Use of a lamtor1 inhibitor in the preparation of an antitumor drug sensitizer

Abstract

The application relates to an application of an LAMTOR1 inhibitor in preparation of an antitumor drug sensitizer. Specifically, it is found for the first time that LAMTOR1 is a binding protein of cGAS, LAMTOR1 inhibits the activation of a cGAS signal channel and the production of IFN-beta by down-regulating the protein level of cGAS. The absence of LAMTOR1 can enhance the infiltration of antitumor T lymphocytes in a tumor microenvironment, thereby inhibiting tumor growth and prolonging the survival period of tumor-bearing mice. A P18 inhibitor is obtained through drug virtual screening, the sensitization effect of antitumor immunity is realized, and the application has a wide application prospect.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese application No. 202410069940X, filed on January 18, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of biopharmaceuticals, specifically to the application of a LAMTOR1 inhibitor in the preparation of antitumor drug sensitizers. Background Technology

[0004] Cancer remains one of the major diseases threatening human life and health, and the mechanisms of tumor development and treatment have always been a key area of ​​research. With the development and advancement of medical technology, current cancer treatments mainly include surgical resection, radiotherapy, chemotherapy, targeted therapy, and immunotherapy. Although cancer treatment methods have become relatively diverse, chemotherapy remains a commonly used clinical treatment. However, chemotherapy resistance significantly limits the effectiveness of chemotherapeutic drugs in killing tumor cells. The mechanisms of chemotherapy resistance mainly include drug expulsion from cells, drug inactivation, epigenetic alterations, and tumor heterogeneity. However, the link between chemotherapy resistance and the tumor microenvironment is rarely reported. Whether tumor cells themselves can affect the efficacy of chemotherapeutic drugs by influencing the tumor microenvironment is also unclear. Cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) is a double-stranded DNA recognition protein identified in recent years, playing a crucial role not only in innate immune responses but also in tumor immune responses. However, the role of cGAS in regulating tumor immunity during cancer chemotherapy is not well understood, and there are few reports on how chemotherapy drugs regulate the activity and function of cGAS. Summary of the Invention

[0005] To fill a gap in existing technologies, the inventors have discovered a novel mechanism of chemotherapy drug resistance for the first time: chemotherapy-induced double-stranded DNA fragments promote the formation of the LAMTOR1-cGAS-p62 complex, which in turn mediates the lysosomal degradation of cGAS, leading to the formation of a suppressive immune microenvironment. Simultaneously, this application also discloses for the first time that LAMTOR1 is a cGAS-binding protein. LAMTOR1 inhibits the activation of the cGAS signaling pathway and the production of IFN-β by downregulating cGAS protein levels. The absence of LAMTOR1 enhances the infiltration of anti-tumor T lymphocytes in the tumor microenvironment, thereby inhibiting tumor growth and prolonging the survival of tumor-bearing mice. Furthermore, through virtual drug screening, a p18 inhibitor was obtained, achieving an anti-tumor immunosensitizing effect, i.e., improving the therapeutic effects of immunotherapy and chemotherapy, including alleviating the inhibition of cGAS-STING by 5-FU, promoting the infiltration of anti-tumor T cells after 5-FU treatment, and reducing the proliferation of suppressor T cells after 5-FU treatment, thereby alleviating the immunosuppressive tumor microenvironment induced by 5-FU treatment and improving the anti-tumor immunotherapy effect.

[0006] Specifically, the present invention provides the following technical solution:

[0007] The first aspect of the present invention provides the use of a LAMTOR1 inhibitor in the preparation of an antitumor drug sensitizer.

[0008] In one embodiment, the antitumor drug is a chemotherapy drug or an immune checkpoint inhibitor.

[0009] In one embodiment, the LAMTOR1 inhibitor is Demethylmurrayanine (DMM).

[0010] In one embodiment, the application is that the LAMTOR1 inhibitor enhances anti-tumor immunity by interfering with the interaction between P18 and cGAS, thereby affecting the degradation of cGAS by P18.

[0011] In a preferred embodiment, the antitumor drug is 5-FU. The immunosensitizing effect of LAMTOR1 inhibitors on 5-FU includes alleviating the inhibition of cGAS-STING by 5-FU, promoting the infiltration of antitumor T cells after 5-FU treatment, and reducing the proliferation of suppressor T cells after 5-FU treatment, thereby alleviating the immunosuppressive tumor microenvironment caused by 5-FU treatment and improving the immunotherapeutic effect of antitumor treatment.

[0012] In one embodiment, the dosage form of the antitumor drug sensitizer is tablets, capsules, microcapsules, injections, or granules.

[0013] In a second aspect, the present invention provides the above-mentioned antitumor drug sensitizer, the main active ingredient of which is a LAMTOR1 inhibitor. Attached Figure Description

[0014] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0015] Figure 1 This section presents the decrease in cGAS protein levels in tumors after chemotherapy. A. Representative images of cGAS immunohistochemical staining in rectal tumor tissue before and after chemotherapy. Scale bar: 5X image is 500μm, 40X image is 50μm. B. Statistical graph of cGAS immunohistochemical staining in rectal tumor tissue before and after chemotherapy. n=17, significance analysis method: two-sided unpaired t-test. **** indicates p<0.0001, error bar is mean ± standard error. C. Representative images of cGAS immunohistochemical staining in breast tumor tissue before and after chemotherapy. Scale bar: 5X image is 500μm, 40X image is 50μm. D. Statistical graph of cGAS immunohistochemical staining in breast tumor tissue before and after chemotherapy. n=34, significance analysis method: two-sided unpaired t-test. *** indicates p<0.001, error bar is mean ± standard error. E. Protein immunoblotting analysis of cGAS protein levels in rectal tumor tissue before and after chemotherapy. F. Protein immunoblotting analysis of cGAS protein levels in breast tumor tissues before and after chemotherapy. *** indicates p<0.001, and the error bar is mean ± standard error.

[0016] Figure 2 This study demonstrates that chemotherapy can downregulate cGAS protein levels in tumor cell lines. After treating (A) HeLa cells, (B) 4T1 cells, and (C) B16F10 cells with 5-fluorouracil (5-FU) for 24 hours, the treatment was withdrawn, and the cells were cultured for 0-3 days. Cells were then collected for protein immunoblotting analysis to detect cGAS protein levels. (D) After treating MC38 (left) or HeLa (right) cells with etoposide for 24 hours, the treatment was withdrawn, and the cells were cultured for 0-3 days. Cells were then collected for quantitative real-time PCR to detect cGAS mRNA levels. n=3, and one-way ANOVA was used for significance analysis. ** indicates p<0.01, *** indicates p<0.001, **** indicates p<0.0001, and the error bar is mean ± standard error.

[0017] Figure 3This study demonstrates that LAMTOR1 promotes the degradation of cGAS. A. After gradient overexpression of LAMTOR1-Flag in HeLa or MCF7 cells, the amount of endogenous cGAS protein in the cells was detected. B. After treating LAMTOR1 knockdown or wild-type HeLa cells with actinomycin (CHX), cells were collected at different time points, and the amount of endogenous cGAS protein in the cells was detected by immunoblotting. C. HeLa cells transfected with LAMTOR1-Flag (2 μg) or empty vector for 24 hours were treated with actinomycin (CHX) for different time points, and cells were scraped at the corresponding time points to detect the amount of endogenous cGAS protein in the cells by immunoblotting. D. After transfecting HeLa cells with a LAMTOR1-Flag gradient for 24 hours, they were treated with 100 μmol / L chloroquine (CQ) for 8 hours, and the cells were collected to detect the cGAS protein level by immunoblotting. E. After transfecting LAMTOR1-HA or empty vector into cells stably expressing cGAS-EGFP for 36 hours, lysosomes were stained with Lysotracker, and the colocalization of cGAS-EGFP with lysosomes was observed. Scale bar: 5 μm. F. After transfecting HT-DNA into LAMTOR1 knockdown and wild-type HeLa cells, cGAS and LAMP2 were immunofluorescently stained and observed 8 hours later. Scale bar: 10 μm.

[0018] Figure 4 This study demonstrates how LAMTOR1 knockdown inhibits tumor growth in immunogenic mice. A and B. Colony formation (A) and cell proliferation (B) of LAMTOR1 knockdown and wild-type B16F10 cells. n=3, one-way ANOVA was used for significance analysis. ns indicates no significant difference, and the error bar is mean ± standard error. C and D. Tumor volume (C) and tumor weight (D) were recorded after subcutaneous inoculation of C57BL / 6J mice with LAMTOR1 knockdown or wild-type B16F10 cells. n=6, one-way ANOVA was used for significance analysis. *** indicates p<0.001, **** indicates p<0.0001, and the error bar is mean ± standard error. E. Tumor volume (E) was recorded after subcutaneous inoculation of nude mice with LAMTOR1 knockdown or wild-type B16F10 cells. n=6, one-way ANOVA was used for significance analysis. ns indicates no significant difference. F. LAMTOR1 knockdown or wild-type B16F10 cells were subcutaneously injected into C57BL / 6J mice, and the survival time of tumor-bearing mice was recorded. n=7, and the statistical method was log-rank test. ns indicates no significant difference, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

[0019] Figure 5This study demonstrates that LAMTOR1 deficiency can remodel the immunosuppressive tumor microenvironment. A and B: LAMTOR1 knockdown or wild-type B16F10 cells were subcutaneously injected into C57BL / 6J mice. On the last day of tumor volume recording, mouse tumors were removed, frozen sections were prepared, and immunofluorescence staining for CD3, CD4, or CD8 was performed (A). The percentage of CD3, CD4, or CD8-positive cells in the field of view was counted (B). n = 8–12, and one-way ANOVA was used for significance analysis. C: LAMTOR1 knockdown, LAMTOR1 knockdown combined with cGAS knockout, cGAS knockout, or wild-type B16F10 cells were subcutaneously injected into C57BL / 6J mice, and mouse tumor volume was recorded. n = 6, and one-way ANOVA was used for significance analysis. D. LAMTOR1 knockdown B16F10 cells were subcutaneously inoculated into C57BL / 6J mice. 200 μg of anti-IFNAR1 monoclonal antibody was injected intraperitoneally into the mice on days 2, 6, and 10, and the tumor volume was recorded. n=6, and one-way ANOVA was used for significance analysis. E and F. RAW264.7 cells were cultured in media containing LAMTOR1 knockdown or wild-type 4T1 cells or B16F10 cells. Cells were collected after 24 hours for qPCR detection of marker genes for M1 and M2 macrophages. n=3, and one-way ANOVA was used for significance analysis. ** indicates p<0.01, **** indicates p<0.0001, and the error bar is mean ± standard error.

[0020] Figure 6 This study demonstrates that the combination of LAMTOR1 knockdown and anti-PD-1 monoclonal antibody significantly enhances the infiltration of anti-tumor T cells and the anti-tumor effect. A. LAMTOR1 knockdown or wild-type B16F10 cells were subcutaneously inoculated into C57BL / 6J mice. Anti-PD-1 monoclonal antibody was injected intraperitoneally into the mice on days 8, 11, and 14 post-inoculation. Tumor volume was recorded (A). At the experimental endpoint, the mouse tumors were harvested and weighed (B). The tumor volume was analyzed by flow cytometry for CD45, CD3, CD4, and CD8, and the proportion of CD3, CD4, and CD8-positive cells to CD45-positive cells was calculated (C). n=6, two-sided unpaired t-test. * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, **** indicates p<0.0001, and the error bar is mean ± standard error.

[0021] Figure 7This study demonstrated that LAMTOR1 knockdown synergistically interacts with chemotherapy drugs in antitumor activity. A. LAMTOR1 knockdown or wild-type MC38 cells were subcutaneously inoculated into C57BL / 6J mice. 5-FU was injected intraperitoneally into the mice on days 5, 8, 11, and 14 post-inoculation, and tumor volume was recorded. The antitumor effect of chemotherapy combined with LAMTOR1 knockdown was significantly different from that of the LAMTOR1 knockdown group. B. Weight of the tumors removed from the mice at the experimental endpoint. n=7, one-way ANOVA was used for significance analysis. * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, **** indicates p<0.0001, and the error bar is mean ± standard error. C. LAMTOR1 knockdown or wild-type MC38 cells were subcutaneously inoculated into C57BL / 6J mice. 5-FU was injected intraperitoneally into the mice on days 5, 8, 11, and 14 post-inoculation. The survival time (E) of tumor-bearing mice was recorded (n=7). The significance analysis method was log-rank test. * indicates p<0.05, ** indicates p<0.01.

[0022] Figure 8 This study demonstrates the screening and functional validation of P18 inhibitors. A. The ability of the three lowest-scoring natural products to interfere with the interaction between P18 and cGAS in MM-GBSA computational analysis. 293T cells were transfected with 6 μg of LAMTOR1 and cGAS plasmids, and 24 hours later, different concentrations (10 μM, 100 μM) of the natural products were administered. Cells were harvested 12 hours later, and CO-IP was used to detect the ability of the natural products to interfere with the interaction between P18 and cGAS. B. 293T cells were transfected with 6 μg of LAMTOR1 and cGAS, and 24 hours later, different concentrations of Demethylmurrayanine (DMM) (10 μM, 30 μM, 90 μM) were administered. Cells were harvested 12 hours later, and CO-IP was used to detect the ability of the natural products to interfere with the interaction between P18 and cGAS. C and D. Activation of cGAS-STING in HeLa cells by the natural product Demethylmurrayanine (DMM). HeLa cells were treated with different concentrations of DMM (12.5 μM, 25 μM, 50 μM, 100 μM), and the cells were collected after 24 hours for protein immunoblotting experiments (C). HeLa cells were treated with 30 μM DMM for different time periods (0d, 1d, 2d, 3d), and the cells were collected for protein immunoblotting experiments (D).

[0023] Figure 9This study demonstrated that DMM can enhance the antitumor efficacy of chemotherapeutic drugs and T-cell infiltration. A. 4T1 cells were treated with 5-FU or 5-FU + DMM, and EdU staining was performed 24 hours later to observe the effect of the drugs on cell proliferation. The results showed that 5-FU inhibited the proliferation of 4T1 cells, while DMM significantly enhanced the effect of 5-FU. B. HeLa cells were treated with 5-FU, and after different recovery times, DMM was administered. The ability of DMM to restore cGAS-STING activity was observed. The results showed that 5-FU inhibited cGAS-STING signaling, but DMM reversed the inhibition of cGAS-STING by 5-FU. C. 4T1 cells were subcutaneously inoculated into C57BL / 6J mice. 5-FU and DMM were injected intraperitoneally into the mice on days 5, 8, 11, and 14 post-inoculation, and the tumors were weighed on the last day. With n=6, one-way ANOVA was used for significance analysis. The results showed that DMM significantly enhanced the inhibitory effect of 5-FU on 4T1 tumors. DE. The proportions of CD3+CD8+ T cells, CD3+CD4+ T cells, and CD3+CD4+Foxp3+ T cells. DMM was found to significantly reduce the chemotherapy-induced decrease in CD4+ T cells and CD8+ T cells, promote the infiltration of anti-tumor T cells (D), and reduce the infiltration of suppressor T cells after 5-FU treatment (E). * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, **** indicates p<0.0001, and the error bar is mean ± standard error. Detailed Implementation

[0024] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention. All tissue samples mentioned herein were obtained after obtaining informed consent from the patients.

[0025] Example 1 Experimental Method

[0026] 1. Subcutaneous tumor formation in mice

[0027] (1) Digest the tumor cells to be inoculated from the cell culture dish and then count the cells.

[0028] (2) Inoculate each mouse with 10 6 Cells. Based on cell density and mouse number, aspirate the appropriate volume of cell suspension, then centrifuge at 700g for 5 minutes.

[0029] (3) Carefully aspirate the supernatant using a pump, and then resuspend the cells in a certain volume of 4°C PBS.

[0030] (4) After disinfecting the tumor inoculation site in mice with povidone-iodine, administer 1*10 6Tumor cells (MC38 mouse colon cancer cell line or B16F10 mouse skin melanoma cell line) were inoculated subcutaneously on both sides of the mouse.

[0031] 2. Drug administration treatment in mice

[0032] Chemotherapy drugs: 200 μL of 5-fluorouracil (100 mg / kg, dissolved in physiological saline) was injected intraperitoneally into mice on days 5, 8, 11 and 14 after tumor inoculation.

[0033] Anti-PD-1 monoclonal antibody: 200 μL of anti-PD-1 monoclonal antibody (BioXCell, Cat#BE0146, 10 mg / kg, dissolved in PBS) was injected intraperitoneally into mice on days 8, 11 and 14 after tumor inoculation.

[0034] Anti-IFNAR1 monoclonal antibody: 200 μL of anti-IFNAR1 monoclonal antibody (Selleck, Cat#A2121, 10 mg / kg, dissolved in physiological saline) was injected intraperitoneally into mice on days 2, 6 and 10 after tumor inoculation.

[0035] 3. Mouse tumor measurement

[0036] Tumor volume measurement:

[0037] Mice were placed belly-up, and the tumor area was wiped with povidone-iodine. The length and width of the tumor were measured and recorded every two days using calipers until approximately day 20 post-inoculation. To ensure animal welfare, excessively large tumor volumes were avoided. The tumor volume was calculated using the formula: length (mm) x width (mm). 2 x 0.5.

[0038] Tumor weight measurement:

[0039] On the last day of tumor volume measurement, mice were sacrificed, subcutaneous tumors were removed, residual hair and tissue on the tumors were cleaned, and the tumors were placed in PBS buffer at 4°C. The tumors were then weighed using an electronic balance.

[0040] 4. Mouse survival analysis

[0041] In the survival analysis of mice, the subcutaneous tumor volume was defined as greater than 1800 mm. 3 Alternatively, mice were euthanized if their body weight decreased by more than 20%. The time from tumor inoculation to euthanasia is the survival time of the mice.

[0042] 5. Flow cytometry detection of tumor-infiltrating T cells

[0043] (1) On the last day of the mouse tumor volume measurement, the mice were sacrificed, the tumors under the skin of the mice were removed, and excess hair and tissue on the tumors were removed.

[0044] (2) Transfer the tumor to a 1.5 mL centrifuge tube containing 1 mL of 4°C PBS buffer, and then use ophthalmic scissors to cut the tumor tissue into small pieces as much as possible. Alternatively, collagenase (1 mg / mL) and deoxyribonuclease (10 U / mL) can be used to digest the tumor tissue at 37°C for 45 minutes.

[0045] (3) The shredded or digested sample was filtered sequentially using 100μm, 70μm, and 40μm cell sieves in conjunction with 50mL centrifuge tubes. During this process, the sample was rinsed with PBS buffer at 4℃ to obtain a single-cell suspension.

[0046] (4) Centrifuge the cell suspension from the previous step at 4°C and 700x g for 5 minutes. Then carefully aspirate the supernatant using a pump, and resuspend the cells in the appropriate volume of 4°C PBS buffer according to the cell volume.

[0047] (5) Count the cells in the resuspended cell suspension and, based on the cell density, aspirate cells containing 2 x 10⁻⁶ cells / mL. 6 The volume required for each cell.

[0048] (6) After centrifuging at 4℃ and 1000x g for 3 minutes, carefully use a pump to aspirate the supernatant.

[0049] (7) Prepare flow cytometry antibody incubation solution. Add flow cytometry antibodies sequentially to PBS buffer (containing 2% fetal bovine serum) at 4℃, mixing well after each addition before adding the next, allowing the antibodies to bind together. Add 4μL CD3-APC, 4μL CD4-PE, 4μL CD8-FITC and 2.5μL CD45-PerCP-Cy5.5 antibody to every 100μL of PBS buffer (containing 2% fetal bovine serum).

[0050] (8) After mixing the antibody incubation solution, take 100 μL to resuspend the cells in (6). Then incubate on ice in the dark for 1 hour, gently tapping the tube wall several times during the incubation period.

[0051] (9) After centrifuging at 4℃ and 1000x g for 3 minutes, carefully aspirate the supernatant with a pump and add 1 mL of 4℃ PBS buffer (containing 2% fetal bovine serum) to resuspend the cells.

[0052] (10) Repeat (9) for a total of 4 washes.

[0053] (11) Resuspend the cells in 400 μL of 4℃ PBS buffer, then add it to the filter screen of a 5 mL flow cytometer tube, and gently tap the bottom of the tube on the table to accelerate the filtration of the liquid from the filter screen into the tube.

[0054] (12) Analyzed using a flow cytometer (FACSCalibur2, BD). Before the formal start of the experiment, a preliminary experiment was conducted using the four antibodies mentioned above with mouse lymph node tissue as a positive control.

[0055] 6. Cell immunofluorescence staining

[0056] (1) After sterilizing the 15 mm cell slide by holding it with tweezers over an alcohol lamp, place it in a six-well plate and add culture medium to the six-well plate after the slide has cooled.

[0057] (2) Add the digested cells to a six-well plate in an appropriate ratio, mix well and then place in a cell culture incubator for culture.

[0058] (3) After the cells adhere to the wall, perform the appropriate cell treatment.

[0059] (4) After cell treatment, use a pump to remove the culture medium from the six-well plate and wash twice with PBS buffer.

[0060] (5) Fixation: Add 750 μL of 4% paraformaldehyde to a six-well plate and let it stand for 15 minutes to fix. Then remove the fixative and wash three times with PBS buffer at 37°C.

[0061] (6) Punching: Add PBS buffer (containing 0.3% Triton X-100) to the six-well plate and let it stand for 15 minutes. Then aspirate the punching solution and rinse 3 times with PBS buffer (containing 0.1% Triton X-100).

[0062] (7) Blocking: Use tweezers to transfer the cell slide from the six-well plate to the humidified chamber, then add 50 μL of blocking solution to the slide, and then place the humidified chamber in a 37°C incubator for 1 hour. Blocking solution: 3% sheep serum and 5% BAS in PBS buffer.

[0063] (8) Primary antibody incubation: After removing the blocking solution from the cell slides using a pump, add the primary antibody, which has been diluted to an appropriate concentration with the blocking solution, to the cell slides. Then, incubate the slides overnight at 4°C. After the primary antibody incubation is complete, use tweezers to remove the cell slides from the humidified chamber and return them to the six-well plate. Then, add PBS buffer (containing 0.1% Triton X-100) to the six-well plate and rinse three times.

[0064] (9) Fluorescent secondary antibody incubation: After rinsing, the cell slides are removed from the six-well plate and placed back into the humidified chamber. Then, 50 μL of fluorescent secondary antibody diluted in PBS buffer (containing 0.1% Triton X-100) is added to the cell slides. The humidified chamber is placed in a constant temperature incubator at 37°C and incubated in the dark for 1 hour. After incubation, the cell slides are removed from the humidified chamber and placed back into the six-well plate with tweezers. Then, the six-well plate is rinsed three times with PBS buffer. This process should be carried out in the dark as much as possible.

[0065] (10) DAPI incubation: Use tweezers to transfer the cell smear from the six-well plate to the humidified chamber, then add 50 μL of DAPI to the smear and incubate at room temperature in the dark for 15 minutes. Afterward, use tweezers to transfer the smear back to the six-well plate, and then add PBS buffer to the six-well plate to rinse three times. This process should be carried out in the dark as much as possible.

[0066] (11) Mounting: Add 3 μL of mounting medium to a clean glass slide, then drain the cell slide on tissue paper and invert it onto the mounting medium. When inverting, touch one end of the slide to the glass slide first, and then slowly lower the other end of the slide to avoid air bubbles.

[0067] (12) Observation and photography were performed using a laser confocal fluorescence microscope (ZEISS LSM 880).

[0068] 7. Immunofluorescence staining of tumor tissue

[0069] (1) Fixation: After removing the tumor from the mouse, the hair and excess tissue were cleaned off, and a portion of the tumor was then immersed in 4% paraformaldehyde and fixed at 4°C for 24 hours. After fixation, the tumor was rinsed three times with PBS buffer.

[0070] (2) Dehydration: Remove the PBS buffer, immerse the tumor tissue in a 30% sucrose solution, and then dehydrate at 4°C for 24 hours.

[0071] (3) Embedding: After dehydration, the tumor tissue was embedded in OCT and stored in a refrigerator at -80°C.

[0072] (4) Sectioning: Prepare 10μm frozen sections from the tissue embedded in OCT in the previous step and attach them to cation-resistant slides.

[0073] (5) Baking the slide: Place the slide with the tumor tissue attached in a 55°C oven for 1 hour.

[0074] (6) Antigen retrieval: Rinse the slide three times with 1x TBS buffer for 5 minutes each time to remove OCT. After shaking off the liquid from the slide, add antigen retrieval solution to the tissue and incubate at room temperature for 15 minutes.

[0075] (7) Blocking: Rinse the slide three times with 1x TBS buffer on a horizontal shaker for 5 minutes each time. Then, shake off the liquid from the slide, circle the tumor tissue with a histochemical pen, place the slide on a humidified chamber, and add blocking solution (containing 1% BSA and 10% sheep serum) to the tissue. Then, place the humidified chamber in a constant temperature incubator at 37°C for 1 hour to block.

[0076] (8) Primary antibody incubation: After the blocking is completed, the primary antibody diluted with TBS buffer (containing 1% BSA and 10% sheep serum) is dropped onto the tumor tissue, and then the humidified chamber is placed in a chromatography cabinet at 4°C for overnight incubation.

[0077] (9) Secondary antibody incubation: Rinse the slide three times with 1x TBS buffer on a horizontal shaker for 5 minutes each time. After that, shake off the liquid from the slide, add the fluorescent secondary antibody diluted with TBS buffer to the tissue, and then place the humidified chamber in a constant temperature incubator at 37°C and incubate in the dark for 1 hour.

[0078] (10) DAPI incubation: Rinse the slide three times with 1x TBS buffer on a horizontal shaker for 5 minutes each time. After that, shake off the liquid from the slide, add DAPI to the tissue, and then incubate in a humidified chamber at room temperature in the dark for 15 minutes.

[0079] (11) Mounting: Add 10 μL of mounting medium to the slide, and then invert the coverslip onto the mounting medium. When inverting, first touch one end of the coverslip to the slide, and then slowly lower the other end of the slide to avoid air bubbles.

[0080] (12) Observation and photography were performed using a laser confocal fluorescence microscope (ZEISS LSM 880).

[0081] 8. Lysosomal staining in cells

[0082] After digestion, the cells were spread in a suitable ratio in a confocal culture dish, gently shaken to mix, and then placed in a cell culture incubator for incubation. Once the cells adhered, appropriate treatments were performed. Before observation using a confocal fluorescence microscope, LysoTracker was added to the culture medium. TM The cells were stained with Red (1:5000) and Hoechst 33342 (1:100) and then placed in a 37°C cell culture incubator for 30 minutes. After washing twice with PBS buffer and replacing with fresh culture medium, the cells were observed and photographed using a laser confocal fluorescence microscope (ZEISS LSM 880).

[0083] 9. Immunohistochemical staining

[0084] Immunohistochemical staining of tumor tissue:

[0085] (1) Colorectal cancer tumor tissue (17 cases) from Peking University People's Hospital and breast cancer tissue (20 cases) from Lishui Central Hospital were fixed and embedded in paraffin.

[0086] (2) Prepare 5μm paraffin sections and attach them to a glass slide to prevent detachment. Then place the slides in a constant temperature incubator at 60℃ for 1 hour.

[0087] (3) Dewaxing: The paraffin sections were soaked in xylene I and II for 15 minutes respectively, in order to remove the paraffin from the tissue.

[0088] (4) Hydration: During the dewaxing process, xylene also enters the tissue. Since the xylene in the tissue is immiscible with the water-soluble staining solution used later, it is necessary to gradually replace the xylene in the tissue by immersing the sections in anhydrous ethanol I and II for 10 minutes each, followed by immersion in 95%, 90%, 80% and 70% ethanol for 5 minutes each. Finally, soak in pure water for 5 minutes.

[0089] (5) Inactivate endogenous peroxides: Soak the slices from the previous step in freshly prepared 3% H2O2 at room temperature for 15 minutes, then pour off the H2O2 and rinse twice with pure water.

[0090] (6) Antigen heat retrieval: Immerse the slices from the previous step in citrate buffer (pH=6.0), then boil them in a water bath at 100°C for 20 minutes, and then cool them to room temperature. The purpose of boiling is to expose the antigen sites.

[0091] (7) Serum blocking: After discarding the citrate buffer, rinse twice with pure water, and then wash three times with PBS buffer, 5 minutes each time. After shaking off the liquid on the slide, draw a circle around the tissue with a histochemical pen, then add serum blocking solution within the circled area, and then incubate in a constant temperature incubator at 37°C for 30 minutes.

[0092] (8) Primary antibody incubation: After blocking, wash the slides three times with PBS buffer for 5 minutes each time. Then dilute the primary antibody to an appropriate concentration with primary antibody dilution buffer, add it to the tissue, and then place the slides on a humidified chamber and incubate overnight at 4°C.

[0093] (9) Secondary antibody incubation: After the primary antibody incubation is completed, wash the slides three times with PBS buffer, 5 minutes each time. Then add the diluted secondary antibody solution to the tissue and incubate in a constant temperature incubator at 37°C for 30 minutes.

[0094] (10) DAB staining: After the secondary antibody incubation is complete, wash the slide three times with PBS buffer, 5 minutes each time. Then, add the diluted DAB working solution to the tissue for staining. During this process, observe the staining of the tissue under a microscope. When the staining degree is suitable, wash the slide in pure water to stop the staining. Finally, rinse the slide with tap water and pure water for 5 minutes each.

[0095] (11) Counterstaining: Immerse the slide in hematoxylin staining solution, stain for 1 minute, rinse with tap water for 5 minutes, and then rinse with pure water for 5 minutes.

[0096] (12) Dehydration: Immerse the counterstained slides in 70%, 80%, 90%, and 95% ethanol for 5 minutes each. Then immerse them in anhydrous ethanol I and II and xylene I and II for 10 minutes each.

[0097] (13) Mounting: Mount the dehydrated tissue with neutral resin to avoid air bubbles.

[0098] (14) Use a pathology scanner to scan and photograph the slides.

[0099] (15) Protein levels are divided into 1-5 grades based on staining intensity. The higher the number, the stronger the staining and the higher the protein level.

[0100] 10. Virtual screening of drugs

[0101] Since the P18 crystal structure lacks a natural ligand, the optimal binding site for the P18 protein was first predicted using the SiteMap module in Schrödinger's algorithm. Then, the Receptor Grid Generation module was used to perfectly enclose the predicted binding site using an optimal enclosing box, and the active site of the protein was obtained based on this. Each processed ligand from six compound libraries was sequentially docked with the active site of the LAMTOR1 protein using HTVS, SP, and XP methods, with progressively increasing docking precision. A lower score indicates a lower binding free energy and higher binding stability between the compound and the protein. The lowest-scoring compound and the active site of the P18 protein were analyzed using MM-GBSA calculations. MM-GBSA ADG Bind can approximate the binding free energy between small molecules and proteins; a lower binding free energy indicates higher binding stability between the ligand and the P18 protein.

[0102] 11. Data Analysis

[0103] Unless otherwise specified, when comparing two groups, the statistical method used is a two-sided unpaired t-test; when comparing more than two groups, one-way ANOVA is used. ns indicates no significant difference, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001, and the error bar is the mean ± standard error.

[0104] Example 2 Experimental Results

[0105] 1. Chemotherapy can downregulate the protein level of cGAS in tumor cells.

[0106] To investigate the effect of chemotherapy drugs on cGAS protein levels in tumor tissues, we collected tumor tissues from colorectal cancer or breast cancer patients before and after chemotherapy and prepared paraffin sections. Immunohistochemical staining was performed using an antibody against cGAS. The immunohistochemical results showed that cGAS staining in tumor tissues from patients who underwent chemotherapy was significantly weaker than in tumor tissues from patients who did not receive chemotherapy. Figure 1 A- Figure 1 D). Western blot analysis of rectal or breast tumor specimens before and after chemotherapy showed that the protein level of cGAS in tumor tissue was lower after chemotherapy. Figure 1 E and Figure 1 F). The above results all indicate that the protein level of cGAS in tumor tissue decreases after chemotherapy in cancer patients.

[0107] To further confirm that chemotherapy drugs can directly affect the protein level of cGAS in tumor cells, we treated various tumor cell lines with different chemotherapy drugs and then detected the protein and mRNA levels of cGAS in the cells. The treatment method was as follows: after treating cells with different chemotherapy drugs for 24 hours, the cells were washed with PBS buffer and then cultured in fresh culture medium for 0-3 days. Subsequently, cells were scraped at different time points for Western blotting or quantitative real-time PCR analysis. Figure 2 The results of protein immunoblotting were consistent with those of tumor-bearing mice treated with chemotherapy drugs; under the continuous action of chemotherapy drugs, the protein level of cGAS in various tumor cell lines generally showed a decreasing trend. Figure 2 Conversely, quantitative real-time PCR results showed that intracellular cGAS mRNA levels maintained an increasing trend throughout the entire course of chemotherapeutic drug action. Figure 2 D). This suggests that chemotherapy drugs downregulate the protein level of cGAS in tumor cells not by reducing the mRNA level of cGAS in cells, but possibly by promoting the degradation of cGAS.

[0108] 2. LAMTOR1 mediates the lysosomal degradation of cGAS

[0109] Conversely, knockdown of LAMTOR1, gradient overexpression of exogenous LAMTOR1 in tumor cells reduced cGAS protein levels, and this reduction was positively correlated with LAMTOR1 expression levels. Figure 3 A). Experiments using cycloheximide (CHX) to determine protein half-life showed that knocking down LAMTOR1 prolonged the half-life of cGAS in tumor cells. Figure 3 B), while overexpression of LAMTOR1 can shorten the half-life of cGAS (B). Figure 3 C). These results indicate that LAMTOR1 can promote the degradation of cGAS.

[0110] The previous results have demonstrated that chemotherapy drugs can promote the lysosomal degradation of cGAS and that LAMTOR1 can negatively regulate cGAS protein levels. Next, we further verified that LAMTOR1 promotes cGAS degradation through the lysosomal pathway. We treated cells with lysosomal pathway inhibitors chloroquine (CQ) or proteasome inhibitor MG132 after LAMTOR1 overexpression. The results showed that CQ could prevent LAMTOR1 overexpression-induced cGAS degradation, while MG132 could not. Figure 3 D), and direct overexpression of LAMTOR1 also promotes the entry of more cGAS into lysosomes (D). Figure 3 E). Conversely, knocking down LAMTOR1 inhibits the process of 5-fluorouracil or HT-DNA-induced cGAS entry into lysosomes (E). Figure 3 F). The above results indicate that LAMTOR1 can mediate the lysosomal degradation of cGAS.

[0111] 3. Knockdown of LAMTOR1 inhibits tumor growth in mice

[0112] Since cGAS activation can inhibit tumor development and progression, we hypothesize that LAMTOR1 can also affect tumor progression by regulating cGAS protein levels. Although clonogenic and cell counting experiments showed that LAMTOR1-knockout tumor cells did not grow differently from wild-type cells in vitro (…),… Figure 4 However, tumorigenesis experiments in mice showed that the growth of tumor cells with knocked-down LAMTOR1 was significantly inhibited in immunogenic mice. Figure 4 .CD). However, when LAMTOR1 knockdown or wild-type B16F10 tumor cells were inoculated into immunodeficient mice, LAMTOR1 deficiency no longer inhibited tumor growth. Figure 4These results indicate that the effect of LAMTOR1 on tumor growth depends on the host's immune system. Survival experiments in immunogenic tumor-bearing mice showed that mice inoculated with LAMTOR1-deficient tumor cells had longer survival times. Figure 4 In summary, knocking down LAMTOR1 in tumor cells can inhibit tumor growth in immune-normal mice.

[0113] 4. LAMTOR1 deficiency can remodel the immunosuppressive tumor microenvironment.

[0114] Numerous studies have demonstrated that cGAS participates in tumor immunity by regulating T lymphocyte recruitment and differentiation. Given that we have already demonstrated that LAMTOR1 negatively regulates cGAS protein levels and activation status, we will further investigate the function of LAMTOR1 in tumor immunity, including the recruitment of anti-tumor T cells and macrophage polarization. Through tissue immunofluorescence and flow cytometry analysis of subcutaneously inoculated B16F10 tumors in mice, we found that tumors lacking LAMTOR1 showed relatively higher levels of CD3+, CD4+, and CD8+ T cell infiltration. Figure 5 (AB). This indicates that LAMTOR1 deficiency can promote the infiltration of anti-tumor T cells in mouse tumors.

[0115] To demonstrate that the tumor growth inhibition induced by LAMTOR1 knockdown depends on the cGAS-STING pathway, we also knocked out cGAS in wild-type and LAMTOR1-knockdown B16F10 cell lines. The constructed cell lines were then subcutaneously seeded into C57BL / 6J mice, and tumor volume and weight were recorded. The results showed that after cGAS knockout, LAMTOR1 knockdown no longer had an inhibitory effect on tumor growth. Figure 5 In addition, we also injected tumor-bearing mice intraperitoneally with a monoclonal antibody targeting interferon alpha and beta receptor subunit 1 (IFNAR1) to block the downstream effects of type I interferon. Tumor growth experiments showed that this monoclonal antibody could also, to some extent, reverse the inhibitory effect of LAMTOR1 deficiency on tumor growth. Figure 5 .D). Furthermore, treating RAW264.7 macrophages with the culture medium of LAMTOR1 knockdown or wild-type 4T1 cells and B16F10 cells for 24 hours revealed that qPCR analysis showed significantly increased levels of markers CXCL10 and TNFα in M1 macrophages, while significantly downregulated levels of markers IL10 and AGR1 in M2 macrophages. Figure 5These results demonstrate that the inhibition of tumor growth by LAMTOR1 deficiency is indeed achieved by activating the cGAS-STING pathway, and that LAMTOR1 knockdown can promote the polarization of M1 macrophages, thereby enhancing anti-tumor immunity.

[0116] 5. LAMTOR1 deficiency can enhance the efficacy of immunotherapy and chemotherapy.

[0117] Subcutaneous xenograft models of LAMTOR1 knockdown or wild-type B16F10 cells were constructed subcutaneously using C57BL / 6J mice, and the cells were treated with anti-PD-1 monoclonal antibody. Results showed that LAMTOR1 knockdown combined with anti-PD-1 monoclonal antibody therapy achieved significant anti-tumor efficacy, with tumor growth in the combined group almost completely halted. Figure 6 .A), and the lowest tumor weight ( Figure 6 .B). Immunofluorescence and flow cytometry were used to detect the infiltration of anti-tumor T cells in LAMTOR1 knockdown or wild-type B16F10 tumors. The results showed that LAMTOR1-deficient tumors, after treatment with anti-PD-1 monoclonal antibodies, had the highest infiltration of anti-tumor T cells in mouse tumors. Figure 6 The results indicate that knocking down LAMTOR1 in combination with anti-PD-1 monoclonal antibody can significantly improve anti-tumor efficacy and T cell infiltration.

[0118] Evidence suggests that cGAS is indispensable for activating the anti-tumor immune response to chemotherapy drugs. Therefore, we hypothesize that LAMTOR1 knockdown tumors, after treatment with chemotherapy drugs, will have a higher level of cGAS-STING activation, and the two can synergistically exert an anti-tumor effect. Mouse tumor growth experiments showed that the growth rate of LAMTOR1 knockdown tumors was significantly slowed after treatment with 5-FU. Figure 7 .AB). Survival experiments in tumor-bearing mice showed that mice with LAMTOR1 knockdown in the tumor combined with 5-FU treatment had the longest survival time. Figure 7 The results above indicate that knocking down LAMTOR1 can significantly enhance the effect of chemotherapy.

[0119] Example 3: Screening of P18 inhibitors and functional verification of their antitumor drug sensitizers

[0120] Based on comprehensive libraries of natural products, antitumor compounds, and the FDA database, molecular docking was performed using key P18 PDBs. The lowest-scoring ligands were analyzed using MM-GBSA calculations to determine their binding stability to the P18 protein. Lower scores indicated higher ligand-P18 protein binding stability. The three compounds with the lowest scores were selected for further validation. Further molecular docking results showed that all three natural products (Demethylmurrayanine, DMM; Tirapazamine, TPZ; and Clausine Z) bound to P18. DMM, as a P18 inhibitor, has been shown to inhibit the binding of P18 to cGAS, thereby inhibiting P18 degradation of cGAS and further activating downstream cGAS-STING. Therefore, we began to investigate the impact of DMM on the antitumor efficacy after chemotherapy and the tumor immune microenvironment.

[0121] By transfecting 293T cells with P18 and / or cGAS plasmids, and adding the selected potential compounds 24 hours later, the interaction between the two was examined 12 hours later. The results showed that the natural product Demethylmurrayanine (DMM) interfered with the interaction between P18 and cGAS. Figure 8 .A), and there is a significant dose dependence ( Figure 8 .B).

[0122] Next, we verified the effect of DMM on the cGAS-STING pathway in tumor cells. First, in cervical cancer cells HeLa ( Figure 8 In .C), we examined the activation levels of P-TBK1 and P-IRF3 in tumor cells by DMM, demonstrating that the activation of cGAS-STING by DMM is dose-dependent. Figure 8 .C) and time dependence ( Figure 8 .D).

[0123] Therefore, we confirmed that the natural product Demethylmurrayanine (DMM) can significantly enhance the activation of cGAS-STING after 5-FU chemotherapy, suggesting that DMM can indeed act as an inhibitor of P18, thereby alleviating the degradation of cGAS by P18 and enhancing anti-tumor immunity.

[0124] Furthermore, we treated 4T1 cells (a breast tumor cell line) with 5-FU or 5-FU + DMM, and performed EdU staining for cell proliferation 24 hours later. The results showed that DMM significantly enhanced the anti-tumor proliferative capacity of 5-FU. Figure 9In addition, in HeLa cells, administration of 5-FU followed by DMM at different recovery times (0, 24h, 48h) showed that DMM could restore the activation of the cGAS-STING pathway to some extent. Figure 9 .B). 4T1 cells were subcutaneously inoculated into C57BL / 6J mice. On days 5, 8, 11, and 14 post-inoculation, mice were intraperitoneally injected with 5-FU or 5-FU + DMM, respectively. Mice were sacrificed at the experimental endpoint, and tumors were harvested. It was found that DMM enhanced the antitumor activity of 5-FU in vivo and inhibited tumor growth. Figure 9 (C). The tumor cells were minced and ground to obtain a suspension. The suspension was stained with antibodies against FITC-CD3, APC-CD4, and PE-CD8, and the proportions of CD3+CD8+ T cells, CD3+CD4+ T cells, and CD3+CD4+Foxp3+ T cells were detected by flow cytometry. It was found that DMM significantly alleviated the chemotherapy-induced decrease in CD4+ T cells and CD8+ T cells, and promoted the infiltration of anti-tumor T cells. Figure 9 .D). Furthermore, we found that the addition of DMM could reduce the proportion of inhibitory Foxp3+ T cells and improve 5-FU-induced immunosuppression (D). Figure 9 (E). All of the above indicate that DMM can enhance the infiltration of anti-tumor T cells and the anti-tumor efficacy after chemotherapy.

[0125] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The use of the combination of demethylmurrayanine (DMM) and 5-Fu in the preparation of medicaments for treating breast cancer, colon cancer, or melanoma, characterized in that, The application involves using demethylmurrayanine (DMM) to interfere with the interaction between P18 and cGAS, thereby affecting the degradation of cGAS by P18.

2. The application as described in claim 1, characterized in that, Demethylmurrayanine alleviates the inhibition of cGAS-STING by 5-FU, promotes the infiltration of anti-tumor T cells after 5-FU treatment, and reduces the proliferation of suppressor T cells after 5-FU treatment, thereby alleviating the immunosuppressive tumor microenvironment caused by 5-FU treatment and improving the efficacy of anti-tumor immunotherapy.

3. The application as described in claim 1, characterized in that, The dosage form of the drug is tablets, capsules, injections, or granules.

4. The application as described in claim 3, characterized in that, The capsule is a microcapsule.

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