa cells were seeded in 6-well culture plates and cultured at 37°C in a 5% CO2 incubator until adherence. The original culture medium was discarded, and the cells were treated with fresh DMEM medium containing different concentrations of Rineterkib and / or different concentrations of 2-fluoroadenine. After incubation for 72 h, the cells were lysed, and the expression level of γH2AX protein was detected and analyzed by Western blot using anti-phosphoH2A.X (Ser139) antibody.

[0051] The results are as follows Figure 7 As shown, Rineterkib monotherapy impairs the DNA damage repair capacity of human cervical cancer HeLa cells, and this damaging effect is more pronounced when used in combination with 2-fluoroadenine.

[0052] Example 7: Western blot analysis of the effect of Rineterkib on the ATR-CHK1 pathway

[0053] Human cervical cancer HeLa cells were seeded in 6-well plates and cultured at 37°C in a 5% CO2 incubator until adherent. The original culture medium was discarded, and the cells were treated with fresh DMEM medium containing different concentrations of Rineterkib and / or different concentrations of 2-fluoroadenine. After incubation for 72 h, the cells were lysed, and Western blot analysis was performed using anti-CHK1 pS345 and anti-WEE1 pS642 antibodies.

[0054] The results are as follows Figure 8As shown, 2-fluoroadenine monotherapy increased the phosphorylation levels of CHK1 and WEE1 in human cervical cancer HeLa cells, indicating that the ATR-dependent DNA damage response was activated. Conversely, Rineterkib monotherapy significantly reduced the basal phosphorylation levels of CHK1 and WEE1, as well as the 2-FA-induced phosphorylation levels, indicating that ATR pathway activity was effectively inhibited.

[0055] Example 8: Western blot determination of the effect of control compound SCH772984 (ERK1 / 2 inhibitor) on the ATR-CHK1 pathway

[0056] Human cervical cancer HeLa cells were seeded in 6-well plates and cultured at 37°C in a 5% CO2 incubator until adherence. The original culture medium was discarded, and the cells were treated with fresh DMEM medium containing different concentrations of SCH772984 and 2-fluoroadenine. After 72 hours of incubation, the cells were lysed, and Western blot analysis was performed using anti-CHK1 pS345 antibody.

[0057] The results are as follows Figure 9 As shown, no significant inhibition of downstream CHK1 phosphorylation of ATR was observed after SCH772984 treatment of cells. This indicates that inhibiting ERK1 / 2 kinase activity is insufficient to block the ATR-CHK1 pathway activation induced by DNA-damaging agents in this experimental system, thus confirming the specificity of Rineterkib's inhibitory effect on this pathway.

[0058] Example 9: Effects of Rineterkib in combination with a DNA damaging agent on the cell cycle of SLFN11-deficient tumor cells

[0059] Cellular DNA content varies at different stages of the cell cycle. Under normal circumstances, G1 / G0 phase cells have diploid DNA content (2N), G2 / M phase cells have tetraploid DNA content (4N), and S phase cells have DNA content between diploid and tetraploid. Propidium iodide (PI) can specifically bind to DNA, and its fluorescence intensity is positively correlated with intracellular DNA content. Therefore, flow cytometry PI staining can be used to distinguish the G1 / G0, S, and G2 / M phases of the cell cycle. The resulting flow cytometry histograms can be used to calculate the cell percentage of each cell cycle phase using FlowJo software.

[0060] Rineterkib can inhibit ATR activation, leading to abnormal cell cycle progression. In this experiment, human cervical cancer HeLa cells were seeded in 6-well plates and treated with fresh DMEM medium containing different concentrations (0 μM, 10 μM) of Rineterkib and / or different concentrations (0 μM, 5 μM) of 2-fluoroadenine. After incubation at 37°C and 5% CO2 for 72 h, cell cycle distribution was determined by flow cytometry.

[0061] The results are as follows Figure 10 As shown, most cells in the control group were in the G0 / G1 phase; after Rineterkib treatment, the cell cycle was arrested in the G2 / M phase, and the use of 2-fluoroadenine did not affect the cell cycle distribution, indicating that Rineterkib can significantly interfere with the cell cycle progression of human cervical cancer HeLa cells and arrest them in the G2 / M phase.

[0062] Example 10: Effect of the combination of control compound SCH772984 (ERK1 / 2 inhibitor) and 2-FA on the cell cycle of SLFN11-deficient tumor cells

[0063] Human cervical cancer HeLa cells were seeded in 6-well culture plates and treated with freshly cultured DMEM containing different concentrations (0 μM, 2 μM) of SCH772984 and / or different concentrations (0 μM, 10 μM) of 2-fluoroadenine. After incubation at 37°C and 5% CO2 for 72 h, cell cycle distribution was determined by flow cytometry.

[0064] The results are as follows Figure 11 As shown. Unlike Rineterkib, the control compound SCH772984 had no significant effect on the cell cycle distribution of HeLa cells and did not cause G2 / M phase arrest. Combined with the results of Example 9, this indicates that interfering with cell cycle progression and causing G2 / M phase arrest is not a common feature of broad-spectrum kinase inhibitors.

[0065] Example 11: Effect of Rineterkib in combination with DNA damaging agents on apoptosis in SLFN11-deficient tumor cells

[0066] To investigate whether the combined treatment of 2-fluoroadenine and Rineterkib exerts its cytotoxic effect by inducing apoptosis in SLFN11-deficient tumor cells, flow cytometry analysis was performed on human cervical cancer HeLa cells after drug treatment. In the experiment, human cervical cancer HeLa cells were seeded in 6-well culture plates and treated with fresh DMEM medium containing different concentrations of Rineterkib and / or different concentrations of 2-fluoroadenine. After incubation at 37°C and 5% CO2 for 72 h, the distribution of apoptosis was determined by flow cytometry.

[0067] The results are as follows Figure 12 As shown in the figure. Compared with the control group, Rineterkib monotherapy can induce apoptosis in human cervical cancer HeLa cells; 2-FA monotherapy has no significant apoptosis-inducing effect, but when combined with Rineterkib, it can significantly enhance the apoptosis-inducing effect of Rineterkib.

[0068] Example 12: Effect of combined use of SCH772984 and DNA damaging agent on apoptosis in SLFN11-deficient tumor cells

[0069] Human cervical cancer HeLa cells were seeded in 6-well culture plates and treated with fresh DMEM medium containing different concentrations (0 μM, 2 μM) of SCH772984 and / or different concentrations (0 μM, 5 μM) of 2-fluoroadenine. After incubation at 37°C and 5% CO2 for 72 h, the distribution of apoptosis was determined by flow cytometry.

[0070] The results are as follows Figure 13 As shown, neither SCH772984 alone nor in combination with 2-fluoroadenine exhibited the significant synergistic pro-apoptotic effect similar to that of Rineterkib in combination, and the apoptosis level was not significantly different from the control group. These results indicate that the potent pro-apoptotic activity of Rineterkib in combination with DNA damaging agents is specific, and pan-ERK1 / 2 kinase inhibition cannot be reproduced.

[0071] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. Application of Rineterkib in combination with DNA damaging agents in the preparation of therapeutic drugs for SLFN11-deficient tumors.

2. The application according to claim 1, characterized in that: The DNA damaging agents include, but are not limited to, 2-fluoroadenine, cisplatin, bleomycin, and etoposide.

3. The application according to claim 1, characterized in that: The tumors include, but are not limited to, cervical cancer, colon cancer, and breast cancer.

4. A pharmaceutical composition for treating SLFN11-deficient tumors, characterized in that: The pharmaceutical composition includes Rineterkib and a DNA damaging agent.

5. The pharmaceutical composition according to claim 4, characterized in that: The DNA damaging agents include, but are not limited to, 2-fluoroadenine, cisplatin, bleomycin, and etoposide.

6. The pharmaceutical composition according to claim 4, characterized in that: The tumors include, but are not limited to, cervical cancer, colon cancer, and breast cancer.

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